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Genetic regulation of formate hydrogenlyase in Escherichia coli

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Title:
Genetic regulation of formate hydrogenlyase in Escherichia coli
Creator:
Maupin, Julie Anne, 1963-
Publication Date:
Language:
English
Physical Description:
xv, 143 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
DNA ( jstor )
Escherichia coli ( jstor )
Formates ( jstor )
Genes ( jstor )
Molybdates ( jstor )
Nitrates ( jstor )
Operon ( jstor )
Plasmids ( jstor )
Proteins ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Microbiology and Cell Science thesis Ph. D
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 120-142).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Julie Anne Maupin.

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The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
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GENETIC REGULATION OF FORMATE HYDROGENLYASE IN ESCHERICHIA COLI


By

JULIE ANNE MAUPIN














A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA


1991














ACKNOWLEDGEMENTS


I would like to sincerely thank my advising professor Dr. K. T. Shanmugam. His continual encouragement, guidance and enthusiasm for science have definitely influenced me for a lifetime. I would like to thank the K. T. S. laboratory group including both old and new. I would also like to extend my thanks to Drs. Richard Boyce, Francis Davis Jr., John Gander, and Lonnie Ingram for their helpful questions and counsel while serving on my committee. Additionally, I would like to thank Drs. A. B6ck and B. Bachmann for providing several strains and plasmids necessary for this study. Finally, special thanks are extended to my family and fiance for their willingness to listen to my ups and downs throughout the course of this work.


ii














TABLE OF CONTENTS

page

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . i

TABLE OF CONTENTS. . . . . . . . . . . . . . . . . . . . . . . . . iii

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . .v

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . vii

ABBREVIATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . .viii

LIST OF GENE SYMBOLS. . . . . . . . . . . . . . . . . . . . . . . . xi

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . .7
Anaerobiosis. . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Nitrate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Formate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Molybdenum. . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Nickel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Selenium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . .. . 30
Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Bacterial Strains and Media. . . . . . . . . . . . . . . . . . . .30
Isolation of Mutants . . . . . . . . . . . . . . *. .. .. . . . 30
Enzyme Activities and their Respective Culture Conditions. . . . .36 Genetic and Molecular Biological Experiments. . . . . . . . . . .41

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . .53

Physiological Properties of an fhlB Mutant, Strain SE-2011 . . . .53 Genetic Characteristics of Strain SE-2011, 0(fhlB'-'lacZ+) . . . .64 Trans-acting Factors of the fhlB Operon. . . . . . . . . . . . . .70
Analysis of the fhlA Gene. . . . . . . . . . . . . . . . . . . . .79


iii








Regulation of the fhlA Gene. . . . . . . . . . . . . . . . . . . .94
Molybdate Metabolism and FHL Activity. . . . . . . . . . . . . . 110

CONCLUSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . .116

REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

BIOGRAPHICAL SKETCH. . . . . . . . . . . . . . . . . . . . . . . . 143









































iv














LIST OF FIGURES
Figure page


1 Model of the genetic regulation of formate hydrogenlyase in
E. coli. . . . . . . . . . . . . . . . . . . . . . . . . . . .4

2 Mixed-acid fermentation of E. coli . . . . . . . . . . . . . . 9

3 Differential rate of synthesis of 0-galactosidase activity by
0(fhlB'-'lacZ+) strain SE-2011 grown in LB medium with
different supplements. . . . . . . . . . . . . . . . . . . . .59

4 Effect of formate on the induction of P-galactosidase
activity by 0(fhlB-lacZ+) strain SE-2011 and a pfl
derivative, strain MJ-9. . . . . . . . . . . . . . . . . . . .61

5 Effect of nitrate supplementation on fhlB gene expression. . .62

6 Differential rate of induction of the 0(fhlB'-'lacZ+)
fusion in strain SE-2011 in the presence and absence of
plasmid pSE-133. . . . . . . . . . . . . . . . . . . . . . . .73

7 Effect of formate concentration on the levels of P-galactosidase activity produced by a 0(fhlB'-'lacZ+) pfl double
mutant, strain MJ-9, in the presence of plasmids pSE-133 and
pSE-133-2 (fhlA::Tn5). . . . . . . . . . . . . . . . . . . . .74

8 Differential rate of synthesis of 0(fdhF'-'lacZ+), strain
M9s, and 0(fhlB'-'lacZ+), strain SE-2011, in LB medium
supplemented with 3 mM formate in the presence and absence
of the fhlA+ gene in a multicopy plasmid (pSE-133-1). . . . . 77

9 Nucleic acid and predicted protein sequences of the
partial hypC gene and complete hydB, hydF and fhlA genes . . .80

10 Alignment of the predicted sequences of E. coli FHL-A,
E. coli NTR-C, K. pneumoniae NIF-A, and P. putida XYL-R
proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .85


v








11 Alignment of the predicted "domain 0" amino acid sequences
of E. coli FHL-A, K. pneumoniae NIF-A, S. typhimurium FLB-D, E. coli NTR-C, P. putida XYL-R, R. leguminosarum DCT-D, and
E. coli TYR-R . . . . . . . . . . . . . . . . . . . . . . . . .88

12 Alignment of potential DNA-binding motifs in the FHL-A,
HYD-G, NTR-C, XYL-R and NIF-A proteins. . . . . . . . . . . . .90

13 Localization of the promoter lac fusions in strains SE-2007
[O(fhlA'-'lacZ+)], SE-2001 [(hyp'-'ZacZ+)1], and SE-2002
[ (hyp -I acZ+)2] . . . . . . . . . . . . . . . . . . . . . . .96

14 Differential rate of synthesis of 0(fhlA'-'lacZ+), strain
SE-2007, and *(hypX'-'lacZ+)1, strain SE-2001, in LB
medium supplemented with 3 mM formate in the presence and
absence of the fhlA+ gene in a multicopy plasmid (pSE-133). . 108

15 Nucleic acid sequence of the mol (chl) operon. . . . . . . . .113


vi














LIST OF TABLES


Table page

1 Strains used in this study. . . . . . . . . . . . . . . . . . .31

2 Plasmids used in this study . . . . . . . . . . . . . . . . . .48

3 Biochemical characterization of strain MC4100 and an fhlB mutant, strain SE-2011. . . . . . . . . . . . . . . . . . 54

4 Effect of media composition on the expression of
*(fhlB'-'lacZ+) in an fhlB mutant, strain SE-2011 . . . . . . .57

5 Plasmid complementation analysis of an fhlB mutant,
strain SE-2011. . . . . . . . . . . . . . . . . . . . . . . . .68

6 Expression of $(fhlB'-'lacZ+) in different genetic backgrounds . . . . . . . . . . . . . . . . . . . . . . . . . .71

7 Effect of dioxygen on the expression of $(fhlA'-'lacZ+) in
an fhlA mutant, strain SE-2007 . . . . . . . . . . . . . . . . 97

8 Effect of media composition on the expression of $(fhlA'-'lacZ+) in an fhlA mutant, strain SE-2007 . . . . . . .99

9 Expression of 0(fhlA'-'lacZ+) in different genetic backgrounds . . . . . . . . . . . . . . . . . . . . . . . . . 100

10 Anaerobic expression of 0(hyp'-'lacZ+), strains SE-2001 and SE-2002, in different genetic backgrounds and culture media. . 104 11 The effect of molybdenum on expression of 0(fhlB'-'lacZ+), mutant strain SE-2011, in chiD and moiR genetic backgrounds. . 111


vii














LIST OF ABBREVIATIONS


A . . . . . . . . Ac-CoA. . . . . .

ATP . . . . . . .

BV . . . . . . .

CAT . . . . . . .

CRP . . . . . . .

CTAB . . . . . . DMS . . . . . . .

DMSO. . . . . . .

FDH-H . . . . . .


FDH-N . . . . . .


FHL FR. HUP

HYD HYD-1 HYD-2 HYD-3 LB. .


Absorbance Acetyl coenzyme A Adenosine triphosphate Benzyl viologen Chloramphenicol acetyltransferase cAMP repressor protein Hexadecyltrimethylammonium bromide Dimethyl sulfide Dimethyl sulfoxide Formate dehydrogenase (linked to H2 evolution)

Formate dehydrogenase (linked to N03 reduction)

Formate hydrogenlyase Fumarate reductase Hydrogen uptake activity Hydrogenase Hydrogenase isoenzyme-1 Hydrogenase isoenzyme-2 Hydrogenase isoenzyme-3 Luria Broth

viii


. . . . . . . . . . .








LBG . . . . . . . . . . . . Luria Broth + glucose

LBGF. . . . . . . . . . . . Luria Broth + glucose + formate

LBF . . . . . . . . . . . . Luria Broth + formate

LBM . . . . . . . . . . . . Luria Broth + maltose

LBN . . . . . . . . . . . . Luria Broth + nitrate

LBNF. . . . . . . . . . . . Luria Broth + nitrate + formate

MES . . . . . . . . . . . . 2-(N-Morpholino)ethanesulfonic acid

Mo. . . . . . . . . . . . . Molybdenum

MOPS. . . . . . . . . . . . 3-(N-Morpholino)propanesulfonic acid

MPT . . . . . . . . . . . . Molybdopterin

MGD . . . . . . . . . . . . Molybdopterin guanine dinucleotide

NAD+, NADH. . . . . . . . . Nicotinamide adenine dinucleotide and its reduced form, respectively OAA . . . . . . . . . . . . Oxaloacetate

ONPG . . . . . . . . . . . Ortho-nitrophenyl--D-galactopyranoside

ORF . . . . . . . . . . . . Open reading frame

PDH . . . . . . . . . . . . Pyruvate dehydrogenase

PEP . . . . . . . . . . . . Phosphoenolpyruvate

PFL . . . . . . . . . . . . Pyruvate formatelyase

PIPES . . . . . . . . . . . Piperazine-N,N'-bis[2-ethanesulfonic
acid]; 1,4-piperazinediethanesulfonic acid PMF . . . . . . . . . . . . Proton motive force

SDS . . . . . . . . . . . . Sodium dodecyl sulfate

TES . . . . . . . . . . . . N-Tris-(hydroxymethyl)methyl-2aminoethanesulfonic acid
TMA . . . . . . . . . . . . Trimethylamine

ix








TMAO. . . . . . . . . . . .

Tris. . . . . . . . . . . .

X-gal . . . . . . . . . . .


Trimethylamine-N-oxide Tris-(hydroxymethyl)-aminomethane 5-Bromo-4-chloro-3-indolyl-o-Dgalactopyranoside


x














LIST OF GENE SYMBOLS

All the genes listed below are from Escherichia coli unless otherwise indicated.

Gene Alternate gene symbols; phenotype affected
symbol

ant See hyc; anaerobic electron transport

arcA Aerobic regulatory control, putative DNA binding
protein of Arc modulon

arcB Aerobic regulatory control, histidine-proteinkinase of Arc modulon

chiA Synthesis of the pterin component of MPT

chlB "Association factor-FA"; synthesis of functional MPT

chIC See narGHJI

chID Peripheral protein of molybdate binding-protein-dependent
transport system

chiE Synthesis of the pterin component of MPT

chIF See fdhGHI

chiG Molybdate-restorable nitrate reductase activity

chlJ' Integral membrane protein of molybdate binding-proteindependent transport

cyd Cytochrome d; high-affinity oxidase

cyo Cytochrome o; low-affinity oxidase

dctD C-4 dicarboxylate transport; Rhizobium leguminosarum

dmsABC DMSO and TMAO reductase activity


xi








ebgA Ancestral gene for O-galactosidase

fdhF FDH-H; formate dehydrogenase (linked to H2 evolution)

fdhGHI FDH-N; formate dehydrogenase (linked to N03 reduction)

fh1A Putative DNA binding protein necessary for transcriptional
activation of the fdhF, hyc and fhlB operons

fhlB Putative modulator of the FHL pathway; necessary for FHL,
FDH-H and total HYD activity

flbD Flagellar synthesis; Caulobacter crescentus

fnr Global regulator of anaerobic respiration; homologous to CRP

frdABCD Fumarate reductase

gyrB DNA gyrase

his Histidine biosynthesis and binding-protein-dependent
transport system

hyb Hydrogenase isoenzyme-2

hyc Hydrogenase isoenzyme-3

hydA Total hydrogenase activity

hydFB hypDE; total hydrogenase activity

hydE hypB; total hydrogenase activity; Ni suppressible

hydC,D Total hydrogenase activity; nickel transport

hyd-17 ORF5 of the hyc operon, hydrogenase isoenzyme-3

hydG Putative DNA binding protein of dihydrogen metabolism
(uptake)

hyp Total hydrogenase activity, operon includes previously
described hydB, E, and F genes

mal Maltose metabolism and binding-protein-dependent transport
system

moIR Molybdate binding-protein-dependent transport system

xii








narL Putative DNA binding protein of nitrate regulation

narX Putative membrane-bound histidine-protein-kinase of
nitrate regulation

narQ Second proposed histidine-protein-kinase of nitrate
regulation

narGHJI Nitrate reductase

nifA Positive activator for nitrogen fixation; Klebsiella
pneumoniae

ntrC ginG, nitrogen metabolism

pf1 Pyruvate formatelyase

pgi oxrC, phosphoglucose isomerase

tyrR Aromatic amino acid biosynthesis and transport

xylR Degradative pathway of aromatic hydrocarbons; Pseudomonas
putida


xiii














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

GENETIC REGULATION OF FORMATE HYDROGENLYASE IN ESCHERICHIA COLI By

Julie Anne Maupin

December, 1991

Chairman: Dr. K. T. Shanmugam
Major Department: Microbiology and Cell Science


A new gene (fh B) whose product regulates the production of

formate hydrogenlyase (FHL) has been identified in Escherichia coli. Biochemical analysis of a mutant, strain SE-2011 [0(fhlB'-'lacZ+)], revealed that this mutant lacks formate dehydrogenase activity associated with FHL (FDH-H) and hydrogenase activity. As a consequence, dihydrogen production and uptake were undetectable in strain SE-2011. Expression of the fhlB gene (measured as 0-galactosidase activity) was increased 2- to 3-fold by anaerobic conditions, and enhanced by formate, but only under anaerobic conditions. Maximum expression of *(fhlB''locZ+) required rpoN, fhlA, chID, and moiR gene products. The concentration of formate required for maximum expression of the fhlB gene was reduced in the presence of fhlA gene in a multicopy plasmid from about 15 mM to 3 mM. The fhlB gene in moiR or chID genetic


xiv








background still retained formate inducibility; however, molybdate was required at high concentrations for expression to wild type levels. DNA sequence analysis of fhlA gene revealed that the FHL-A protein is homologous to other known transcriptional activators. It is proposed that formate, in association with the FHL-A protein, initiates transcription of the fhlB, hyc, hyp and fdhF operons. Analysis of mutant strain SE-2007 [4(fhlA'-'lacZ+)] indicated that the transcription of the fhlA gene, located at the 3'-end of the hyp operon, is constitutive. This is in contrast to the hyp operon transcription which has dual controls: formate-dependent and FNR-dependent. The DNA sequence of the mol operon (essential for molybdate transport, FHL activity and fhlB expression) was also determined to investigate the role of molybdate metabolism in the regulation of FHL in E. coli. These studies revealed that the fermentative dihydrogen production is regulated by a complex series of reactions involving the availability of formate, Mo and absence of 02. It is postulated that the FHL-B protein is one of the modulators of this control system.


xv














INTRODUCTION


The production of dihydrogen by the members of the

Enterobacteriaceae family has been known since 1901 (166). Although much has been learned since that time, many avenues of research remain to be explored. It is now understood that the formate hydrogenlyase complex is composed of FDH-H, HYD-3, and associated electron transport proteins (10, 27, 72, 173, 193, 215, 243). This complex catalyzes the oxidation of formate to H2 and C02 (2, 166) and is found only in anaerobically growing cells. The reaction catalyzed by the FHL complex operates at a standard free energy (AGO') of zero and does not appear to be involved in cellular energy production (2). It is presumed that the FHL system plays an important role in later stages of the microorganism's fermentative growth when organic acids such as formate accumulate and lower the pH of the culture medium. The cell apparently synthesizes the FHL complex in order to modulate the external pH, thus facilitating growth (77).

The FHL pathway is an attractive research field due to the

potential of harnessing dihydrogen production as an alternative energy source. Current events, including the Persian Gulf War and concern over the greenhouse effect, have aroused the public's attention. Although lobbyists continue to solicit Congress for the oil industry, the


1








2

American population is more aware of this crisis and open to environmentally and economically sound options. Research in these fields must continue in order for alternative fuels to become more competitive. Unfortunately, historical events, i.e. the "Hindenberg", have curbed the use of dihydrogen as an energy source. When properly controlled, dihydrogen is a clean burning fuel. The end product being H20, instead of CO, C02, S02 and nuclear waste (the current waste products of energy production in our country).

Additionally, the question of how an organism responds to various environmental stimuli such as dioxygen and regulates the metabolism by activating or repressing specific genes remains to be fully elucidated. By studying the FHL system of Escherichia coli, one can not only integrate regulatory similarities from this system to other pathways, but also study modes of control which are unique to FHL in order to gain a clear understanding of gene expression. Associating this with the physiological role of anaerobic H2 and C02 production is also of interest. The metabolic fate of the end products of FHL are poorly understood.

Following the advent of DNA sequencing techniques and computer programs designed to organize the sequences generated, studies on protein homology have enabled the researcher to identify apparent function from the primary structure. It is now understood that there are two categories of proteins involved in signal transduction: sensor and receiver. Generally, the sensor is a membrane-bound, histidine-








3

protein-kinase. Appropriate environmental stimulation results in sensor autophosphorylation cascading to phosphorylation of the receiver protein. Once in an active form, the receiver is a positive regulator of specific modulon transcription (163, 183, 222).

Much remains to be understood about how the "appropriate

environmental stimulation" is transmitted to the sensor protein. It has been proposed that the FHL system is regulated by five environmental signals which include anaerobiosis, intracellular formate, pH, molybdate, and nickel. Although the genes encoding the structural components of the FHL complex [fdhF (FDH-H) and hyc (HYD-3) operons] are located approximately 33 minutes apart in the E. coli chromosome, their transcription appears to be coordinated (11, 24, 25, 171).

In order to decipher the mode by which these signals are

transmitted to the transcription of the formate modulon, a series of promoter lac fusion strains were constructed and characterized for anaerobic induction and dihydrogen related enzyme activities. Mutational analysis revealed a unique gene, later denoted as the fhlB gene, encoding a product essential for FHL activity including FDH-H and total hydrogenase activity. However, FHL-B protein was not necessary for expression of the fdhF gene and is therefore currently assumed modulate transcription of the FHL pathway (Fig. 1). Transcription of the fhlB gene was repressed by high-redox potential electron acceptors (dioxygen and nitrate) and required formate for maximum anaerobic induction. Further analysis revealed that rpoN (encoding the a54








4


(fhiX)?


Sensor?


SfhIA


Inactive
r Formate


(+) M Active

X e FHLA
- ~P- fhlB
URS? y
modulator?


hyc T ' fdhF
I I I I
* URS URS +
HYD3]- - --FDH-H
FHL
FORMATE b H2 + CO2


Figure 1. Model of genetic regulation of formate hydrogenlyase in E.
coli.








5

subunit of RNA polymerase), fhlA, chlD and molR gene products were necessary for optimum fhlB gene transcription (Fig. 1).

Subsequent investigation of the fhlB gene transcription

demonstrated that high cytoplasmic concentrations of FHL-A protein (using multicopy plasmid pSE-133) decreased but did not eliminate the formate requirement. Because of these observations, the DNA sequence of the fhlA gene was determined to identify the characteristics of the gene and its product. The FHL-A protein was found to have sequence homology with putative a54-dependent transcriptional activators (receivers) of the two-component regulatory system. However, the amino terminus of the FHL-A protein was unique and did not contain the conserved secondary structure or aspartate and lysine residues which have been shown to be phosphorylated by the respective histidine-protein-kinase (sensor). This would suggest that phosphorylation is not required for active FHL-A protein, and a sensor protein may not be involved. Interestingly, transcription of the fhlA gene was shown to be from the weak "-35 and -10" fhlA promoter (constitutive) and not from the upstream FNR- or a 4-dependent promoters of the hyp operon. It is therefore possible that the FHL-A protein is synthesized in an inactive form and activated in the presence of formate (Fig. 1).

During investigation of the molybdate requirement for FHL, it was determined that the fhlB gene in moIR or chID genetic backgrounds still retained formate inducibility in the presence of multiple copies of the fhlA+ gene (exogenous molybdate was required for expression to wild type








6

levels). This indicates that FHL is regulated by a dual regulatory system, one involving molybdate and a second formate-dependent pathway (Fig. 1). The results presented show that fermentative dihydrogen production is regulated by a complex series of reactions involving the availability of formate, molybdate and the absence of oxygen.














LITERATURE REVIEW


Facultative anaerobes, such as Escherichia coli, have evolved respiratory networks organized such that the most energetically favorable electron transport pathway is utilized for the specific redox state of the environment. E. coli is capable of using a variety of electron acceptors which include in order of decreasing potential: 1/202/H20 (E'= +0.82 V), N037/N02~ (E0'= +0.42 V), N02/NH4+ (E' +0.36 V), DMSO/DMS (E0'= +0.16 V), TMAO/TMA (E0'= +0.13 V), fumarate/succinate (E0'= +0.03 V), pyruvate/lactate (E0= -0.19 V), acetaldehyde/ethanol (E'= -0.2 V), H+/H2 (Eo'= -0.42 V; ref. 16, 228). Pathway preference is determined by the difference between the standard oxidation-reduction potential of the initial electron-donor (NADH) and terminal acceptor system (AEO') available in the growth medium. The standard free energy change (AGO') of a reaction is given by AGO '= -nFAEO'

where n is the number of electrons transferred and F is Faraday's constant. Therefore the greater the AE0', the more free energy can be harnessed by the organism for biosynthetic purposes.

This establishes a hierarchy with aerobic respiration being the most energetically favorable for the cell's survival. Thus, other


7








8

electron transport pathways are not synthesized in the presence of dioxygen. Furthermore when nitrate is present in an anaerobic environment, the nitrate respiratory pathway is induced and alternate respiratory systems are repressed (for review, see 82, 102, 139, 175). Presumably, a complex interconnected regulation involving transcription, translation, protein processing, and allosteric effectors is conceivable.

Respiration involves the generation of a proton motive force

(PMF), consisting of a pH gradient (ApH) and electrochemical gradient

(At), by electron transport (for review, see 84, 160). In general, respiratory components are organized into "modules" or pathways. These include a substrate dehydrogenase, which transfers electrons to a quinone pool, and ultimately a membrane-bound reductase; thus coupling substrate oxidation to the reduction of an electron acceptor (139). A fermenting cell, on the other hand, produces endogenous electron acceptors and derives most of its energy from substrate-level phosphorylation (42). Although it is generally believed that the FHL system is a fermentative process discrete from anaerobic respiration, the precise function of the FHL pathway remains unclear.

E. coli is a mixed-acid fermentor producing acetate, lactate, formate, ethanol, succinate, H2 and C02 (Fig. 2). The ratio of these major end products is regulated (conceivably by redox-mediated modulation) to balance the reducing equivalents generated during glycolysis (3, 206). All of these fermentation products are derived









9


GLUCOSE ADP 2(H]
ATP PEP ADP 2[H] ATP

LACTATE 1 PYRUVATE - ' OAA
12


2[H]

10

ACETALYDEHYD 2[H]- A11

ETHANOL


AC


C02 24+
5 - FORMATE 6 ,,
I MAL
2[H]
ETYL-CoA %
8 i HS.
CoA , FUMA
ACETYL-P

9 ADP2[H]1- 4
ATP


ACETATE


Figure 2. Mixed-acid fermentation of E. coli. 1, Phosphoenolypyruvate carboxylase; 2, malate dehydrogenase; 3, fumarase; 4, fumarate reductase; 5, pyruvate formatelyase; 6, formate hydrogenlyase complex (formate dehydrogenase-H, hydrogenase isoenzyme-3, and associated electron transport proteins); 7, hydrogenase isoenzyme-2; 8, phosphotransacetylase; 9, acetate kinase; 10, acetaldehyde dehydrogenase; 11, alcohol dehydrogenase; 12, lactate dehydrogenase. Redrawn with modifications from Alam and Clark (3).


- 2[H] ATE


RATE


SUCCINATE








10

from pyruvate. It has been proposed that PDH is inhibited by the high [NADH]/[NAD+] ratio reached upon shift to anaerobic conditions (83). Therefore anaerobically active PFL, encoded by the pfl operon, plays a central role in glucose metabolism (122, 170, 191). The PFL complex catalyzes the nonoxidative cleavage of pyruvate leading to transfer of "acetyl" group to coenzyme A (Ac-CoA) and formate (75). Consequently, the C02 and reduced NADH produced aerobically by PDH is replaced anaerobically by formate. This is critical since the cell must maintain an acceptable ratio of [NADH]/[NAD+] (42). The FHL catalyzes the

oxidation of formate to H2 and C02. This minimizes the accumulation of formic acid which would otherwise lower the pH (77). The metabolic fates of the gaseous products, H2 and C02, are poorly understood. It is hypothesized that FHL is the major anaerobic enzyme producing C02 for the synthesis of oxaloacetate (OAA) from phosphoenol pyruvate (PEP; ref. 42). OAA can be further metabolized to succinate or 2-oxoglutarate, all three of these metabolites being essential for biosynthetic reactions such as amino acid production. E. coli can utilize the H2 produced from formate oxidation by FHL to reduce fumarate to succinate (HUP; ref. 148). This energy yielding pathway is HYD-2 dependent (12, 151, 193). This would link FHL to the membrane-bound fumarate reductase and thus a respiratory process which provides the cell with 1 mole of ATP per mole of fumarate reduced, presumably by the generation of PMF (116). Therefore, FHL appears to be linked to both fermentative and respiratory pathways.








11

Formate hydrogenlyase is composed of FDH-H, HYD-3, and redox carriers linking the two enzymes (27, 77, 173, 193). The fdhF gene (92.4 min) encodes the 80 kDa selenopolypeptide constituent of FDH-H (11, 243). Additionally, the FDH-H protein requires molybdenum (molybdopterin-guanylate) for activity (33, 73, 112). The hyc operon (59 min) consists of 8 ORFs and is presumed to encode the remaining FHL components. Five of these ORFs code for electron carriers (previously termed ant and hyd-17; ref. 11, 27, 171, 242). The ORF5 (hyd-17) shows significant sequence homology with the large subunit from other Ni/Fe hydrogenases and is the presumptive structural gene for HYD-3.

Numerous mutants have been isolated with defects in H2 metabolism (Fhl~), the majority of which have been deficient in all three hydrogenase isoenzymes (35, 37, 76, 107, 128, 135, 167, 171, 231). Through the characterization of mutant strains, DNA sequence analysis and mapping of the genes affected, FHL regulation can be more clearly understood. It is proposed that the FHL system is regulated by signals which include anaerobiosis, nitrate, intracellular formate, low pH, molybdate, nickel, and selenium. How these signals are transmitted to the transcription, translation and post-translational modification of the components of the FHL complex remains poorly understood.

Anaerobiosis

The presence or absence of terminal electron acceptors, such as dioxygen or nitrate, play a major role in regulating genes involved in respiration and fermentation. It was estimated, by using 2-dimensional








12

protein electrophoresis, that 18 or more proteins were induced by anaerobiosis in E. coli (205). Later studies using a physiological approach with lac gene fusions, estimated that 50 genes were induced when dioxygen and alternative electron acceptors were absent, 20% of these were H2 repressible, and only a small number of mutants failed to grow microaerobically on glucose minimal medium (41).

The FHL pathway has been shown to be induced anaerobically by a variety of methods. Initially, FHL induction was monitored by an increase in specific activity of FHL and the enzymes of the pathway (10, 57, 78, 127, 203, 235, 236). Later, transcription of the fdhF and hyc (hyd-17) operons (structural genes of FHL), measured as P-galactosidase activity using gene fusion strains, was found to be anaerobically inducible (171). These studies demonstrate that anaerobically growing organisms require the synthesis of additional proteins to generate energy and maintain redox balance. Unfortunately, little is known about how the organism senses the onset of anaerobiosis and activates or represses specific pathways.

Many earlier studies have suggested that anaerobic electron transport systems are subject to a "dual control" in which an unidentified effector molecule somehow senses the "redox" status of the environment and activates FNR protein, a pleiotropic transcriptional activator and repressor. In combination with this global regulatory mechanism, specific transcriptional effectors responded to terminal electron acceptors such as nitrate or fumarate (36, 47, 197, 198). With








13

the identification and characterization of the Arc system and other forms of respiratory control, current research forms a much more complex picture of regulation (105, 106, 212).

Available evidence suggests that this aerobic-anaerobic switch may involve several genes including arcA, arcB, fnr and pgi (involved in DNA-supercoiling); mutants deficient in any one of these genes have pleiotropic phenotypes. The Arc modulon is a two-component sensorregulator system (105, 106). Through DNA sequence analysis, ARC-B is presumed to be a membrane-bound, histidine-protein kinase. Autophosphorylation of ARC-B occurs in response to dioxygen-limitation (possibly redox control). The signal is then transmitted to ARC-A by phosphorylation to produce a transcriptional activator of the cytochrome d (a high-affinity oxidase) gene (cyd). ARC-A is also a repressor of "aerobic" enzyme synthesis (i.e. succinate dehydrogenase and cytochrome o; ref. 68, 105). Cytochrome o, having a lower affinity for dioxygen, is only synthesized aerobically; where as cytochrome d is stimulated under microaerobic conditions as the terminal electron carrier (for review, see 175).

The FNR protein, likewise, acts as both a transcriptional

activator and repressor (for review, see 211, 212). Mutations in the fnr gene were originally isolated as strains deficient in nitrate reductase and fumarate reductase (133). It is now known that several other anaerobically inducible enzymes require FNR for transcription and include nitrite reductase (both cyt552 and NADH-linked; ref. 38, 45,








14

159), DMSO:TMA0 reductase (216, 232), PFL (190, 191), FDH-N (19, 25); anaerobic glycerol-3-phosphate dehydrogenase (130), fumarase B (18, 237), aspartase, and asparaginase II (109). The FNR protein represses its own synthesis (autoregulation; ref. 209) as well as cytochrome o (low-affinity oxidase; ref. 50, 68) and NADH dehydrogenase II (213). Recent evidence is conflicting on FNR regulation at the cyd promoter. By monitoring cyd expression at varying levels of dissolved dioxygen concentrations, Fu et al. (68) suggest that FNR, a weaker transcriptional activator at this operon, outcompetes ARC-A for operator binding as the culture shifts from microaerobic to anaerobic conditions.

The FNR protein has been shown through sequence analysis to be homologous to cAMP receptor protein (CRP) in the helix-turn-helix and nucleotide binding domains (199). However, the FNR amino-terminus differs. It is cysteine rich and three of the four cysteine residues are proposed to be involved in redox-sensitive iron-binding (210). The addition of ferrozine, an iron specific chelator, to the growth medium reduced the level of transcription of the frd operon (encoding fumarate reductase). Since this operon is positively regulated by FNR, iron in association with FNR may be involved in this regulation. In support of this possibility, Green et al. (79) purified FNR in the presence of externally added iron and demonstrated specific in vitro binding of the pure protein to a synthetic (postulated) FNR-binding site by both DNaseI and methylation-protection (DMS) footprinting. Although direct evidence is still lacking, parallels can be drawn with the thoroughly








15

investigated, metal-dependent regulation of FUR (iron uptake) and MER-R (mercury resistance) systems (for review, see 90). It is possible that molybdenum and nickel (necessary for transcriptional repression of chlD and hydC genes, respectively) be also involve a metal-protein complex.

Formate hydrogenlyase synthesis is independent of the ARC system, and FNR-dependence appears to be indirect (44, 105, 171). The transcription of the pfl gene which is essential for the production of formate is partially FNR dependent (190, 191). The gene coding for nickel transport (hydC) is also FNR dependent (239). The hyp operon, whose products presumably process the nickel into forms suitable for hydrogenase, appears to be FNR regulated when the intracellular formate concentration is limiting (this study).

DNA supercoiling has been suggested to be essential for

transcription from several promoters including the FNR-independent promoters of the FHL pathway. Early studies have shown that mutants lacking the DNA gyrase activity are also impaired in anaerobic growth (241). Additionally, FHL activity is absent and plasmid supercoiling is altered in a glucose-grown pgi (oxrC) mutant. Phosphoglucose isomerase, a glycolytic enzyme encoded by the pgi (oxrC) gene, is necessary for the synthesis of FDH-H, peptidase T, tripeptide permease, HYD-1 and HYD-3 in Salmonella typhimurium (107, 108). Recently, Hsieh et al. (98) have compared changes in [ATP]/[ADP] ratios and negative supercoiling of both chromosomal and plasmid DNA upon shifting E. coli from aerobic to anaerobic conditions. They monitored the effect of dioxygen tension on








16

supercoiling activity in wild type and gyrB (DNA gyrase) mutants. It was concluded that the respiratory state of the organism influenced DNA supercoiling. Furthermore, this was mediated by DNA gyrase since, changes in supercoiling paralleled the [ATP]/[ADP] ratios. This is analogous to in vitro studies which demonstrated that the [ATP]/[ADP] ratio influences gyrase-mediated DNA supercoiling (233). Therefore, the energy status of the organism may be an environmental signal affecting DNA supercoiling. In general, the state of chromosomal supercoiling is presumed to be a mechanism of transcriptional control; however, verification of this hypothesis has been difficult.

Nitrate

Anaerobically, nitrate supplementation induces the nitrate-formate respiratory system and represses other respiratory and fermentative pathways of lower redox potential. The narGHJI (nitrate reductase; ref. 36, 62, 137, 216) and fdhGHI (FDH-N; ref. 19) operons, both required for nitrate respiration, are induced. Whereas, the frdABCD operon (fumarate reductase; ref. 104, 115), the dmsABC operon (DMSO and TMAO reductase; ref. 51, 104), the pfl gene (190), the fdhF gene and the hyc operon (25, 171) are repressed in the presence of the high redox potential acceptor, nitrate.

Current evidence suggests that a two-component regulatory system mediates transcriptional control of the narGHJI, fdhGHI, frdABCD, and pfl operons. The NAR-X protein (and possibly NAR-Q) displays sequence homology to membrane-bound histidine-protein-kinases (sensors) and NAR-L








17

to other known DNA-binding proteins (receivers; ref. 81, 164, 220). The NAR-Q protein was proposed by Egan and Stewart (60) as a second protein kinase which could also activate NAR-L via phosphorylation at aspartate residue 59; however, this role has not been confirmed. Mutations in the narX gene confer variable phenotypes. In-frame deletions are phenotypically indistinguishable from the wild type (59), while narX::Tn5 and point mutants relieve (to varying levels) nitrate inhibition at the frd operon (60, 118). Kalman and Gunsalus (118) have sequenced three such point mutants which no longer require nitrate for repression and have localized these single-amino acid changes to an 11residue domain. Molybdate is required for frd operon repression in one out of the three narX strains tested, suggesting that NAR-X may have an additional role in molybdenum sensing. The nafrL gene function is less ambiguous, since all mutants studied to date render frd operon expression nitrate insensitive (104, 117).

When cells are grown in buffered, rich medium with glucose, pfl

operon expression is repressed approximately 2.5-fold in the presence of nitrate. The repression is directly meditated by NAR-L and not relieved by the addition of formate (190). The physiological significance of this partial NAR-L repression is unclear since formate is the substrate for nitrate respiration. The NAR-L protein may serve as a modulator of formate levels at the site of pfl transcription to prevent formic acid accumulation during nitrate respiration. Transcription of the hyc (hyd17) and fdhF genes are repressed in the presence of nitrate (171);








18

therefore, FHL activity is also reduced. However, this control does not appear to be mediated by the NAR-X, NAR-L cascade (218). High concentrations of formate relieve both nitrate and fumarate repression of the FHL structural genes (171). Interestingly, mutations in narL, narK (hypothetical nitrate transport gene) and narGHJI partially relieved nitrate inhibition at the level of hyc and fdhF operon expression when tested in the absence of formate (218). Likewise, strain WL24, deficient in the FDH-N selenopeptide encoded by the fdhGHI operon, is derepressed for the synthesis of FDH-H when grown anaerobically in the presence of nitrate (192). These results suggest that the nitrate effect at the fdhF and hyc level may be a consequence of formate (an obligate inducer) being channeled to nitrate respiration.

Formate

Irrespective of whether high redox potential electron acceptors (dioxygen or nitrate) are present in the environment, glucose is actively transported into the cytoplasm by the phosphotransferase system (PTS) and catabolized to pyruvate (for review, see 75). Therefore, the pivotal metabolic step signalling anaerobiosis could potentially be pyruvate degradation by PFL to formate which occurs in the absence of dioxygen (42). Pyruvate dehydrogenase (PDH) and pyruvate formatelyase (PFL) are tightly controlled by dioxygen both transcriptionally and allosterically (123, 124, 205). Anaerobiosis represses and inhibits PDH while inducing PFL synthesis and catalytic activity (83, 170). Pyruvate formatelyase constitutes up to 3% of the cytoplasmic protein in








19

anaerobically growing cells (122). Lactate dehydrogenase (LDH), responsible for the oxidation of pyruvate to lactic acid, is presumed to be only fully active at high concentrations of pyruvate (17).

The pathway of formate oxidation is determined by the presence or absence of nitrate. Anaerobically, in the absence of nitrate, both formate and low pH enhance FHL activity (17, 46, 78). Additionally, formate and dihydrogen are known inducers of hydrogenase synthesis (128, 135, 171). Formate is considered an obligate inducer for expression of the fdhF and hyc (hyd-17) operons. The DNA sequence of both operons has been determined (27, 145, 243), and the structural genes appear to have comparable regulatory patterns in various media and genetic backgrounds tested, as measured by the amount of P-galactosidase activity produced by the appropriate lac fusion mutants (25, 171). DNA sequence analysis suggests a common upstream regulatory sequence (URS) positioned between bases -101/-142 (fdhF) and -53/-79 (hyc) relative to the transcription start site. The URS exhibits several characteristics comparable to eukaryotic enhancer elements (121). The fdhF URS consists of two tandem conserved hexanucleotide sequences (GTCACG; ref. 22). Deletion analysis of the fdhF upstream region show that the URS is a cis-acting DNA element essential for formate induction and dioxygen/nitrate repression

(22). Construction of chimeric promoter regions, exchanging the Klebsiella pneumoniae nif upstream activating sequence (UAS for nitrogen fixation genes) with the fdhF URS and vice versa, established that the cis-acting element mediated complete regulatory control of the fdhF gene








20

and not the spacer region (23). The hyc operon, on the other hand, is divergently transcribed with the hyp operon. The 210 bp intragenic region contains 3 central hexanucleotide repeats (GTCGAC) which are comparable to the fdhF URS (145).

Further analysis of both promoters revealed sequence homology to the "-24 and -12" promoter consensus (TGGCAC-N5-TTGC) which is recognized by the o5 subunit of RNA polymerase (24, 227). This subunit is encoded by the constitutive rpoN (ntrA or glnF) gene and is required for expression of a number of operons whose physiological roles are apparently diverse although many are involved in nitrogen metabolism (for review, see 131). The rpoN gene product was originally identified as a trans-acting factor required for glutamine synthetase transcription in enteric bacteria and is now known to encode a unique sigma factor (69, 132), distinct both structurally and functionally from the major 070 subunit of RNA polymerase. Based on DNA sequence analysis, no significant amino acid homology between the a54 subunit and the major family of sigma subunits was observed (for review, see 89). However, a helix-turn-helix motif can be predicted in the C-terminal end of both sigma factors (152). Mutagenesis coupled with DNA footprinting has shown that the second helix is probably involved in promoter recognition

(48, 70, 204). All o5-dependent promoters, which have been analyzed, are transcriptionally controlled by activators (homologous to the receiver proteins of the two-component systems) which commonly bind to enhancer-like upstream elements. The NTR-C and NIF-A proteins,








21

transcriptional activators which have been most thoroughly investigated, are essential for catalysis of isomerization from closed to open promoter complexes. This would suggest that the fdhF and hyc operons require a common trans-acting, DNA-binding protein for transcription.

Sankar et al. (186) reported the presence of a putative regulatory element encoded by the fhlA gene which was required for transcription at both promoters. This was subsequently verified by Schlensog et al. (196). The fhlA mutant isolated was phenotypically deficient in Fhl activity (FDH-H and HYD-3) which would be expected for a positive regulator of the FHL modulon. Transduction and plasmid complementation analysis revealed that fhlA was linked to the hydEFB gene cluster (hyp operon) and adjacent to the hydB gene. "Maxicell" experiments established the apparent molecular weight of FHL-A protein as 78 kDa and suggested transcription occurred both aerobically and anaerobically (186). This present study includes DNA sequence analysis of the fhlA gene and predicted amino acid sequence of FHL-A, now known to be homologous to DNA-binding proteins found in two-component regulatory systems. By studying regulation of the fhlB operon, it was shown that both formate and FHL-A were necessary for its transcription. Hypothetically, formate could activate the FHL-A protein or an unidentified protein which positively interacts with FHL-A during transcription.

Molybdenum

Five redox enzymes of E. coli are known to contain molybdenum (Mo) as MPT: FDH-H, FDH-N, nitrate reductase, TMAO/DMSO reductase, and








22

biotin-sulfoxide reductase (55, 92, 111, 140, 141, 202, 219; for review, see 217). Molybdopterin is a complex composed of a nonprotein organic moiety (6-alkyl-pterin) with a Mo atom (111, 113). Initially, Pateman et al. (169) linked anaerobic chlorate-resistance to nitrate respiration in Aspergillus nidulans by demonstrating that the nitraterespiration defective mutant is unable to reduce chlorate to toxic chlorite. The chlorate-resistance (chl) genes exhibited pleiotropic phenotypes and were later identified as essential for molybdenum transport, regulation and formation of functional molybdopterin containing enzymes (for review, see 94). Complementation analysis and deduced sequence homology to other periplasmic binding and transport systems estimated at least 11 chl genes mapping at 5 loci which are necessary for MPT biosynthesis in E. coli (110, 178).

The chlA operon, consisting of three complementation groups (18 min), and the chlE operon, composed of two (18 min), are presumed to be necessary for synthesis of the organic portion of the MPT (11, 113, 178, 219). Mutations in either one of these operons give rise to defects in pterin biosynthesis (114). Baker and Boxer (14) constructed merodiploids containing chlA+ / 0(chlA'-'lacZ+) to study the transcription of the chlA locus. Various chl mutations (chlA, chlB, chlD, chlG and chiE) were introduced into the merodiploid strains. These results suggested that the chiA operon is anaerobically inducible and repressed in the presence of MPT. These investigators concluded








23

that chlA repression is probably mediated by a complex of MPT and a MPTbinding protein which has been reported previously (6, 189).

The constitutively synthesized chlB gene (86 min) product has been


purified (Mr = 35 kDa) and is denoted a is essential for synthesis of function this gene inhibits insertion of the MP1 association factor-FA may have a direct step of apoprotein maturation (71, 146) developed a more sensitive fluorescence extracts for MPT and MGD derivatives. to wild type, it was concluded the chlb


s "association factor-FA" which il MPT (143, 180). A mutation in into the apoprotein; therefore, role in mediating the terminal . Johnson et al. (112) recently technique to analyze cell By comparing chlB mutant extracts gene was required for the


formation of MGD, a step occurring late in the MPT maturation process. The chlC (27 min) and chlF (32 min) operons code for nitrate reductase and FDH-N respectively. Since neither are necessary for MPT biosynthesis, they have been renamed narGHJI (chiC) and fdnGHI (chiF) (11, 19, 219). Mutations in both the chlG (0 min) and chlD operons (17 min) are suppressed by Mo supplementation of the growth medium (11, 207, 219). Phenotypically, the chlG mutants lack nitrate reductase activity, even though detectable levels of the protein were produced in the original isolates, and retain wild type levels of Fhl activity (219). It is currently postulated that the CHL-G proteinfunctions to incorporate Mo into molybdoenzymes and Mo-binding proteins (94).

Mutations in the chlD operon can be phenotypically distinguished from the chiG gene. Dihydrogen production is lacking in chlD mutants








24

due to a deficiency in both FDH-H and HYD-3 activities (33, 73, 76, 196). The partial sequence of the chlD operon discloses significant homology to binding-protein-dependent transport systems (110). Generally, this type of transport is a unidirectional process. It is now known that this process requires ATP for energy instead of acetylphosphate (20, 95, 97, 100), and the substrate does not undergo modification during transport (4). In the systems which have been analyzed, the number and location of proteins involved in the transport process are conserved (for review, see 5, 52). These proteins include:

(i) a high-affinity, substrate-binding protein located in the periplasmic space which is released upon cold osmotic shock (i.e. HIS-J, MAL-E), (ii) two integral membrane proteins present in lower concentration than the binding protein (i.e. HIS-Q, HIS-M, MAL-F, MALG), and (iii) another membrane-associated protein, presumed to be peripheral, which contains a nucleotide-binding domain with a conserved amino acid sequence (i.e. HIS-P, MAL-K). The central domain of the complete CHL-D sequence revealed extensive homology to peripheral transport proteins (110). The partial CHL-J sequence suggested that this protein is an integral membrane component of Mo transport (110).

Both the chlD gene and the molR gene, recently described by Lee et al. (136) are essential for fdhF and hyc (ant, hyd-17) operon expression as measured by 0-galactosidase activity (134, 196). This implies that transcription of these FHL operons either directly or indirectly requires Mo or a Mo-derivative. Interestingly, both moiR and chlD genes








25

are located in the same operon as determined by transduction and plasmid complementation analysis (Shanmugam, unpublished results; this study). However, MOL-R is expressed constitutively (134) while CHL-D synthesis is Mo repressible (155) thus suggesting at least two promoters for this operon.

Nickel

E. coli synthesizes three distinct nickel (Ni) containing hydrogenase isoenzymes (12, 193). The HYD-1 protein and an enzymatically active fragment of HYD-2 have been purified to homogeneity (1, 13, 66, 168, 194). Biochemical characterization of these proteins revealed that HYD-1 and HYD-2 contain nonheme iron, inorganic sulfur and nickel. The HYD-3 protein, responsible for dihydrogen evolution, is electrophoretically labile and has remained elusive to purification (193). DNA sequence analysis of the large subunit of HYD-3 (hyc ORF5), however, displays significant homology to the "nickel-amino acid" consensus sequence of other NiFe-hydrogenases (27, 195, 214). The large subunits of all sequenced NiFe-hydrogenases contain the consensus R-X-CG-X-C-X3-H in the amino-terminus and D-P-C-X2-C-X2-H at the carboxyterminus (177). Apparently the conserved sites at both termini are somehow involved in liganding nickel at the active site (61, 87).

Several genes required for total hydrogenase activity (thus Fhl activity) have been characterized and are presumed to be involved in nickel transport, nickel processing, or hydrogenase regulation. These include hydA (59 min), hydB or hypE (59 min), hydC (77.6 min), hydD








26

(77.6 min), hydE or hypB (59 min), and hydF or hypD (59 min; ref. 35, 119, 128, 135, 185, 187, 188, 231, 238). Although the hyp operon has been sequenced, the DNA did not display any detectable homology to the DNA available in Genbank data base and thus possible function could not be deduced (144). Mutations in both the hydC and hydE genes are suppressed by high concentrations of nickel in the growth medium (231, 238). This would suggest that both are essential for transport of the divalent metal ion. Transcription of the FNR-controlled hydC is fully repressed by approximately 0.2 mM NiCl2 which is correlated to the Nidependent restoration of hydrogenase activity (239). Hypothetically, a nickel-protein complex could function as a transcriptional repressor at the hydC gene operator. The hydE or hypB gene, now known to be a component of the hyp operon, does not appear to be nickel repressible at either of its two promoters (FNR or a54-dependent), as measured by RNA transcript levels and 0-galactosidase activity of hyp gene fusions (144, this study). It is now believed that this gene product is required for activation or processing of nickel. The other hyd genes may also process the nickel for insertion into hydrogenase or regulate the expression of all three isoenzymes. Sankar et al. (187, 188), using maxicells, determined that both hydB (hypE) and hydF (hypD) expression was rpoN dependent, anaerobically inducible, and nitrate repressible. Fumarate and TMAO supplementation had no significant effect on transcription of either gene. This is in correlation with hyp-lac gene








27

fusion expression and transcript levels under the same conditions (144; this study).

Recent studies have established that nickel is not essential for transcription of the electrophoretically stable HYD-1 and HYD-2 isoenzymes, but necessary for hydrogenase activity. Lutz et al. (144) reported that mutations in hypBCDE did not alter hydrogenase transcription as monitored by immunoblotting analysis with anti-HYD-1 and HYD-2 antibodies; however, the electrophoretic mobility was altered in a 10% SDS-polyacrylamide gel. Potentially the lack of nickel processing and insertion could lead to a modified hydrogenase conformation and proteolytic susceptibility. Menon et al. (151) obtained similar results for HYD-1 in a hydE (hypB) mutant. Although HYD-1 transcription was comparable to wild type in these mutants in the absence of Ni, activity was absent. Additionally, the hydE gene product appeared essential for membrane localization of HYD-1, and this requirement was nickel repressible. Labile HYD-3 is probably regulated by a similar post-translational mechanism requiring nickel incorporation for Fhl activity.

Selenium

A number of enzymes from both prokaryotic and eukaryotic organisms, including FDH-H, require selenium in the form of selenocysteine for activity (172; for review, see 26). DNA sequence analysis of the fdhF gene [coding for the 701 amino acid FDH-H subunit (Mr=79 kDa)] established that a UGA "nonsense" codon at amino acid








28

position 140 with an associated unique stem loop structure is essential for selenocysteinyl-tRNA incorporation during translation (15, 64, 65, 243, 244). By constructing a plasmid in which the first 39 amino acids of FDH-H were fused to P-galactosidase, it was determined that selenium was not required for anaerobic transcription of the fdhF gene (243). Selenium regulation of Fhl activity is evidently at the translational level of the fdhF gene.

In summary, the FHL system is presumed to be regulated by a number of elements including anaerobiosis, nitrate, formate, low pH, molybdate, nickel, and selenium. The absence of dioxygen induces the transcription of fdhF, hyc, and other operons whose products are required for FHL activity. Additionally, enzymes necessary for dihydrogen production are often irreversibly inactivated by the presence of dioxygen thus enabling tighter metabolic control. Nitrate repression of FHL appears to be mediated by the NAR-X and NAR-L, two-component regulatory system at the level of pfl gene transcription. Therefore, it is possible that the nitrate repressive effect on the transcription of FHL structural components is indirectly a result of the absence of the obligate inducer, formate as well as low pH. Currently it is unknown whether molybdenum is directly or indirectly required for Fhl transcription. Mutations in both moIR and chiD genes abolish expression of fdhF and hyc operons and are reversed by high molybdate. Nickel is essential for HYD-3 activity at the post-translational level; whereas, selenium is required for translation of the selenopolypeptide, FDH-H.








29

In this study, the mechanism by which formate and molybdenum

regulate the FHL pathway was investigated. This includes isolation of a specific mutant, strain SE-2011 [O(fhlB'-'lacZ+)], physiological characterization of this fhlB mutant, and trans-acting factors regulating the fhlB gene. These trans-acting factors were found to be the FHL-A protein of the formate-dependent pathway and an uncharacterized component of the molybdate-dependent pathway.













MATERIALS AND METHODS

Materials

Biochemicals were purchased from Sigma Chemical Co. Analyticalgrade inorganic and organic chemicals were from Fisher Scientific.

Bacterial Strains and Media

The bacterial strains are derivatives of E. coli K-12 and are listed in Table 1. Basal minimal, dihydrogen/fumarate and glycerol/fumarate media and LB were prepared as described previously (135). Cultures were grown at 37*C in LB which was supplemented with glucose (0.3%) or sodium formate (0.1% to 0.5%), as needed. Ampicillin (100 pg/ml), kanamycin (50 pg/ml), streptomycin (100 pg/ml), tetracycline (15 pg/ml), chloramphenicol (5 pg/ml) or X-gal (20-40 pg/ml) were added as needed.

Isolation of Mutants

Strain MC4100, grown in LB+maltose (0.3%; LBM) medium was

mutagenized with AplacMu53 and ApMu507, as described by Bremer et al.

(29). Kanamycin resistant mutants were transferred by replica plating techniques to LB + X-Gal medium (156) and incubated under aerobic or anaerobic conditions. Mutants which are Lac+ only under anaerobic growth conditions were identified and inoculated into 1 ml of LBG medium in 12 x 75 mm tubes. These tubes were sealed with serum stoppers and


30








31

Table 1. Bacterial strains used in this study


Relevant Genotype or Phenotype


Strain

E. coli

BW545 CSH26 JRG780


JRG861a LCB898


LS853 M2508 M9s

MC4100 MJ-2 MJ-3


MJ-4 MJ-5


MJ-6 MJ-7


Source or Reference


A(lacU)169, rpsL ara, A(lac-pro), thi trpA9761, frdAll, trpR72, gal-25, rpsL195

gal, trpA9761, iciR, trpR, rpsL, fnr thr-1, leuB6, pfl-1, thi-1, lacYl, rpsL175, tonA21

trpA9605, his-85, cya-2, trpR55 Hfr, relAl, spoT1, metBi, melA7 MC4100, 0(fdhF'-'lacZ+) araD139, A(argF-lacU)205, ptsF25, relA1, rpsL150, deoC1, flb5301 0(fhlB'-'lacZ+), hydF102, cys::TnlO MC4100, 0(fhlB'-'lacZ+)


BW545, 0(fhlB'-'lacZ+) 0(fhlB'-'lacZ+), rpoN::TnlO 0(fhlB'-'lacZ+), fnr, zcj-5::TnlO


0(fhZB'-'lacZ+), narL215::TnlO


G. Walker (242) Laboratory collection CGSC #5916


J. Guest CGSC #6161


CGSC #5381 CGSC #4926 A. B6ck (171) CGSC #6152


P1 transduction (SE-2011 x SE-67-1) P1 transduction (MC4100 x SE-2011) P1 transduction (BW545 x SE-2011) P1 transduction (SE-2011 x YMC18) P1 transduction (SE-2011 x SE-1188) P1 transduction (MJ-4 x RK5278)








32


Table 1. continued

Strain Relevant Genotype or Phenotype Source or Reference


MJ-8 MJ-9 MJ-18 MJ-19


MJ-20 MJ-21


MJ-40 MJ-50


MJ-101 MJ-102 MJ-103 MJ- 107 MJ-108 MJ-109


0(fhlB'-'lacZ+), cya-2, zif-4::TnlO 0(fhZB'-'lacZ+), pfl-1, zbo-6::Tnl0 MJ-19, Hfr PO(fhB) CSH26, 0(fhlB'-'lacZ+) 0(fhlB'-'lacZ+), fhlA::TnlO SE-1000, metB1, melA7, thr+, arg+, leu+, F 0(fhlB'-'lacZ+), moIR SE-1000, 0(fhlB'-'lacZ+) 0(fhlA '-'lacZ+), rpoN: :TnlO 0(fhlA'-'lacZ+), narL215::TnlO 0(fhlA'-'lacZ+), fnr, zcj-5::TnlO


*(fhlA'-' lcZ+), cya-2, zif-4::TnlO 0(fhlA'-'lacZ+), moiR $(fhlA'-'locZ+), pfl-1, zbo-6::Tn1l


P1 transduction (SE-2011 x SE-1162)

P1 transduction (SE-2011 x SE-1265)

Conjugation (MJ-19 x TT627)

P1 transduction (CSH26 x SE-2011)

P1 transduction (SE-2011 x SE-1174)

Conjugation (SE-1000 x M2508)

P1 transduction (SE-2011 x SE-1704)

P1 transduction (SE-1000 x SE-2011)

P1 transduction (SE-2007 x YMC18)

P1 transduction (SE-2007 x RK5278)

P1 transduction (SE-2007 x SE-1188)

P1 transduction
(SE-2007 x SE-1162)

P1 transduction (SE-2007 x SE-1704)

P1 transduction (SE-2007 x SE-1265)








33


Table 1. continued

Strain Relevant Genotype or Phenotype Source or Reference


thr-20,
argI60, tonA48,


leu-32, proA35, argF58, lacYl, gal-6, rpsL125, tsx-70, supE44


narL215::TnlO


PC0287



RK5278

SE-1000




SE-67-1


SE-1100 SE-1162 SE-1174 SE-1188 SE-1265 SE-1300 SE-1651 SE-1652 SE-1654


CGSC #5404


V. Stewart


Laborotory collection



P1 transduction
(SE-67 x SE-1300)

Laboratory collection (136)

Laboratory collection


Laboratory collection


(186)


cysC-43, srl-3000::TnlO, thr-1, leu-6, thi-1, lacY-1, galK2, ara-14, xyl-5, mtl-1, proA2, his-4, argE3, rpsL31, tsx-33, supE44 hydF102, cys::Tn1O BW545, 0(molR'-'laczi) LS853, zif-4::TnlO fhlA: :TniO


JRG861a, zcj-5::TnlO LCB898, zba-6::TniO BW545, cys::TniO 0(hyp'-'lOcZ+)1, fnr, zcj-5::TnlO 0(hyp'-'lacZ+)2, fnr, zcj-5::TniO 0(hyp'-'lacZ+)1, narL215::TniO


Laboratory collection

Laboratory
collection

Laboratory collection

P1 transduction
(SE-2001 x SE-1188)

P1 transduction
(SE-2002 x SE-1188)

P1 transduction
(SE-2001 x RK5278)








34


Table 1. continued


Strain SE-1655 SE-1657 SE-1658 SE-1659 SE-1660 SE-1704 SE-1714


SE-1760 SE-1761 SE-1762 SE-2001 SE-2002 SE-2007 SE-2009 SE-2011 VJS720


Relevant Genotype or Phenotype 0(hyp'-'lacZ+)2, narL215::TnlO 0(hyp'-'lacZ+)1, rpoN::TnlO


*(hyp'-'lacZ+)1, rpoN::TnlO 0(hyp'-'lacZ+)2, rpoN::TnlO 0(hyp'-'lacZ+)2, rpoN::TnlO molR::Tn5, zgg::TnlO


0(fhlB'-'lacZ+), chlD::TnlO 0(hyp'-'lacZ+)1, chlD::TnlO 0(hyp'-'lacZ+)2, chlD::TnlO 0(fhlA'-'lacZ+), chID MC4100, 0(hyp'-'lacz+)1 MC4100, $(hyp'-'lacZ+)2 MC4100, $(fhlA'-'lacZ+) MC4100, 0(hydC'-'lac+) MC4100, 0(fhlB'-'lacZ+) chiD::TnlO


Source or Reference P1 transduction (SE-2002 x RK5278) P1 transduction (SE-2001 x YMC18) P1 transduction (SE-2001 x YMC18) P1 transduction (SE-2002 x YMC18) P1 transduction (SE-2002 x YMC18) Laboratory collection P1 transduction (SE-2011 x VJS720) P1 transduction (SE-2001 x VJS720) P1 transduction (SE-2002 x VJS720) P1 transduction (SE-2007 x VJS720) This study This study This study This study This study V. Stewart








35


continued

Relevant Genotype or Phenotype

endA, thi, hsdR, A(lacU)169,
rpoN: TnlO

a typhimurium
strAl, pyrC7/F'ts114 zzf::TnlO


Source or Reference B. Magasanik J. Roth (40)


Table 1. Strain YMC18


Salmonel I
TT627








36

the gas phase was replaced with dinitrogen. After 16 hr of incubation at 37*C, dihydrogen in the gas phase of the culture tubes was determined using a gas chromatograph (Varian; Model 920) fitted with a 50 nm molecular sieve column. From a total of 68 mutants, 13 were found to be defective in dihydrogen production (Fhl~) and were analyzed further.



Enzyme Activities and their Respective Culture Conditions

s-galactosidase activities and culture conditions. For anaerobic induction of P-galactosidase activity in mutant strains SE-2001, SE-2002 and SE-2011, 120 ml of medium in a 160 ml "Wheaton" bottle was inoculated (1% V/V) with a 1.5 hr old aerobic culture, grown at 37*C, in a shaker, at 250 rpm. However, with strain SE-2007 or derivatives thereof, alternate procedures were used to maximally aerate the culture at low cell density before starting the experiment. A 2 hr old aerobic culture (370C; 250 rpm) was transferred to fresh LB medium (1% V/V) and grown again in the shaker for 1 hr. This culture was used to inoculate the experimental medium at 10% (V/V) and then grown under anaerobic conditions in a "Wheaton" bottle. The bottles were closed with rubber stoppers and secured with aluminum seals. The gas phase was replaced with argon. Samples were removed at different time periods with a syringe and needle and growth of the culture and -galactosidase activity of the cells were determined. In another set of experiments, the aerobic cultures were used to inoculate (1% V/V) the appropriate medium in 13 x 100 mm screw cap tubes filled to the top. Cells from








37

these cultures were harvested after 4 hr of incubation at 37C (standing) and used for enzyme assays.

The amount of 0-galactosidase present in cells was determined as described by Miller (156), after permeabilization with SDS and chloroform. The specific activity of the enzyme is expressed as nanomoles ortho-nitrophenol produced per min per mg cell protein. The differential rate of synthesis of -galactosidase activity was calculated as units of activity per pg cell protein produced by the culture and represents the increase in the amount of 0-galactosidase activity produced by the culture in relation to the increase in total cell protein.

Dihydrogen related metabolic activities and culture conditions.

Whole cells were utilized for all biochemical determinations (135). Cells were inoculated (5% V/V) from overnight LB-grown cultures into fresh LBG medium in 250 ml screw cap bottles or 20 ml screw cap tubes (16 x 150 mm) filled to the top. Cultures were grown anaerobically for 4 hr at 37*C. Cells were collected by centrifugation (3,000 x g for 10 min) at 40C, washed with half volume of phosphate buffer (0.1 M sodium phosphate, pH 7.0; 1 mM glutathione; 0.1 mg/ml chloramphenicol). The cell pellet was resuspended in 1.0 ml buffer and diluted to 1.75 mg cell protein per ml, as determined in a Spectronic 710 spectrophotometer

(Baush and Lomb) in which 1 A420nm unit equalled 350 pg cell protein per ml. Samples were maintained under N2 at 4*C and assayed immediately.








38

Total hydrogenase activity, measured as tritium exchange (7, 138), was determined using 50 pg cell protein adjusted to 0.2 ml volume in sodium phosphate buffer (0.1M; pH 7.0). Assays were carried out in 12 x 75 mm thick walled test tubes, sealed with 11 x 17 mm serum stoppers. The gas phase was replaced with helium. The reactions were initiated with 0.7 ml of dihydrogen and 0.55 pCi tritium gas (11.2 mCi/mmol; New England Nuclear Corp.) and terminated after 1 hr incubation at room temperature. After removing the stopper, the reaction mixture was agitated with a vortex mixer and allowed to stand for 10 min. Tritium in a 50 pl sample in 2.5 ml Scintiverse-E scintillation fluid was determined, and total hydrogenase activity was calculated as nanomoles of tritiated water produced per min per mg cell protein.

Hydrogen uptake (HUP) was measured both as the ability to reduce benzyl viologen (BV) (96, 167) and fumarate with dihydrogen as electron donor (148). The reduction of BV was monitored in 13 x 100 mm test tubes with "subaseal" stoppers. The assay mixture (0.1 M sodium phosphate buffer, pH 7.0; 4 mM BV) was degassed, the gas phase replaced with dihydrogen and reduced with sodium diothionite until a faint purple color appeared. The reaction was initiated by the addition of whole cells to a final volume of 2.5 ml. The rate of BV reduction was correlated to AA550nm using a DW2C spectrophotometer (SLM Instruments; Urbana, Illinois). The HUPBV activity was expressed as nanomoles BV reduced per min per mg cell protein. Fumarate reduction was measured as the rate of dihydrogen consumption from the gas phase using a gas








39

chromatograph as described above. Assay mixture consisted of 0.1 M phosphate buffer (pH 7.0) and 50 mM fumarate brought to a final volume of 1.0 ml with cells. "Wheaton" vials (10 ml) with serum stoppers and aluminum seals were used to establish anaerobic conditions (10% H2/90% N2 gas phase). The HUPfumarate activity was calculated as nanomoles of H2 consumed per min per mg cell protein.

Formate-dependent reduction of BV (FDH-H, FDH activity associated with FHL), was assayed in an assay mixture containing 0.33 M sodium phosphate buffer (pH 7.0), 6.5 mM BV, 40 mM formate and cells in a final volume of 4.0 ml (135). Test tubes (13 x 100 mm) with "subaseal" stoppers were used to monitor the reaction in a N2 atmosphere. Assay mixture was reduced with sodium dithionite until a faint purple color developed. Reaction was initiated by adding whole cells and the

AA550nm was correlated to BV reduction. The FDH-H activity was calculated as nanomoles BV reduced per min per mg of cell protein.

Formate hydrogenlyase activity was measured in 70 mM sodium

phosphate buffer (pH 6.5) containing 0.1 M formate (final volume of 1.0 ml with cells; 96). " Wheaton" vials (10 ml) with serum stoppers and aluminum seals were used to monitor the production of dihydrogen from formate in a dinitrogen atmosphere. Dihydrogen evolution was quantified using a gas chromatograph. The FHL activity was calculated as nanomoles of H2 produced per min per mg of cell protein.

Fumarate reductase activity was assayed in 92.5 mM sodium

phosphate buffer (pH 7.0), 30 mM fumarate, and 0.35 mM BV under a N2








40

atmosphere. Activity was measured as fumarate-dependent oxidation of

BVred* Assay mixture was reduced with sodium dithionite to 2.0 absorbance units at 550 nm, whole cells were added to a final volume of

5.0 ml, and oxidation of reduced BV was monitored as AA550nm* Activity was expressed as nanomoles BVred oxidized per min per mg cell protein (208).

Chloramphenicol acetyltransferase (CAT) assay and culture

conditions. Plasmid pSV208 (fdhF'::'cat) was generously provided by Dr. A. B6ck (196). Cell extract was prepared according to Brosius and Lupski (30) with modifications. Transformants were inoculated into 1.0 ml LB + ampicillin (50 pg/ml) medium and grown to stationary phase. This was used as an inoculum for 20 ml (5% V/V) of the same medium supplemented with glucose (0.3%), formate (0.2%), and trace metals (FeSO4.7H20, 0.01 mg/ml; NaMoO4.2H20, 0.01 mg/ml; NaSeO3.5H20, 0.263 ng/ml). The cultures were grown anaerobically (16 x 150 mm screw cap tubes filled to top) to approximately 5 x 108 CFU/ml (4 hr; 37*C). Cells were harvested by centrifugation at 5,000 rpm for 10 min at 40C, washed with 20 ml of assay buffer (50 mM Tris-HCl, pH 7.8; 30 pM dithiothreitol), and resuspended in 1.0 ml of same buffer. This was transferred to 1.5 ml plastic centrifuge tubes and placed at -70*C for 1 hr. Cells were thawed at 37*C and then disrupted by sonication (one 20 seconds pulse at full power; Heat Systems sonifier with microprobe) after dilution to 2.0 ml volume in conical-bottomed glass test tubes in an ice-water bath. Cellular debris were removed by centrifuging in








41

"Eppendorf" micro-centrifuge for 5 min at 4C. Supernatant was collected and kept on ice for immediate assay using a modified procedure described by Shaw (200). The reaction mixture was freshly prepared by dissolving 4 mg of 5,5' dithiobis-2-nitrobenzoic acid (DTNB) in 1.0 ml Tris-HCl (pH 7.8), adding 0.2 ml of 5 mM acetyl-CoA, and then making the total volume up to 10 ml. After measuring the rate of change of absorbance at 412 nm with 900 ul of reaction mixture and 80 pl of extract, the reaction was started with 20 p1 of chloramphenicol (Cm; 5 mM in 70% ethanol) added to a final concentration of 0.1 mM. The

difference in the rate of change at A412nm with and without Cm was calculated. Protein concentration of the extract was determined using Coomassie brilliant blue (28). The CAT activity was expressed as nanomoles of free 5-thio-2-nitrobenzoate produced per min per mg cell protein.



Genetic and Molecular Biological Experiments

Bacteriophage (P1 and lambda) preparation by plate lysis. Host strain was grown to stationary phase in LB medium for P1 infection. Cells were sedimented by centrifugation (3,500 x g) at 25*C and resuspended in an equal volume of P1 adsorption medium (5 mM CaCl2.2H20; 10 mM MgCl2.6H20). Bacteriophage P1 (105 to 106 PFU) was added to 0.2 ml of host cells and incubated for 5 min at 250C. Then 3 ml of LCTG soft agar (5g/L yeast extract; 10 g/L each of NaCl and trypticase peptone; 2.5 mM CaCl2 . 2H20; 25 mg/ml thymine; 60 mM glucose; 0.6%








42

agar) at 50*C was added to the mixture and after mixing was overlayed on LB+thymine (25 pg/ml) plates. After 9 hr of incubation at 420C, the overlay from the plates with confluent lysis was harvested after addition of 2.5 ml P1-diluent (10 g/L trypticase peptone; 10 mM MgCl2.6H20). Chloroform (0.5 ml) was added to the lysate and mixed with a glass pipet. This was centrifuged (12,100 x g) for 15 min at 4*C. The supernatant was again extracted with choloroform, and the final supernatant was stored at 40C with 0.1 ml chloroform. Two consecutive series of P1 infection were done to ensure enrichment of host strain mutation.

Procedures implemented for replication of bacteriophage A were comparable to P1 with some critical exceptions. Strain LE392 (supF, supE) was used as the host strain and was grown in LBM medium. Cells were pelleted and resuspended in an equal volume of 10 mM MgSO4.7H20. Water-Thy agar (0.6% agar; 0.1 mg/ml thymine) was used as the soft agar overlay, and plates were incubated at 37*C. Lambda diluent (10 mM TrisHCl, pH 7.5; 10 mM MgSO4.7H20; 50 mM NaCl; 0.1% gelatin) was used to titer and harvest phage.

Transduction using bacteriophage P1 Cm clrlOO. Transduction experiments were carried out according to Miller (156) with modifications. Two milliliters of a "mid-log phase" culture of recipient cells were centrifuged (3,500 x g) for 5 min at 25C and resuspended in 1.0 ml of P1 adsorption medium. Then 0.2 ml of the resuspension was infected at a M.0.I. of 1 to 10 with the appropriate








43

phage preparation. After 30 min at 25*C, 1 ml of P1 diluent was added and the bacteria-phage mixture was vortexed. This was then centrifuged (3,500 x g) for 5 min at 250C, resuspended in 0.1 ml of LB and incubated for 1 hr at 25C before plating on selective medium for transductants (25*C). Tranduction of donor mutation was confirmed prior to and after curing recipients off bacteriophage at 420C.

Conjugation. The F' complementation analysis was performed

according to Miller (156) using F'143-1 (57 to 60 min), F'126 (17 to 30 min), F'116 (60 to 65 min), F'128 (6 to 8 min; lacZ::Tn1O), F'112 (90 to 98 min) and F'104 (0 to 7 min) provided by Dr. B. Bachmann. The Hfrmediated transfer of chromosomal DNA was accomplished by aerobically (250 rpm) growing the donor and recipient strains to approximately 5 x 108 CFU/ml at 370C. Mating was initiated by mixing 1.0 ml of donor with

1.0 ml of recipient in 3.0 ml of fresh LBG (125 ml flask; 50 rpm). Immediately upon removal, samples were vortexed, diluted in minimal medium and centrifuged (3,500 x g) for 5 min at 25*C. Exconjugants were then selected on appropriate media.

Transformation by CaCl -heat shock method. Routine transformation was performed by two methods, depending on the amount of competent cells required. Both were a modification of the calcium chloride-heat shock method described by Mandel and Higa (149) which was later established for transforming episomal elements (43). The first method was basic and expedient. An overnight LB culture of the desired host strain was inoculated into fresh medium in 13 x 100 mm tubes (0.02 ml into 2.0 ml)








44

and incubated for 2 hr standing at 37'C. Cells were sedimented (3,500 x g; 5 min; 25*C) and resuspended in 0.4 ml of 0.1 M CaCl2.2H20. DNA (about 50 ng) was added to 0.2 ml of cell suspension and incubated on ice for 20 min. This was transferred to 42*C for 2 min and then returned to ice for 10 min. Fresh LB medium (1.0 ml) was added, and cells were incubated at 370C for 1 to 2 hr and then plated on applicable selection medium. If more cells were needed, the overnight culture was inoculated into 10 ml LB medium and aerobically grown at 300C for 1.5 to 2 hr. This was transferred to 370C for 30 min and then harvested (3,500 x g; 5 min; 250C). After washing with 0.1 M NaCl, the cells were resuspended in equal volume of 0.1 M CaCl2.2H20 and incubated for 20 min at 25*C. This was then pelleted and resuspended in CaCl2 solution at approximately one-fifth the original volume. Similar procedures were then followed to that described above.

For high efficiency transformation, competent cells were prepared by a modified procedure and stored at -700C (Sankar, personal communication). An overnight culture was used to inoculate 50 ml of fresh LB medium. This was grown to early-log phase (2 x 108 CFU/ml) aerobically at 370C. The cells were centrifuged (3,000 x g; 5 min; 4C), washed in 0.1 M cold MgCl2'6H20, resuspended in equal volume of

0.1 M cold CaCl2.2H20, and incubated on ice 20 min. Treated cells were then pelleted (3,000 x g; 5 min; 4*C) and resuspended in one-tenth the starting volume with 0.1 M CaCl2 containing 15% glycerol. This was aliquoted and stored at -700C for later use. Cells were thawed slowly








45

on ice and 0.1 ml was transferred to a tube for transformation. DNA (approximately 50 ng) was added to the competent cells. This was incubated on ice for 20 min, transferred to 37*C for 5 min, and returned to ice for 2 min. Transformants were preincubated in SOC medium (20 g/L trypticase peptone; 5 g/L bacto-yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2.6H20; 10 mM MgSO4.7H20; 20 mM glucose) with aeration at 370C for 1 hr prior to plating on selection medium.

Transposon Tn5 mutagenesis of cloned genes in plasmid DNA. The two transposon Tn5 derivatives of plasmid pSE-133 were constructed as described before (188). Plasmid pSE-133 which carries the hydB+ and fhlA+ genes was described previously (187). Strain MBM7014 (supF) was utilized as the host strain for plasmid DNA (pSE-133) Tn5 mutagenesis. A transformant was aerobically grown (250 rpm) in LBM medium (20 ml) to mid-log phase (3 x 108 CFU/ml) at 370C, the culture was centrifuged (3,500 x g; 25"C), and the pellet was resuspended in 1.0 ml of 10 mM

MgSO4.7H20. Cells were infected with ANK421 (Tn5) at a M.0.I. of 10 for 30 min at 250C. The infected cells were then vortexed, centrifuged (3,500 x g; 250C), washed with 5.0 ml of LB medium, and resuspended in final volume of 10 ml. This was subcultured (2.0 ml inoculum) in 10 ml fresh LBG medium supplemented with 10 mM sodium citrate and aerobically grown (250 rpm) at 37*C for 30 min and then shifted to 300C for 1 hr. Kanamycin (50 pg/ml) and ampicillin (100 pg/ml) were added after the hr. Then the culture was grown to stationary phase (18 hr), and plasmid DNA was extracted by the alkaline lysis method described below (150).








46

Plasmids carrying the transposon Tn5 in the gene of interest were

selected as Kmr Ampr transformants and then screened for lack of complementation in the appropriate mutant. Location of the transposon Tn5 was determined by analyzing "restriction endonuclease" digests of the DNA. The relevant genotype of plasmid pSE-133-1 is hydB::Tn5, fhlA+ and pSE-133-2 carries the transposon in the fhlA gene (hydB+, fhlA::Tn5).

Plasmid and chromosomal DNA preparations. Small and large scale plasmid isolation were carried out following standard alkaline lysis protocol with some modifications (150). The cesium chloride gradient consisted of (final concentration) 1 g/ml cesium chloride and 0.625 mg/ml ethidium bromide. This was centrifuged at 50,000 rpm (160,000 x g), for 18 hr, at 180C. Plasmid band was recovered with syringe fitted with a 22G x 1" needle, and ethidium bromide was removed with water saturated 1-butanol. Extracted plasmid solution was diluted with 2 volumes of deionized water and then ethanol precipitated at -20C for 18 hr. After centrifugation (12,100 x g; 30 min; 4C), the pellet was washed with 70% cold ethanol and vacuum dried. This was resuspended in a small volume of sterile H20.

Chromosomal DNA was prepared according to Ausubel et al. (9) with modifications. A culture of the E. coli strain of interest (1 liter) was grown to mid-log phase (3 x 108 CFU/ml). The cells were harvested by centrifugation at 2,300 x g, for 10 min at 40C. The pellet was resuspended in 95 ml of TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0).








47

Sodium dodecyl sulfate and proteinase K were added to a final concentration of 0.5% and 0.1 mg/ml, respectively. This was incubated for 1 hr at 37*C. The NaCl concentration of the sample was then adjusted to 0.7 M, mixed thoroughly and CTAB/NaCl solution (10% hexadecyl trimethylammonium bromide in 0.7 M NaCl) was added to a final concentration of 1% CTAB. This was heated to 65*C for 10 min, extracted with equal volume of chloroform: isoamyl alcohol (24:1) and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The DNA was precipitated with isopropanol and washed with 70% ethanol. The pellet was vacuum-dried, resuspended in TE buffer (7.5 ml) and centrifuged in a cesium chloride gradient (1 mg/ml cesium chloride; 0.625 mg/ml ethidium bromide) using similar parameters to the large scale plasmid preparation. Chromosomal DNA was removed from the gradient using a syringe fitted with a 16G x J" needle and then extracted and precipitated as with plasmid DNA. Concentration was determined by agarose gel electrophoresis with known DNA standards and fluorometrically with Hoechst 33258 dye (using the TKO 100-dedicated Mini-Fluorometer; Hoeffer Scientific). Concentration as well as the purity of the DNA was determined from the absorption spectrum between the wavelengths 200 nm and 300 nm.

DNA sequence determination. DNA sequence was determined using the Sanger dideoxy method with double stranded plasmid DNA (85, 184). The plasmids used are listed in Table 2. Plasmids pSE-130, pSE-132 and pSE133 used in the fhlA gene sequencing experiments were described








48


Table 2. Plasmids used in this study


Plasmid Relevant Genotype or Phenotype Reference


Cmr, 14 kb ClaI-BamHI fragment with the complete hyd-17 gene cluster from MC4100 in pACYC184

Apr, 14.7 kb Sau3A1 fragment with the complete hyd (hyp) and partial hyc operon in pBR322

Tcr, 2.8 kb SaIlI fragment from pSE-22 with partial hypC and fhlA genes and complete hypDE (hydFB) genes in pBR322

Tcr, 0.8 kb SalI-ClaI fragment from pSE-125 with partial hypCD (hydXF) genes in pBR322


Apr , 6.5 kb SalI-Sau3A1 fragment from pSE-22 with partial hypC (hydX) gene and complete hypDE (hydFB) and fhlA genes in pBR322

Apr, 4.7 kb PstI fragment with partial hypA and fhlA genes and complete hypBCDE (hydEXFB) genes in pBR322

Apr , 2.0 kb KpnI-SalI fragment from L
pSE-125 with partial hypD (hydF) and fhlA genes and complete hypE (hydB) gene in pUC19

Apr , 3.8 kb ClaI fragment with partial hydF gene and complete hydB and fhlA genes


As pSE-133, hydB::Tn5 As pSE-133, fhlA::Tn5


Apr, 1.0 kb KpnI-SalI fragment from with partial hypCD (hydXF) genes in pUC19

Apr, 1.3 kb SalI-PstI fragment from pSE-111 with partial fhlA gene in pUC19


A. Bock (27)



Laboratory collection (185) Laboratory collection (187)


pRBH pSE-111 pSE-125 pSE-125-1


Laboratory collection



Laboratory collection


(185) (186)


aboratory collection (187)


aboratory collection (187)


This study This study


Laboratory collection (138)

Laboratory collection (188)


This study


pSE-128 pSE-130 pSE-132 pSE-133 pSE-133-1 pSE-133-2


pSE-137 pSE-190








49


Table 2. continued

Plasmid Relevant Genotype or Phenotype Reference


pSE-1009 Apr, 2.9 kb KpnI-EcoRV fragment from
pSE-1007, complementing moiR and chID
mutants, in pUC19


pSE-1009 Exo #1-13


Apr, ExoIII deletions from KpnI, in pUC19


pSE-1004 Apr, 4.7 kb PvuII-CloI fragment from
pSE-1001, complementing moIR and chiD
mutants, in pBR322

pSE-1213 Apr, 6.5 kb Sau3A1 fragment with
partial hyb operon in pBR-322


33pBR pSV208


Tcr, 6.2 kb PstI-EcoRI fragment with complete hyb operon

Apr , Cmr , EcoRI-BamHI promoter fragment from pBN208 [Apr, 0(fdhF'-'lacZ+)hyb39, 240 bp upstream of fdhF] into pKK232-8


Laboratory collection (136)


This study


Laboratory collection (136) Laboratory collection A. E. Przybyla A. Bock (196)








50

previously (187, 188). Plasmid pSE-190 carries a 1.0 kb SalI-PstI internal fragment of fhlA gene from previously described plasmid pSE-128 (187) in vector plasmid pUC19. Sequencing of the hydX'FB (hypC'DE) genes, upstream of the fhlA gene, was accomplished using plasmid pSE137, a SalI-KpnI fragment containing the partial hydX (hypC) and hydF (hypD) genes in vector plasmid pUC19, pSE-132 which was described previously, and plasmid pSE-125-1 which was constructed as a 2.38 kb ClaI deletion of previously described plasmid pSE-125 (185). The mol operon was sequenced using previously described plasmid pSE-1009 (134, 136) and exonuclease III-generated deletion derivatives from the KpnI (the 5'-end of the open reading frames) towards the EcoRV site (88). Procedures from Promega "Erase-a-base system" technical manual were followed in these experiments. Plasmid pSE-1004 was also used to establish the EcoRV to ClaI DNA sequence. Both strands of chromosomal DNA present in these plasmids were sequenced using appropriate primers. New oligonucleotide primers were synthesized, as needed, based on the partial DNA sequence of the genes, by the DNA synthesis core laboratory, Interdisciplinary Center for Biotechnology Research, and by Dr. F.C. Davis, Department of Microbiology and Cell Science, University of Florida. Commercially available sequencing primers were obtained from US Biochemical Corporation, Pharmacia-LKB or New England BioLabs. DNA sequence was determined using T7 DNA polymerase (Sequenase), obtained from either US Biochemical Corporation or Pharmacia-LKB, and 51-dATP was supplied by DuPont-New England Nuclear. The protocols supplied by








51

the manufacturers were followed. The DNA sequence was manipulated and homology with other known sequences in the Genbank and EMBL library was determined using the computer programs provided by Genetics Computer Group, University of Wisconsin (56, 234) and Genepro (Hoeffer Scientific).

DNA sequencing gels (both 21 x 50 cm and 38 x 50 cm) were poured

and run according to the procedures supplied by BioRad Laboratories with modifications. A 6% polyacrylamide gel (acrylamide:bis 19:1) in 1 X TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) with 8 M urea was used to separate the reaction products. For increasing the length of DNA sequence determined, a variety of successful techniques were implemented. These included the use of wedge spacers provided by BioRad laboratories, "double loading", and the addition of sodium acetate (final concentration of approximately 0.1 M) to the bottom chamber in order to form an electrolyte gradient (201).

Southern transfer and hybridization. Restriction endonuclease

digested chromosomal DNA was separated by electrophoresis in a vertical 1.0% agarose (Sigma type 1: low electroendosmosis grade) gel. The DNA was stained with ethidium bromide and photographed for calculating the fragment size of the hybridizing fragments. The DNA was depurinated (0.25 M HCl for 10 min) and denatured (0.5 N NaOH; 1 M NaCl for 30 min), and then the gel was neutralized (0.5 M Tris-HCl pH 7.4; 3 M NaCl for 30 min). Southern transfer to Zeta-Probe membranes (Bio-Rad Laboratories) was implemented using standard blotting techniques with 10 X SSC (1.5 M








52

NaCl; 0.15 M trisodium citrate) buffer (150). Membranes were dried under vacuum at 80*C for 3 hr and stored dry between two pieces of "Whatman" 3MM filter paper in plastic bags until used for hybridization. DNA probes were labeled by random primed incorporation of digoxigeninlabeled deoxyuridine-triphosphate (Dig-dUTP), hybridized under stringent conditions to the transferred DNA, and immunologically detected using an alkaline phosphatase linked antibody-conjugate. Procedures provided with the Boehringer-Mannheim Biochemicals "Genius"-kit were performed to carry out these experiments.

For localization of the lac fusion in strain SE-2007, an internal fhlA gene fragment (1.3 kb SalI-PstI) from plasmid pSE-190 was labeled and used to probe chromosomal DNA from strains SE-2007 and MC4100 (parent) digested with either a single or two restriction endonucleases (SailI and BglI, Sail and EcoRI, SalI and ClaI, or ClaI). Similarly Zac fusion strain SE-2001 (strains MC-4100 and SE-2009 were used as controls) chromosomal DNA was digested with endonucleases KpnI and SalI, ClaI and BglI, BglI, or SalI and EcoRI and probed with a 0.7 kb SalIKpnI fragment from plasmid pSE-137 (carrying the partial hypCD genes). Further localization of the lac fusion was done on both mutant strains, SE-2001 and SE-2002, by digesting chromosomal DNA with PstI and then using 4.0 kb and 4.6 kb PstI fragments from plasmid pSE-111 as probes.














RESULTS AND DISCUSSION


Physiological Properties of an fhlB Mutant, Strain SE-2011

Biochemical characteristics of an fhlB mutant, strain SE-2011.

Using AplacMu53 mutagenesis (29), strain SE-2011 was isolated as a lac operon fusion derivative of strain MC4100 which produced 0-galactosidase activity anaerobically and was deficient in fermentative dihydrogen production (Fhl-). Upon detailed biochemical analysis, this strain was found to be affected in the production of hydrogenase, formate dehydrogenase-H, and fumarate reductase activities (Table 3). The lack of tritium exchange activity in strain SE-2011 shows that all three HYD isoenzymes are absent in this strain. As a consequence of this defect, both FHL, which requires active HYD-3, and hydrogen uptake, mediated by HYD-2, activities were not detectable in this strain. The FDH-H activity in strain SE-2011 was less than 10% of the levels observed in the parent, strain MC4100. On the basis of this property, strain SE2011 can be distinguished from all known hyd mutants, which produced FDH-H activity. Similarly, the SE-2011 phenotype can be readily distinguished from FDH-H mutants using the hydrogen uptake characteristics of the other strains. In experiments which are similar to the ones described in Table 3, strain M9s, a known fdhF mutant (171), produced 75 to 85% of hydrogen uptake activity of the parent, strain 53








54

Table 3. Biochemical characteristics of strain MC4100 and an fhlB mutant, strain SE-2011


Specific activity

Enzyme MC4100 SE-2011
(parent) 0(fhlB'-' lacZ+)



Hydrogenasea
(3H2-exchange) 1,200 44

Hydrogen uptake
(H2 to BV)b 700 29

(H2 to fumarate)c 290 UD9

Formate hydrogenlyased 170 UD

Formate dehydrogenase-He 490 38

Fumarate reductasef 600 100


aExpressed as nanomoles of 3H20 produced per minute per milligram of rotein.
Expressed as nanomoles of BV reduced per minute per milligram of rotein.
Expressed as nanomoles of H2 consumed per minute per milligram of rotein.
Expressed as nanomoles of H2 produced per minute per milligram of rotein.
Expressed as nanomoles of BV reduced per minute per milligram of protein.
Expressed as nanomoles of BVred oxidized per minute per milligram of protein.
UD, Undetectable


cell cell cell cell cell cell








55

MC4100, measured either as BV or fumarate reduction. The hydrogenase activity of this strain, measured as tritium exchange was close to 100% of the parent. Strain M9s produced elevated levels of FDH-N (6.6-fold) and lower levels of fumarate reductase (28%) as compared with its parent, strain MC4100. These values which are comparable to the phenotype described by Pecher et al. (171) are quite distinct from the properties of strain SE-2011 (Table 3).

Strain SE-2011 is normal for nitrate respiration which requires active FDH-N, but has not been tested for the recently described third FDH isoenzyme which is presumed to be a major component of formate oxidase, expressed both aerobically and anaerobically (192). The level of fumarate reductase activity in strain SE-2011 was also lower (less that 20% of the parent value). This deficiency is probably the reason for the growth characteristics of strain SE-2011. The aerobic and anaerobic growth of this mutant was comparable to that of the parent, strain MC4100, in LB supplemented with different sugars. However, strain SE-2011 failed to grow in glucose-minimal medium and to produce succinate as a fermentation product. Since succinate is a necessary precursor for biosynthesis, this would account for the poor growth of the organism in defined medium. As will be discussed later, the same phenotype was observed when this mutation was transduced into strain MC4100 or other lac deletion mutants of E. coli (strains CSH26 and BW545), indicating that the pleiotropic effect is due to a single gene defect in these genetic backgrounds. The altered gene is termed fhlB








56

since the gene is formate inducible (see below) and thus probably plays a major role in the production of FHL activity. However, defects in the production of all three HYD isoenzymes and FR can be readily detected in strain SE-2011.

Formate requirement for expression of the fhlB gene. In order to better understand the transcriptional control of the FHL pathway, expression of the fhlB gene was monitored by measuring the levels of Sgalactosidase activity produced by strain SE-2011 from the fhlB promoter. When cultured under strictly aerobic conditions, strain SE2011 produced about 100 U of 0-galactosidase activity in all media tested (Table 4). Upon transfer to anaerobic conditions, the 1galactosidase activity of the LB culture increased approximately 2.5fold after a 4 hr incubation period. Fumarate had no effect on this anaerobic induction. Nitrate, a high redox potential electron acceptor, had a repressive effect on anaerobic expression. Both glucose and formate supplementation elevated fhlB expression anaerobically by about 2-fold and 5-fold, respectively. The glucose enhancement was due to the endogenous production of formate which was verified (see below) by the lack of glucose induction in a pf1 background, deficient in pyruvate formatelyase activity. Acidic pH has been shown in the past to increase FHL activity (17, 78). Formate-dependent induction was reduced by 50% when the culture medium was buffered at pH 7.0. This parallels the physiological data which suggests a role for FHL in pH stabilization during the fermentative growth of E. coli.








57

Table 4. The effect of media composition on the expression of
0(fhlB'-lacZ+) in an fhlB mutant, strain SE-2011


Medium composition


Luria Broth + Nitrate (1.0%) + Fumarate (0.5%) + Glucose (0.3%) + Formate (0.1%) + Formate and Glucose + Formate and bufferb


R-Galactosidase activitya


Aerobic

110 90

110 92

170 190 NDc


Anaerobic

280

120 250 580 1,300

1,400 700


Cells were grown as described in the "Methods" section.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram of protein.
0.1 M Phosphate buffer at pH 7.0 cND, not determined








58

Upon detailed analysis of fhlB gene expression, monitored at specific time intervals, it was found that there was an exponential increase in P-galactosidase activity which paralleled the growth. The specific activity of the culture reached maximum value during early stationary phase and remained constant over an additional 8 hr of incubation (data not shown). In LB medium, maximum activity observed was about 250 U (Fig. 3). In this medium, the increase in specific activity of the enzyme was coupled to growth and the differential rate of induction was about 1.0. In LB medium supplemented with glucose, the differential rate of P-galactosidase production increased exponentially during growth, probably due to continued production of formate by the growing culture (since the amount of formate produced by the culture is proportional to the cell density). The maximum activity reached was about 600 units during the early stationary phase of growth when the cell density was about 100 pg protein/ml. With the addition of formate to the medium, the growth rate and final cell yield decreased, although the differential rate of synthesis of -galactosidase was enhanced to as high as 130 units/pg cell protein. In this medium, the maximum activity produced by the culture increased to about 1,200 units. In LB medium supplemented with both glucose and formate, the differential rate of induction of -galactosidase activity was similar to the values obtained with LB-formate cultures but the total amount of the enzyme produced by strain SE-2011 was slightly higher (about 1,400 units). The final cell yield of the culture in the latter two media was comparable. These








59


1500


>M




E-1
U


as 'C C.



*1


1000 F


500


0


0 20 40 60 80 100


PROTEIN (pg)








Figure 3. Differential rate of synthesis of 0-galactosidase activity by 0(fhlB'-'lacZ+) strain SE-2011 grown in LB medium with different supplements. LBG, LBF, and LBGF represent LB-glucose, LB-formate, and LB-glucose-formate media, respectively. R-Galactosidase and protein activities are expressed as units per ml and pg per ml, respectively.


-LBGF
LBF


C0




- LBG

LBLB
' '








60

experiments clearly show that the transcription of the fhlB operon is dependent on formate, either produced internally or added externally to the medium.

The amount of -galactosidase activity produced by the $(fhlB''lacZ+) strain increased linearly with increasing formate concentration up to about 5 mM (Fig. 4). The activity continued to increase at a lower rate until the maximum was reached at about 15 mM formate in the medium. For these experiments, strain SE-2011 was grown under anaerobic conditions, in LB-formate medium, and the cells were harvested after 4 hr for enzyme assays. Similar results were also obtained with strain MJ-9 [$(fhlB'-'lacZ+), pfl] which lacks the ability to produce formate internally due to a loss of pyruvate formatelyase activity. At 15 mM formate, strain MJ-9 produced only about 60% of the P-galactosidase activity observed in the pfl+ parent strain. At higher formate concentrations (about 30 mM), the specific activity of -galactosidase detected in strain MJ-9 was comparable to the pfl+ strain. These results suggest that the internally produced formate plays a significant role in the transcription of the fhlB operon.

Nitrate repression of fhlB gene expression. Further analysis of the repressive effect of nitrate on fhlB operon expression, was carried out by monitoring the P-galactosidase activity and nitrite produced by strain SE-2011 over a 6 hr growth period in an anaerobic environment. The fhlB mutant was grown in LB, LB-formate, LB-nitrate and LB-formatenitrate medium (LB, LBF, LBN and LBFN respectively; Fig. 5). Nitrite








61


E-4
Q

14 Pq


I
a.


1200 1000


800 600


400 200


0


0


10


20


30


40


FORMATE (mM)








Figure 4. Effect of formate on the induction of 0-galactosidase activity by $(fhlB-lacZ+) strain SE-2011 and a pfZ derivative, strain MJ-9. Specific activity represents the maximum value observed at each formate concentration.


. ,SE-2011 0


MJ-9



-









62


100


0


a



2


10-I




102


1500 1000


Qi 02)

0


U



2

2

.3
2
0
a
U
a


100


30


A


LBN

LBNF










1 2 3 4 5 6

TIME (hours)


0 1 2 3 4 5 6

TIME (hours)




Figure 5. Effect of nitrate supplementation on fhlB gene expression. Cultures were grown in LB, LB-formate, LB-nitrate, and LB-nitrateformate media (LB, LBF, LBN and LBNF, respectively). A. Nitrate respiration (as measured by nitrite produced) in fhlB mutant strain SE2011 grown in LB and LBNF. B. Rate of induction of 0-galactosidase activity by 0(fhlB'-'lacZ+) strain SE-2011.


- B . . LBF
S LBNF.





LB


LBN


10-1


200 F








63

production was measured in the cultures supplemented with nitrate to estimate the rate of nitrate respiration. Results of these experiments indicate that significant levels of nitrate respiration are not evident until 2 hr after initiation of anaerobiosis and growth (Fig. 5A). After this initial lag, nitrite accumulated in the medium throughout the remaining time of the experiment. In the absence of exogenous formate, nitrate repression of fhlB gene transcription (as measured by 3galactosidase activity) paralleled the initiation of nitrate respiration (LBN; Fig. 5B). Formate supplementation (73 mM) partially suppressed this repressive effect, and a narL mutation did not affect the fhlB operon regulation. This is comparable to the regulatory patterns observed for other genes of the FHL pathway (hyd-17 and fdhF genes; 171). These results suggest that both formate and anaerobiosis are necessary for maximal induction of fhlB operon transcription. Although nitrate and neutral pH repress this expression, this effect can be overcome by formate.








64

Genetic Characteristics of Strain SE-2011, 0(fhlB'-'lacZ+)



F' complementation analysis. Initial complementation studies were performed using F'143-1, an episomal element which carried DNA from 57 to 61 min in the E. coli chromosome. This was done since the previously described hyd and fhlA genes, which demonstrated pleiotropic phenotypes for dihydrogen metabolism, mapped to the 58 to 59 min region (185, 186, 187, 188). Mutant strain MJ-1, a cys::TnlO derivative of SE-2011, was used as a recipient for F'143-1 transfer. Exconjugants were selected for Cys+ phenotype and then screened for FHL activity. Approximately 20% of the Cys+ clones were Fhl+. Among those complemented for dihydrogen production, HUP activity was not restored (as measured by H2 to BV). It is possible that the 20% complementation for FHL activity was because the entire region necessary for restoration of strain SE-2011 to parental phenotype was not completely transferred or that deletions may have occurred in the plasmid during growth with uracil, a needed nutrient. Therefore, strain MJ-50 [0(fhiB'-'lacZ+) cys, srl] was used to select a F' element carrying the entire region between cys+ and srl+ genes. A Fhl+ exconjugant (5% of the clones tested) was isolated and used as a F'143-1 donor strain to select for Cys+ in recipient strain MJ-1, $(fhlB'-'lacZ+) cys. Of the exconjugants analyzed, 100% were Fhl+ and Hup+ which suggested that the mutation mapped in this region.

P1 transduction. Similar results were obtained with P1

transduction experiments using strain BW545 as the donor. Of the 72








65

Cys+ clones tested, 12.5% were restored for FHL activity. One lysogen from this experiment tested positive for HUP activity but the clone cured of the lysogenic phage was deficient in both FHL and HUP activities.

Hfr mediated conjugation analysis of the fhlB gene. Because of these inconclusive results the approximate map location of the altered gene in the E. coli chromosome was determined by Hfr-mediated conjugation analysis. For these experiments, an Hfr-derivative of strain SE-2011 in which the origin of DNA transfer is the fhlB gene was constructed, using lac homology, as described before (40, 136). In a 30 min conjugation period, strain MJ-18, transferred argI gene (96.6 min; 11) and not frdA gene (94.4 min; 11) indicating that the fhlB gene was located between these two genes. Both thr and leu were also transferred at high frequency during this 30 min duration. Since the orientation of the lac operon with respect to the origin of transfer is known (40), results of these experiments were also used to determine the direction of transcription of the fhlB gene. According to the results, it appeared that the fhlB gene was transcribed in a clockwise direction, towards argl, thr and leu.

The accuracy of utilizing this procedure in certain lac fusion strains is currently under investigation (Shanmugam, personal communication). Therefore after reevaluating the physiological data and mapping results of SE-2011, it appeared probable that the localization of fhlB gene to the 96.6 min position was due to homology of the








66

chromosomal region (i.e. melAB at 93 min) to the episomal lac DNA sequences used in the construction of the Hfr (PO-fhlB). Comparable mapping complications were encountered in another study with strain SE1100, a 0(molR'-'lacZ+) fusion. The moIR gene was originally mapped to 66 min using similar Hfr procedures. Phage P1-mediated transduction experiments later mapped the molR gene at 17 min in the E. coli chromosome. Interestingly, a second ancestral gene for 0-galactosidase, the ebgA gene, which is homologous to the lacZ gene was found to map in this region (68 min; ref. 8, 11, 32, 225, 226, 230).

Stability of the 0(fhlB'-'lacZ+) mutation. The pleiotropic nature of the fhlB mutation led to the question of whether there was one (or multiple) chromosomal mutation(s) leading to the observed phenotype in strain SE-2011. Initially, the same biochemical characteristics as well as operon expression (0-galactosidase activity) were observed when the mutation from strain SE-2011 was transduced into various E. coli strains, including strains MC4100, BW545 and CSH26. Even upon several years of maintaining the mutant strains at -700C in 20% glycerol, the original characteristics of strain SE-2011 remained stable. However, in recent experiments, the majority of transductants which were selected for Lac+ (X-gal+) and Kmr were no longer analogous to the fhlB mutation previously described. Only about 1 to 5% of the transductants were Fhl~. Dihydrogen uptake activity of these strains was comparable to wild type levels of activity. Many of these mutants were also altered in fhlB operon expression. Basal level anaerobic expression, in the








67

absence of formate was increased 2- to 3-fold. The significance of these results remains to be determined.

Plasmid complementation. Previously described plasmid pSE-133, which carries the partial hydF and complete hydB, fhlA genes (186), restored FHL activity of strain SE-2011 to low levels (data not shown). As later experiments would show, this effect is due to a physiological effect of the FHL-A protein, a transcriptional activator for the FHL system.

It is now known that the two divergently transcribed operons in the 58 to 59 min region (the hyc and hyp operons) span approximately 15.5 kb of DNA. Plasmid pRBH, which carries the complete hyc operon on low copy vector pACYC184 restored the Fhl+ activity of strain SE-2011 to parental levels. Although the H2 to BV activity was increased, this activity was attributable to HYD-3 (Table 5). Additionally, the 1galactosidase activity produced by strain SE-2011/pRBH was 2-fold lower than strain SE-2011 (LB-formate; 10 mM). Lutz et al. (144) have recently reported that the first gene of the hyc operon is a putative repressor and this is probably responsible for this decrease in fhlB expression.

Interestingly, multicopy plasmid pSE-1213 which carries the

partial hyb operon (encoding HYD-2) and complements hyb mutants restored strain SE-2011 to parental levels of HUP and low levels of FHL activity (10% of wild type levels). Multicopy plasmid 33pBR which carries the complete hyb operon complemented strain SE-2011 for both FHL and HUP








68

Table 5. Plasmid complementation analysis of fhlB mutant, strain SE-2011


Specific activity

Strain/plasmid FHLa HUPb HUPC
(H2 to BV) (H2 to fumarate)

BW545 100% 694 44

SE-2011 UDd UD UD

SE-2011/pRBH 100% +e NDf

SE-2011/pSE-1213 10% 1,017 63

SE-2011/33pBR 75% 616 91

aExpressed as % wild type levels of H produced.
bExpressed as nanomoles of BV reduceA per minute per milligram of cell rotein.
Expressed as nanomoles of H2 consumed per minute per milligram of cell rotein.
UD, Undetectable.
eMainly HYD-3 activity.
fND, Not determined.








69

activities to 75% and 100% of wild type levels, respectively. This suggests that the mutation is more complex than anticipated. Further analysis is currently being done to determine the extent of the hyb operon on the Sau3A1 fragment which is carried by plasmid pSE-1213. Also, the exact location of the fhlB mutation in strain SE-2011 is under investigation using polymerase chain reaction (PCR) procedures combined with *(fhlB'-'lacZ+) cloning methods.








70

Trans-acting Factors of the fhlB Operon



Genetic regulation of the fhlB operon. Expression of the enzymes in dihydrogen metabolism requires the products of several genes and these include hyd, fnr, fhlA, rpoN, moiR and chiD gene products (25, 35, 135, 136, 171, 185, 186, 187, 188, 196, 223, 224, 231, 238, 242). In order to

study the role of these gene products on the expression of 0(fhlB''lacZ+), appropriate double mutant strains were constructed in which one of the putative regulatory genes is defective. Analysis of these double mutant strains revealed that the fhlA, rpoN, chiD and moiR gene products

are needed for the anaerobic formate-dependent induction of 0(fhlB''lacZ+) (Table 6). The differential rate of expression of 0-galactosidase activity in the double mutants, strains MJ-5, MJ-20 and MJ-40 (rpoN, fhlA and moiR, respectively) was unity indicating that the induction and cell growth are coupled and the enhancing effect of formate was absent. In an

fnr mutant (strain MJ-6), although the differential rate of expression was comparable to strain SE-2011, the maximum activity was reduced by about 35%. The reduction in the amount of P-galactosidase activity produced by the fhlB, fnr double mutant could be a consequence of lower cell yield of the culture since the production of P-galactosidase activity by 0(fhZB''lac7+) required growth of the organism. HydF and cya mutations had no apparent effect on the expression of 0(fhlB'-'locZ+) operon with and without formate supplementation. Introducing norL::TnlO (217) into strain








71


Table 6. Expression of 0(fhlB'-'lacZ+) in different genetic backgrounds



Strain Relevant P-Galactosidase Activitya
Genotype
Differential Maximum
Rate of Inductionb Activity
(U/pg Protein)


SE-2011 0(fhlB-lacZ+) 130 1,300

MJ-5 0(fhlB-lacZ+), rpoN 1 110

MJ-20 * (fhlB-lacZ+), fhlA 1 150

MJ-2 0(fhlB-lacZ+), hydF NDc 1,200

MJ-6 *(fhlB-lacZ+), fnr 110 840

MJ-8 0(fhlB-lacZ+), cya 160d 1,300

MJ-40 @(fhlB-ZacZ+), moiR 1 130

SE-1714 0(fhlB-lacZ+), chID ND 160


aExpressed as nmoles ONP produced per min per mg protein. All cultures were grown anaerobically at 370C in LB + formate medium except strain MJ-6 which was grown in LB medium with formate and glucose to enhance cell gield.
The differential rate of induction of -galactosidase activity was calculated as the amount of enzyme activity produced by the culture in relation to the increase in total cell protein. CND, Not Determined.
dPoor cell growth.








72

SE-2011 did not alter the amount of -galactosidase activity produced by

the culture both in the presence and absence of nitrate (data not shown).

Effect of multiple copies of the fhlA gene on fhlB transcription.

The effect of increasing the copy number of the fhlA gene on the expression of $(fhlB'-'lacZ+) was investigated using plasmid pSE-133 which carries the complete fhlA+ and hydB+ genes (186, 187). In the presence of plasmid pSE-133, the differential rate of production of fgalactosidase activity by 0(fhlB'-'lacZ+) was about 700 units / pg cell protein in LB-formate medium (Fig. 6). This value is greater than 5 times the rate of about 130 units / pg cell protein for strain SE-2011 cultured in the same medium. The maximum activity produced by strain SE-2011/pSE-133 is also greater than 2 times the values obtained with strain SE-2011 and this increase was detected immediately after establishing anaerobic conditions. The maximum activity observed in strain SE-2011/pSE-133, grown in LB medium without formate supplementation, was also increased to about 1,100 units of fgalactosidase activity. This level of activity is comparable to the values obtained with strain SE-2011 grown in LB medium with 30 mM formate (about 1,000 units) although the differential rate of induction observed with strain SE-2011/pSE-133, in LB medium, was lower. Inserting transposon Tn5 into the fhlA gene in the plasmid (pSE-133-2; Fig. 7) abolished this enhancing effect of plasmid pSE-133 while a hydB::Tn5 mutation in the plasmid (pSE-133-1) had no effect indicating that the plasmid-mediated increase is due to the fhlA gene. Transferring








73


A


a

a


2500


2000 1500


1000 500


0


0 10 20 30 40 50 60


PROTEIN (gg)








Figure 6. Differential rate of induction of the 0(fhlB'-'lacZ+) fusion in strain SE-2011 in the presence and absence of plasmid pSE-133. LB, LB medium; LBF, LB medium supplemented with 30 mM formate. -Galactosidase and protein activities are expressed as units per ml and pg per ml, respectively.


LBF + pSE133






LBF LB + pSE133



LB
- - - - - - ' - - -








74


1500


a
-S
C


1000 500


W





0
E-4


0 1 2 3 4 5


FORMATE (mM)









Figure 7. Effect of formate concentration on the levels of fgalactosidase activity produced by a 0(fhlB'-'lacZ+) pfl double mutant, strain MJ-9, in the presence of plasmids pSE-133 and pSE133-2 (fhlA::Tn5). Cultures were grown for 4 hr in LB medium with appropriate concentrations of formate under anaerobic conditions before the assay.


0


MJ-9/pSE133








MJ-9
MJ-9/pSE133-2








75

an F' element carrying fhlA+ gene (F'143-1) into strain SE-2011, did not significantly alter the rate or the level of 0-galactosidase activity. These results suggest that increasing the copy number of the fhlA+ gene either decreased the concentration of formate required for transcription of 0(fhlB'-'lacZ+) or eliminated the need for formate.

Formate is required for FHL-A activation of the fhlB operon. In order to distinguish between the two possibilities, plasmid pSE-133 was transferred to strain MJ-9 [O(fhlB'-'lacZ+), pfl] and the amount of 0galactosidase activity produced by the culture was determined after culturing the cells in either LB or LB-formate medium. The strain MJ9/pSE-133 produced about 100 units of 0-galactosidase activity when grown in LB medium and about 1,300 units when grown in LB-formate medium (Fig. 7). The minimum amount of formate needed for this transcription was about 3 mM. At formate concentrations higher than 3 mM, the amount of 0-galactosidase activity produced by strain MJ-9/pSE-133 increased slowly reaching a maximum value of about 1,500 units at about 30 mM. The amount of 0-galactosidase activity produced by strain MJ-9/pSE-133-2 (fhlA::Tn5) was actually lower than strain MJ-9 itself and under the conditions used in these experiments, this activity never exceeded 300 units. These results show that in the presence of multiple copies of fhlA gene, the concentration of formate required for optimum expression of fhlB gene is considerably reduced but is still a needed inducer.

The FHL-B protein is cytoplasmic. With 3 mM formate in the

medium, the differential rate of synthesis of -galactosidase activity








76

by strain SE-2011 was enhanced from about 20 units/pg protein to about 330 units/pg protein if the fhlA gene was also present in a multicopy plasmid (pSE-133-1) (Fig. 8). Immediately after anaerobic conditions were established, the fhlB-mediated P-galactosidase activity increased to about 1,200 units within a generation time. During the second generation, this activity decreased to about 75% of the observed peak value. The P-galactosidase activity of the culture reached about 1,000 units and was maintained at that level. On the other hand, strain M9s [0(fdhF'-'lacZ+)] carries the lac fusion in FDH-H, a known membrane protein. With the same plasmid, this strain (M9s/pSE-133-1) produced 1galactosidase activity after an initial lag. The differential rate of synthesis was about 10% of the values of strain SE-2011/pSE-133-1 and was only about 3-fold higher than strain M9s without the plasmid. The levels of 0-galactosidase activity produced by strains SE-2011 and M9s, both in the presence and absence of the plasmid pSE-133-1, was not altered by including Mo or Se to the LB medium, although the FDH-H is a Mo, Se-containing protein. The amount of formate needed for optimum expression of 0(fdhF'-'lacZ+)/pSE-133-1 was about 3 mM also which is similar to that required for strain MJ-9/pSE-133. This rapid induction of fhlB gene in the presence of multiple copies of fhlA+ gene, which is in contrast to the fdhF gene, suggests that the FHL-B protein is cytoplasmic. Its synthesis is apparently not constrained by the availability of membrane proteins and membrane synthesis.








77


H

H
U




0
H
U


1200

1000


C S.,
-C

C


800 600

400 200


0


0


20


40


60


80


PROTEIN (gg)









Figure 8. Differential rate of synthesis of 0(fdhF'-'locZ+, strain M9s, and $(fhlB'-'ZacZ+), strain SE-2011, in LB medium supplemented with 3 mM formate in the presence and absence of the fhlA+ gene in a multicopy plasmid (pSE-133-1). O-Galactosidase and protein activities are expressed as units per ml and pg per ml, respectively.


SE-201 1/pSE133-1

WA M9S/pSE133-1
SE-2011.

M9S



' ' '








78

Expression of 0(fdhF'-'cat) in an fhlB mutant, strain SE-2011.

The FDH-H activity was undetectable in strain SE-2011. In order to determine whether this effect is at the transcriptional or posttranscriptional level, a plasmid carrying a 'CAT' gene fusion in the fdhF gene was transformed into strain SE-2011 and the effect of the fhlB mutation on fdhF gene mediated CAT activity was determined. Cells were grown anaerobically (4 hr) in LB medium supplemented with glucose and formate for maximal expression. Due to the multicopy nature of the expression vector, trace metals (Se, Mo, and Fe) were included to insure saturation. The fdhF transcription measured as CAT activity in strain SE-2011 [$(fhlB'-'lacZ+)] was comparable to parent strain MC-4100 at 140 and 110, units respectively. Whereas, in strain SE-2007 [$(fhlA''lOcZ+)] expression was reduced over 5-fold, to about 20 units confirming the need for FHL-A protein. This suggests that FHL-B protein is not required for transcription of fdhF gene but is required for the production of active FDH-H (Table 3).








79

Analysis of the fhlA Gene



Primary structure of the fhlA gene. Previous experiments

identified the fhlA gene product as a putative regulatory element of both fdhF and hyd-17 genes (185, 187, 196). The results presented above (Table 6, Fig. 6-8) also show that the FHL-A protein is a needed regulatory element for the fhlB operon. Because of these observations, the DNA sequence of the fhlA gene was determined to identify the characteristics of the gene and its product. The fhlA gene (coding region position 2421 to 4497; Fig. 9) codes for a protein of 692 amino acids with an anhydrous molecular weight of 78,467 Da which is comparable to the apparent molecular weight of 78,000 Da obtained by other experiments (144, 185). This protein did not contain any significant hydrophobic region indicating that the primary location of this protein is the cytoplasm. Eight base pairs from the end of translational stop codon (position 4497), the coding region is followed by an inverted repeat (underlined in Fig. 9; positions 4508 to 4517 and 4524 to 4533) which can produce a 10 base pairs stem and a 6 bases (positions 4518 to 4523) loop structure. This region is followed by a stretch of 6 thymine residues at a distance of 14 bases (positions 4548 to 4553). Five more thymine residues can be found 9 bases from the first set of thymines (positions 4563 to 4567). This segment of DNA, in appropriate configuration may function as a p-independent transcription termination site. The 5'-end of the putative coding region is preceded








80


SalI
GTCGACGTCTGCGGCATTCAGCGCGATGTCGATTTAACGTTAGTCGGCAGCTGCGATGAA 60 V D V C G I Q R D V D L T L V G S C D E AACGGTCAGCCGCGCGTGGGCCAGTGGGTACTGGTACACGTTGGCTTTGCCATGAGCGTA 120 N G Q P R V G Q W V L V H V G F A M S V ATTAATGAAGCCGAAGCACGCGACACTCTCGACGCCTTACAAAACATGTTTGACGTTGAG 180 I N E A E A R D T L D A L Q N M F D V E CCGGATGTCGGCGCGCTGTTGTATGGCGAGGAAAAATAATGCGTTTTGTTGATGAATATC 240 P D V G A L L Y G E E K *** M R F V D E Y R
GCGCGCCGGAACAGGTGATGCAGTTAATTGAGCATCTGCGCGAACGTGCTTCACATCTCT 300
A P E Q V M Q L I E H L R E R A S H L S
CTTACACCGCCGAACGCCCTCTGCGGATTATGGAAGTGTGTGGCGGTCATACCCACGCTA 360
Y T A E R P L R I M E V C G G H T H A I
TCTTTAAATTCGGCCTCGACCAGTTACTGCCGGAAAACGTTGAGTTTATCCACGGTCCGG 420
F K F G L D Q L L P E N V E F I H G P G
GGTGCCCGGTGTGCGTACTGCCGATGGGTAGAATCGACACCTGCGTGGAGATTGCCAGCC 480
C P V C V L P M G R I D T C V E I A S H
ATCCGGAAGTCATCTTCTGTACCTTTGGCGACGCGATGCGCGTGCCGGGGAAACAGGGAT 540
P E V I F C T F G D A M R V P G K Q G S
CGCTGTTGCAGGCAAAAGCACGCGGTGCCGATGTGCGCATCGTTTACTCGCCGATGGATG 600
L L Q A K A R G A D V R I V Y S P M D A
CGTTGAAACTGGCGCAGGAGAATCCAACCCGCAAAGTGGTGTTCTTCGGCTTAGGTTTTG 660
L K L A Q E N P T R K V V F F G L G F E
AAACCACTATGCCGACCACCGCTATCACTCTGCAACAGGCGAAAGCGCGTGATGTGCAGA 720
T T M P T T A I T L Q Q A K A R D V Q N
ATTTTTACTTCTTCTGCCAGCACATTACGCTTATCCCGACGTTGCGCAGTTTGCTGGAAC 780
F Y F F C Q H I T L I P T L R S L L E Q
Clal
AGCCGGATAACGGTATCGATGCGTTCCTCGCGCCGGGTCACGTCAGTATGGTTATCGGCA 840
P D N G I D A F L A P G H V S M V I G T
CCGACGCCTATAATTTTATCGCCAGCGATTTTCATCGTCCGCTGGTGGTTGCTGGATTCG 900
D A Y N F I A S D F H R P L V V A G F E
AACCCCTTGATCTACTACAAGGCGTGGTCATGCTGGTGCAGCAGAAAATAGCGGCCCACA 960
P L D L L Q G V V M L V Q Q K I A A H S KpnI
GCAAGGTAGAGAATCAGTATCGTCGAGTGGTACCGGATGCCGGTAACCTGCTGGCGCAAC 1020
K V E N Q Y R R V V P D A G N L L A Q Q

Figure 9. Nucleic acid and predicted amino acid sequences of the partial hypC gene and complete hydB, hydF and fhlA genes. The termination codons are indicated by three asterisks. The "ShineDalgarno" sequences and the weak "-35 and -10" region of the fhlA gene are double underlined. Restriction sites for some of the enzymes are highlighted. The inverted triangle between positions 4,404 and 4,405 represents the position of transposon Tn5 in plasmid pSE133-2, as determined by DNA sequence analysis.








81

AGGCGATTGCCGATGTGTTCTGTGTCAACGGCGACAGCGAATGGCGCGGCTTAGGCGTGA 1080
A I A D V F C V N G D S E W R G L G V I
TTGAATCTTCTGGCGTGCACCTGACGCCGGATTATCAACGATTCGATGCCGAAGCACATT 1140
E S S G V H L T P D Y Q R F D A E A H F
TCCGCCCGGCACCGCAGCAGGTCTGCGATGACCCGCGCGCGCGTTGTGGTGAGGTATTAA 1200
R P A P Q Q V C D D P R A R C G E V L T
CGGGCAAATGTAAGCCGCATCAATGCCCGCTGTTTGGTAACACCTGTAATCCTCAAACCG 1260
G K C K P H Q C P L F G N T C N P Q T A
CGTTTGGTGCGCTGATGGTTTCCTCCGAAGGAGCGTGCGCCGCGTGGTATCAGTATCGTC 1320
F G A L M V S S E G A C A A W Y Q Y R Q
AGCAGGAGAGTGAAGCGTGAATAATATCCAACTCGCCCACGGTAGCGGCGGCCAGGCGAT 1380
Q E S E A *** M
GCAGCAATTAATCAACAGCCTGTTTATGGAAGCCTTTGCCAACCCGTGGCTGGCAGAGCA 1440 Q Q L I N S L F M E A F A N P W L A E Q GGAAGATCAGGCACGTCTTGATCTGGCGCAGCTGGTAGCGGAAGGCGACCGTCTGGCGTT 1500 E D Q A R L D L A Q L V A E G D R L A F CTCCACCGACAGTTACGTTATTGACCCGCTGTTCTTCCCTGGCGGTAATATCGGCAAGCT 1560 S T D S Y V I D P L F F P G G N I G K L GGCGATTTGCGGCACAGCCAATGACGTTGCGGTCAGTGGCGCTATTCCGCGCTATCTCTC 1620 A I C G T A N D V A V S G A I P R Y L S CTGTGGCTTTATCCTCGAAGAAGGATTGCCGATGGAGACACTGAAAGCCGTAGTGACCAG 1680 C G F I L E E G L P M E T L K A V V T S CATGGCAGAAACCGCCCGCGCGGCAGGCATTGCCATCGTTACTGGCGATACTAAAGTGGT 1740 M A E T A R A A G I A I V T G D T K V V GCAGCGCGGCGCGGTAGATAAACTGTTTATCAACACCGCTGGCATGGGCGCAATTCCGGC 1800 Q R G A V D K L F I N T A G M G A I P A GAATATTCACTGGGGCGCACAGACGCTAACCGCAGGCGATGTATTGCTGGTGAGCGGTAC 1860 N I H W G A Q T L T A G D V L L V S G T ACTCGGCGACCACGGGGCGACTATCCTTAACCTGCGTGAGCAGCTGGGGCTGGATGGCGA 1920 L G D H G A T I L N L R E Q L G L D G E ACTGGTCAGCGACTGCGCGGTGCTGACGCCGCTTATTCAGACGCTGCGTGACATTCCCGG 1980 L V S D C A V L T P L I Q T L R D I P G CGTGAAAGCGCTGCGTGATGCCACCCGTGGTGGTGTAAACGCGGTGGTTCATGAGTTCGC 2040 V K A L R D A T R G G V N A V V H E F A GGCAGCCTGCGGTTGTGGTATTGAACTTTCAGAAGCGGCACTGCCTGTTAAACCTGCCGT 2100 A A C G C G I E L S E A A L P V K P A V GCGTGGCGTTTGCGAATTGCTGGGACTGGACGCCCTGAACTTTGCCAACGAAGGCAAACT 2160
R G V C E L L G L D A L N F A N E G K L AGTAATAGCTGTTGAACGCAACGCGGCAGAGCAAGTGCTGGCAGCGTTACATTCCCATCC 2220 V I A V E R N A A E Q V L A A L H S H P ACTGGGGAAAGACGCGGCGCTGATTGGTGAAGTGGTGGAACGTAAAGGTGTTCGTCTTGC 2280 L G K D A A L I G E V V E R K G V R L A CGGTCTGTATGGCGTGAAACGAACCCTCGATTTACCACACGCCGAACCGCTTCCGCGTAT 2340
G L Y G V K R T L D L P H A E P L P R I


Figure 9--continued.








82

ATGCTAATAAAATTCTAAATCTCCTATAGTTAGTCAATGACCTTTTGCACCGCTTTGCGG 2400
C ******
TGCTTTCCTGGAAGAACAAAATGTCATATACACCGATGAGTGATCTCGGACAACAAGGGT 2460
M S Y T P M S D L G Q Q G L TGTTCGACATCACTCGGACACTATTGCAGCAGCCCGATCTGGCCTCGCTGTGTGAGGCTC 2520
F D I T R T L L Q Q P D L A S L C E A L
TTTCGCAACTGGTAAAGCGTTCTGCGCTCGCCGACAACGCGGCTATTGTGTTGTGGCAAG 2580
S Q L V K R S A L A D N A A I V L W Q A
CGCAGACTCAACGTGCGTCTTATTACGCGTCGCGTGAAAAAGACACCCCCATTAAATATG 2640
Q T Q R A S Y Y A S R E K D T P I K Y E
AAGACGAAACTGTTCTGGCACACGGTCCGGTACGCAGCATTTTGTCGCGCCCTGATACGC 2700
D E T V L A H G P V R S I L S R P D T L
TGCATTGCAGTTACGAAGAATTTTGTGAAACCTGGCCGCAGCTGGACGCAGGTGGGCTAT 2760
H C S Y E E F C E T W P Q L D A G G L Y
ACCCAAAATTTGGTCACTATTGCCTGATGCCACTGGCGGCGGAAGGGCATATTTTTGGTG 2820
P K F G H Y C L M P L A A E G H I F G G
GCTGTGAATTTATTCGTTATGACGATCGCCCCTGGAGCGAAAAAGAGTTCAATCGTCTGC 2880
C E F I R Y D D R P W S E K E F N R L Q HpaI
AAACATTTACGCAGATCGTTTCTGTCGTCACCGAACAAATCCAGAGCCGCGTCGTTAACA 2940
T F T Q I V S V V T E Q I Q S R V V N N
SalI
ATGTCGACTATGAGTTGTTATGCCGGGAACGCGATAACTTCCGCATCCTGGTCGCCATCA 3000
V D Y E L L C R E R D N F R I L V A I T
CCAACGCGGTGCTTTCCCGCCTGGATATGGACGAACTGGTCAGCGAAGTCGCCAAAGAAA 3060
N A V L S R L D M D E L V S E V A K E I
TCCATTACTATTTCGACATTGACGATATCAGTATCGTCTTACGCAGCCACCGTAAAAACA 3120
H Y Y F D I D D I S I V L R S H R K N K
AACTCAACATCTACTCCACTCACTATCTTGATAAACAGCATCCCGCCCACGAACAGAGCG 3180
L N I Y S T H Y L D K Q H P A H E Q S E
AAGTCGATGAAGCCGGAACCCTCACCGAACGCGTGTTCAAAAGTAAAGAGATGCTGCTGA 3240
V D E A G T L T E R V F K S K E M L L I
TCAATCTCCACGAGCGGGACGATTTAGCCCCCTATGAACGCATGTTGTTCGACACCTGGG 3300
N L H E R D D L A P Y E R M L F D T W G
GCAACCAGATTCAAACCTTGTGCCTGTTACCGCTGATGTCTGGCGACACCATGCTGGGCG 3360
N Q I Q T L C L L P L M S G D T M L G V
TGCTGAAACTGGCGCAATGCGAAGAGAAAGTGTTTACCACTACCAATCTGAATTTACTGC 3420
L K L A Q C E E K V F T T T N L N L L R
GCCAGATTGCCGAACGTGTGGCAATCGCTGTCGATAACGCCCTCGCCTATCAGGAAATCC 3480
Q I A E R V A I A V D N A L A Y Q E I H
ATCGTCTGAAAGAACGGCTGGTTGATGAAAACCTCGCCCTGACCGAGCAGCTCAACAATG 3540
R L K E R L V D E N L A L T E Q L N N V
TTGATAGTGAATTTGGCGAGATTATTGGCCGCAGCGAAGCCATGTACAGCGTGCTTAAAC 3600 D S E F G E I I G R S E A M Y S V L K Q


Figure 9--continued.








83


AAGTTGAAATGGTGGCGCAAAGTGACAGTACCGTGCTGATCCTCGGTGAAACTGGCACGG 3660
V E M V A Q S D S T V L I L G E T G T G
GTAAAGAGCTGATTGCCCGTGCGATCCATAATCTCAGTGGGCGTAATAATCGCCGCATGG 3720
K E L I A R A I H N L S G R N N R R M V
TCAAAATGAACTGCGCGGCGATGCCTGCCGGATTGCTGGAAAGCGATCTGTTTGGTCATG 3780
K M N C A A M P A G L L E S D L F G H E
AGCGTGGGGCTTTTACCGGTGCCAGCGCCCAGCGTATCGGTCGTTTTGAACTGGCGGATA 3840
R G A F T G A S A Q R I G R F E L A D K
AAAGCTCCCTGTTCCTCGACGAAGTGGGCGATATGCCACTGGAGTTACAGCCGAAGTTGC 3900
S S L F L D E V G D M P L E L Q P K L L
TGCGTGTATTGCAGGAACAGGAGTTTGAACGTCTCGGCAGCAACAAAATCATTCAGACGG 3960
R V L Q E Q E F E R L G S N K I I Q T D
ACGTGCGTCTAATCGCCGCGACTAACCGCGATCTGAAAAAAATGGTCGCCGACCGTGAGT 4020
V R L I A A T N R D L K K M V A D R E F
TCCGTAGCGATCTCTATTACCGCCTGAACGTATTCCCGATTCACCTGCCGCCACTACGCG 4080
R S D L Y Y R L N V F P I H L P P L R E
AGCGTCCGGAAGATATTCCGCTGCTGGCGAAAGCCTTTACCTTCAAAATTGCCCGTCGTC 4140
R P E D I P L L A K A F T F K I A R R L
TGGGGCGCAATATCGACAGCATTCCTGCCGAGACGCTGCGCACCTTGAGCAACATGGAGT 4200
G R N I D S I P A E T L R T L S N M E W
GGCCGGGTAACGTACGCGAACTGGAAAACGTCATTGAGCGCGCGGTATTGCTAACACGCG 4260
P G N V R E L E N V I E R A V L L T R G
Pst I
GTAACGTGCTGCAGCTGTCATTGCCAGATATTGTTTTACCGGAACCTGAAACGCCGCCTG 4320
N V L Q L S L P D I V L P E P E T P P A
CCGCAACGGTTGTCGCCCTGGAGGGCGAAGATGAATATCAGTTGATTGTGCGCGTGCTGA 4380
A T V V A L E G E D E Y Q L I V R V L K Tn5
v
AAGAAACCAACGGCGTGGTTGCCGGGCCTAAAGGCGCTGCGCAACGTCTGGGGCTGAAAC 4440
E T N G V V A G P K G A A Q R L G L K R
GCACGACCCTGCTGTCACGGATGAAGCGGCTGGGAATTGATAAATCGGCATTGATTTAAC 4500
T T L L S R M K R L G I D K S A L I ***
TGCAAATTGCCGGACAGATCTGCCTGTCCGGCATACTATTCATGAGGTTTTTTCGGACGA 4560
ClaI
TATTTTTCCGGCAGTTCTGGCACCGGACGCTTGTCATCGAT 4601


Figure 9--continued.








84

by a typical ribosome binding site (GGA, starting at position 2410; ref. 126). A weak "-10 and -35" 070 promoter consensus sequence is also indicated in Fig. 9 (positions 2312 to 2317 and 2338 to 2343; ref. 86). Based on the DNA sequence, the fhlA gene resides between 2,867 and 2,870 kb of the E. coli chromosomal DNA as described by Kohara et al. (125) and the direction of transcription is clockwise towards cys operon at 59 min (11).

FHL-A protein is a transcriptional activator. The FHL-A protein has sequence homology with known transcriptional activators like NTR-C protein of E. coli, NIF-A protein of Klebsiella pneumoniae, and XYL-R protein of Pseudomonas putida (Fig. 10; and ref. 58, 103, 157). The overall homology is about 30% between the four proteins although the FHL-A protein is considerably larger than the other three. It is 224, 168, and 126 amino acids larger than NTR-C, NIF-A and XYL-R proteins, respectively. Significant regions of the FHL-A protein were also found to be homologous with other transcriptional activators of the twocomponent regulatory systems including the recently described LEV-R protein of Bacillus subtilis (53), DCT-D protein of Rhizobium leguminosarum (181), FLB-D protein of Caulobacter crescentus (176), TYRR protein (49) and HYD-G protein of E. coli (224). Homology with this class of proteins, combined with physiological data, suggest that the FHL-A protein functions as a transcriptional activator of the 054dependent promoters of the FHL pathway (fhlB, fdhF and hyc operons; for review, see 80, 222).









85


FHL-A MSYTPMSDLGQQGLFDITRTLLQQPDLASLCEALSQLVKRSALADNAAIVLWQAQTQRAS 60 FHL-A YYASREKDTPIKYEDETVLAHGPVRSILSRPDTLHCSYEEFCETWPQLDAGGLYPKFGHY 120 FHL-A CLMPLAAEGHIFGGCEFIRYDDRPWSEKEFNRLQTFTQIVSVVTEQIQSRVVNNVDYELL 180 NIF-A MIHKSDSDTTV 11
XYL-R MSLTYKPKMQHEDMQDLSSQIRFVAAEGKIWLG 33

FHL-A CRERDNFRILVAITNAVLSRLDMDELVSEVAKEIHYYFDIDDISIVLRSHRKNKLNIYST 240 NIF-A RRFDLSQQFTAMQRISVVLSRATEASKTLQEVLSVLHNDAFMQHGMICLYDSQQEILSIE 71 XYL-R EQRMLVMQLSTLASFRREIISLIGVERAKGFFLRLGYQSGLMDAELARKLRPAMREEEVF 93

FHL-A HYLDKQHPAHEQSEVDEAGTLTERVFKSKEMLLINLHERDDLAPYERM-LFDTWGNQIQT 299 NTR-C MQRGIVWVVDDDSSIRWVLERALAGAGLTCTTFENGAEVLEA-LASKTPDVLLS 53
NIF-A ALQQTEDQTLPGSTQIRYRPGEGLVGTVLAQGQSLVLPRVADDQRFLDRL-SLYDYDLPF 130 XYL-R LAGPQLYALKGMVKVRLLTMDIAIRDGRFNVEAEWIDSFEVDICRTELGL-MNEPVCWTV 152

FHL-A LCLLPLMSGDTMLGVLKLAQCEEKVFTTTN-LNLLRQIA---ERVAIA-VDNALAYQEIH 354 NTR-C DIRMPGMDGLALLKQIKQRHPMLPVIIMTA-HSDLDAAVSAYQQGAFDYLPKPFDIDEAV 112 NIF-A IAVPLMGPHSRPIGVLAAHAMARQEERLPA-CTRFLETV---ANLIAQ-TIRLMILPTSA 185 XYL-R LGYASGYGSAFMGRRIIFQETSCRGCGDDKCLIVGKTA---EEWGDVSSFEAYFKSDPI- 208

FHL-A RLKER--LVDENLALTEQLN-NVDSEFGEIIGRSEAMYSVLKQVEMVAQSDSTVLILGET 411 NTR-C ALVER--AISHYQEQQQPRNVQLNGPTTDIIAKP-AMQDVFRIIGRLSRSSISVLINGES 169 NIF-A AQAPQ--QSPRIERPRACTP-SRGFGLENMVGKSPAMRQIMDIIRQVSRWDTTVLVRGES 242 XYL-R -VDERYELQTQVANLRNRLK-gYDGQYYG-IGHSPAYKRICETIDKAARGRVSVLLLGET 265

FHL-A GTGKELIARAIHNLSGRNNRRMVKMNCAAMPAGLLESDLFGHERGAFTGASAQRIGRFEL 471 NTR-C GTGKELVAHALHRHSPRAKAPFIALNMAAIPKDLIESELFGHEKGAFTGANTIRQGRFEQ 229 NIF-A GTGKELIANAIHHNSPRAAAAFVKFNCAALPDNLLESELFGHEKGAFTGAVRQRKGRFEL 302 XYL-R GVGKEVIARSVHLRSERAEQPFVAVNCAAIPPDLIESELFGVDKGAYTGAVNARAGRFER 325

FHL-A ADKSSLFLDEVGDMPLELQPKLLRVLQEQEFERLGSNKIIQTDVRLIAATNRDLKKMVAD 531 NTR-C ADGGTLFLDEIGDMPLDVQTRLLRVLADGQFYRVGGYAPVKVDVRIIAATHQNLEQRVQE 289 NIF-A ADGGTLFLDEIGESSASFQAKLLRILQEGEMERVGGDETLRVNVRIIAATNRHLEEEVRL 362 XYL-R ANGGTIFLDEVIELTPRAqATLLRVLQEGELERVGGDRTRKVDVRLITATNENLEEAVKM 385

FHL-A REFRSDLYYRLNVFPIHLPPLRERPEDIPLLAKAFTFKIARRLGRNIDSIPAETLRTLSN 591 NTR-C GKFREDLFHRLNVIRVHLPPLRERREDIPRLARHFLQVAARELGVEAKLLHPETEAALTR 349 NIF-A GHFREDLYYRLNVMPIALPPLRERQEDIAELAHFLVRKIAHSQGRTL-RISDGAIRLLME 421 XYL-R GRFRADLFFRLNVFPVHIPPLRERVEDIPLLVEHFLRRHHKEYGKKTLGLSDRAMEACLH 445

FHL-A MEWPGNVRELENVIERAVLLTRG-NVLQLSLPDIVLPE-PETPPAATVVALE-G--EDEY 646 NTR-C LAWPGNVRQLENTCRWLTVMAAGQEVLIQDLPGELFES-TVAESTSQMQPDSWA--TLLA 406 NIF-A YSWPGNVRELENCLERSAVLSES-GLIDRDVILFNHRDNPPKALASSGPAED-G------ 473 XYL-R YQWPGNIRELENALERGVILTES-N--ESINVESLFPG-LATATEGDRLSSE-GRLEEES 500

FHL-A QLIVRVLKETNGVVAG---------------------PKGAAQRLGLKRTTLLSRMKRLG 685
NTR-C QWADRALRSGHQNLLSEAQPELERTLLTTALRHTQG LG 466
NIF-A -WLDNSLDERQRLIAALEKAGWV-------------- IMD 518
XYL-R GDSW--FRQI--IDQGVSLEDLEAGLMRTAMDRCGQNGKLD 556

FHL-A IDKSALI* 692
NTR-C ME* 468
NIF-A ITMPRL* 524
XYL-R PSLSVKAMGR* 566
Figure 10. Alignment of the predicted sequences of E. coli FHL-A, E. coli NTR-C, K. pneumoniae NIF-A, and P. putida XYL-R proteins. Identical and functionally similar amino acids are highlighted. Underlined regions represent those residues of which two out of the three aligined amino acids are homologous to FHL-A protein. The helixturn-helix motif is double underlined.




Full Text
89
polar mutations in "domain D" of NTR-C abolish the positive (not
negative) control of the protein (147). Deletions in NIF-A suggest that
only this central domain is necessary for transcriptional activation at
the nif promoters (99). However, the other NIF-A domains are probably
essential for specific regulation. Due to the high degree of homology,
the central region of the FHL-A protein probably serves a similar
function.
The final region IV (amino acids 663 to 682) of 20 amino acids is
the suggested DNA binding domain (helix-turn-helix; ref. 39, 165) of the
receiver proteins (58, 222). Both NTR-C and NIF-A have been shown to
bind to upstream promoter sites by DNA footprinting (158, 179). This
final segment of FHL-A protein is 50%, 45%, 40% and 30% homologous with
the HYD-G, NTR-C, XYL-R and NIF-A proteins respectively (Fig. 12).
Critical amino acids, Ala-5, Gly-9, Leu-15 and hydrophobic amino acids
at positions 4, 8 and 10 (165) can be detected starting at position 663
(PKGAA QRLGL KRTTL LSRMK). This segment of the protein is crucial for
the biological activity of the FHL-A protein. Inserting transposon Tn5
between the amino acids 662 and 663 (between the DNA bases G and G at
positions 4404 and 4405 in plasmid pSE-133-2; Fig. 7) completely
abolished the formate dependent expression of lacZ*). Thus,
the helix-turn-helix motif is necessary in vivo for specific activation
of genes with FHL-A binding sites, potentially the UAS described by
Birkmann and Bock (23). The R. meliloti NIF-A studies suggest that the
C-terminal domain can be deleted without abolishing in vivo activation;


21
transcriptional activators which have been most thoroughly investigated,
are essential for catalysis of isomerization from closed to open
promoter complexes. This would suggest that the fdhF and hyc operons
require a common trans-acting, DNA-binding protein for transcription.
Sankar et al. (186) reported the presence of a putative regulatory
element encoded by the fhlA gene which was required for transcription at
both promoters. This was subsequently verified by Schlensog et al.
(196). The fhlA mutant isolated was phenotypically deficient in Fhl
activity (FDH-H and HYD-3) which would be expected for a positive
regulator of the FHL modulon. Transduction and plasmid complementation
analysis revealed that fhlA was linked to the hydEFB gene cluster (hyp
operon) and adjacent to the hydB gene. "Maxicell" experiments
established the apparent molecular weight of FHL-A protein as 78 kDa and
suggested transcription occurred both aerobically and anaerobically
(186). This present study includes DNA sequence analysis of the fhlA
gene and predicted amino acid sequence of FHL-A, now known to be
homologous to DNA-binding proteins found in two-component regulatory
systems. By studying regulation of the fhlB operon, it was shown that
both formate and FHL-A were necessary for its transcription. Hypo
thetically, formate could activate the FHL-A protein or an unidentified
protein which positively interacts with FHL-A during transcription.
Molybdenum
Five redox enzymes of E. coli are known to contain molybdenum (Mo)
as MPT: FDH-H, FDH-N, nitrate reductase, TMAO/DMSO reductase, and


LBG Luria Broth + glucose
LBGF Luria Broth + glucose + formate
LBF Luria Broth + formate
LBM Luria Broth + maltose
LBN Luria Broth + nitrate
LBNF Luria Broth + nitrate + formate
MES 2-(N-Morpholino)ethanesulfonic acid
Mo Molybdenum
MOPS 3-(N-Morpholino)propanesulfonic acid
MPT Molybdopterin
MGD Molybdopterin guanine dinucleotide
NAD+, NADH Nicotinamide adenine dinucleotide and its
reduced form, respectively
OAA Oxaloacetate
ONPG Ortho-nitrophenyl-p-D-galactopyranoside
ORF Open reading frame
PDH Pyruvate dehydrogenase
PEP Phosphoenolpyruvate
PFL Pyruvate formatelyase
PIPES Piperazine-N,N'-bis[2-ethanesulfonic
acid]; 1,4-piperazinediethanesulfonic acid
PMF Proton motive force
SDS Sodium dodecyl sulfate
TES N-Tris-(hydroxymethyl)methyl-2-
aminoethanesulfonic acid
TMA Trimethyl amine
ix


40
atmosphere. Activity was measured as fumarate-dependent oxidation of
BVred. Assay mixture was reduced with sodium dithionite to 2.0
absorbance units at 550 nm, whole cells were added to a final volume of
5.0 ml, and oxidation of reduced BV was monitored as AA550nm. Activity
was expressed as nanomoles BVred oxidized per min per mg cell protein
(208).
Chloramphenicol acetyltransferase (CAT) assay and culture
conditions. Plasmid pSV208 (fdhF'cat) was generously provided by Dr.
A. Bock (196). Cell extract was prepared according to Brosius and
Lupski (30) with modifications. Transformants were inoculated into 1.0
ml LB + ampicillin (50 pg/ml) medium and grown to stationary phase.
This was used as an inoculum for 20 ml (5% V/V) of the same medium
supplemented with glucose (0.3%), formate (0.2%), and trace metals
(FeS04.7H20, 0.01 mg/ml; NaMo04.2H20, 0.01 mg/ml; NaSe03.5H20, 0.263
ng/ml). The cultures were grown anaerobically (16 x 150 mm screw cap
tubes filled to top) to approximately 5 x 108 CFU/ml (4 hr; 37C).
Cells were harvested by centrifugation at 5,000 rpm for 10 min at 4C,
washed with 20 ml of assay buffer (50 mM Tris-HCl, pH 7.8; 30 pM
dithiothreitol), and resuspended in 1.0 ml of same buffer. This was
transferred to 1.5 ml plastic centrifuge tubes and placed at -70C for 1
hr. Cells were thawed at 37C and then disrupted by sonication (one 20
seconds pulse at full power; Heat Systems sonifier with microprobe)
after dilution to 2.0 ml volume in conical-bottomed glass test tubes in
an ice-water bath. Cellular debris were removed by centrifuging in


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20
and not the spacer region (23). The hyc operon, on the other hand, is
divergently transcribed with the hyp operon. The 210 bp intragenic
region contains 3 central hexanucleotide repeats (GTCGAC) which are
comparable to the fdhF URS (145).
Further analysis of both promoters revealed sequence homology to
the "-24 and -12" promoter consensus (TGGCAC-N5-TTGC) which is
recognized by the a54 subunit of RNA polymerase (24, 227). This subunit
is encoded by the constitutive rpoN (ntrA or glnF) gene and is required
for expression of a number of operons whose physiological roles are
apparently diverse although many are involved in nitrogen metabolism
(for review, see 131). The rpoN gene product was originally identified
as a trans-acting factor required for glutamine synthetase transcription
in enteric bacteria and is now known to encode a unique sigma factor
(69, 132), distinct both structurally and functionally from the major
o70 subunit of RNA polymerase. Based on DNA sequence analysis, no
significant amino acid homology between the o54 subunit and the major
family of sigma subunits was observed (for review, see 89). However, a
helix-turn-helix motif can be predicted in the C-terminal end of both
sigma factors (152). Mutagenesis coupled with DNA footprinting has
shown that the second helix is probably involved in promoter recognition
(48, 70, 204). All o54-dependent promoters, which have been analyzed,
are transcriptionally controlled by activators (homologous to the
receiver proteins of the two-component systems) which commonly bind to
enhancer-like upstream elements. The NTR-C and NIF-A proteins,


19
anaerobically growing cells (122). Lactate dehydrogenase (LDH),
responsible for the oxidation of pyruvate to lactic acid, is presumed to
be only fully active at high concentrations of pyruvate (17).
The pathway of formate oxidation is determined by the presence or
absence of nitrate. Anaerobically, in the absence of nitrate, both
formate and low pH enhance FHL activity (17, 46, 78). Additionally,
formate and dihydrogen are known inducers of hydrogenase synthesis (128,
135, 171). Formate is considered an obligate inducer for expression of
the fdhF and hyc {hyd-17) operons. The DNA sequence of both operons has
been determined (27, 145, 243), and the structural genes appear to have
comparable regulatory patterns in various media and genetic backgrounds
tested, as measured by the amount of p-galactosidase activity produced
by the appropriate lac fusion mutants (25, 171). DNA sequence analysis
suggests a common upstream regulatory sequence (URS) positioned between
bases 101/142 {fdhF) and 53/79 {hyc) relative to the transcription
start site. The URS exhibits several characteristics comparable to
eukaryotic enhancer elements (121). The fdhF URS consists of two tandem
conserved hexanucleotide sequences (GTCACG; ref. 22). Deletion analysis
of the fdhF upstream region show that the URS is a cis-acting DNA
element essential for formate induction and dioxygen/nitrate repression
(22). Construction of chimeric promoter regions, exchanging the
Klebsiella pneumoniae nif upstream activating sequence (UAS for nitrogen
fixation genes) with the fdhF URS and vice versa, established that the
cis-acting element mediated complete regulatory control of the fdhF gene


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Lonnie 0. Ingram
Professor of Microbiology and
Cell Science
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December, 1991
at- <^/vy
Dean, college of Agriculture
Dean, Graduate School


12
protein electrophoresis, that 18 or more proteins were induced by
anaerobiosis in E. coli (205). Later studies using a physiological
approach with lac gene fusions, estimated that 50 genes were induced
when dioxygen and alternative electron acceptors were absent, 20% of
these were H2 repressive, and only a small number of mutants failed to
grow microaerobically on glucose minimal medium (41).
The FHL pathway has been shown to be induced anaerobically by a
variety of methods. Initially, FHL induction was monitored by an
increase in specific activity of FHL and the enzymes of the pathway (10,
57, 78, 127, 203, 235, 236). Later, transcription of the fdhF and hyc
(hyd-17) operons (structural genes of FHL), measured as 0-galactosidase
activity using gene fusion strains, was found to be anaerobically
inducible (171). These studies demonstrate that anaerobically growing
organisms require the synthesis of additional proteins to generate
energy and maintain redox balance. Unfortunately, little is known about
how the organism senses the onset of anaerobiosis and activates or
represses specific pathways.
Many earlier studies have suggested that anaerobic electron
transport systems are subject to a "dual control" in which an
unidentified effector molecule somehow senses the "redox" status of the
environment and activates FNR protein, a pleiotropic transcriptional
activator and repressor. In combination with this global regulatory
mechanism, specific transcriptional effectors responded to terminal
electron acceptors such as nitrate or fumarate (36, 47, 197, 198). With


71
Table 6. Expression of Q(fhlB'-'lacZ*) in different genetic backgrounds
Strain
Relevant
Genotype
0-Galactosidase
Activity3
Differential
Rate of Inductionb
(U/pg Protein)
Maximum
Activity
SE-2011
(fhlB-lacZ+)
130
1,300
MJ-5
QifhlB-lacZ*), rpoN
1
110
MJ-20
QifhlB-lacZ*), fhlA
1
150
MJ-2
Q^fhlB-lacZ*), hydF
NDC
1,200
MJ-6
QifhlB-lacZ*), fnr
110
840
MJ-8
QifhlB-lacZ*), cya
160d
1,300
MJ-40
^{fhlB-lacZ^), molR
1
130
SE1714
QtfhlB-lacZ*), chlD
ND
160
Expressed as nmoles ONP produced per min per mg protein. All cultures
were grown anaerobically at 37C in LB + formate medium except strain MJ-6
which was grown in LB medium with formate and glucose to enhance cell
vield.
The differential rate of induction of 0-galactosidase activity was
calculated as the amount of enzyme activity produced by the culture in
relation to the increase in total cell protein.
^ND, Not Determined.
dPoor cell growth.


28
position 140 with an associated unique stem loop structure is essential
for selenocysteinyl-tRNA incorporation during translation (15, 64, 65,
243, 244). By constructing a plasmid in which the first 39 amino acids
of FDH-H were fused to p-galactosidase, it was determined that selenium
was not required for anaerobic transcription of the fdhF gene (243).
Selenium regulation of Fhl activity is evidently at the translational
level of the fdhF gene.
In summary, the FHL system is presumed to be regulated by a number
of elements including anaerobiosis, nitrate, formate, low pH, molybdate,
nickel, and selenium. The absence of dioxygen induces the transcription
of fdhF, hyc, and other operons whose products are required for FHL
activity. Additionally, enzymes necessary for dihydrogen production are
often irreversibly inactivated by the presence of dioxygen thus enabling
tighter metabolic control. Nitrate repression of FHL appears to be
mediated by the NAR-X and NAR-L, two-component regulatory system at the
level of pfl gene transcription. Therefore, it is possible that the
nitrate repressive effect on the transcription of FHL structural
components is indirectly a result of the absence of the obligate
inducer, formate as well as low pH. Currently it is unknown whether
molybdenum is directly or indirectly required for Fhl transcription.
Mutations in both molR and chlD genes abolish expression of fdhF and hyc
operons and are reversed by high molybdate. Nickel is essential for
HYD-3 activity at the post-translational level; whereas, selenium is
required for translation of the selenopolypeptide, FDH-H.


26
(77.6 min), hydE or hypB (59 min), and hydF or hypD (59 min; ref. 35,
119, 128, 135, 185, 187, 188, 231, 238). Although the hyp operon has
been sequenced, the DNA did not display any detectable homology to the
DNA available in Genbank data base and thus possible function could not
be deduced (144). Mutations in both the hydC and hydE genes are
suppressed by high concentrations of nickel in the growth medium (231,
238). This would suggest that both are essential for transport of the
divalent metal ion. Transcription of the FNR-controlled hydC is fully
repressed by approximately 0.2 mM NiC12 which is correlated to the in
dependent restoration of hydrogenase activity (239). Hypothetically, a
nickel-protein complex could function as a transcriptional repressor at
the hydC gene operator. The hydE or hypB gene, now known to be a
component of the hyp operon, does not appear to be nickel repressive at
either of its two promoters (FNR or o54-dependent), as measured by RNA
transcript levels and pgal actos i dase activity of hyp gene fusions (144,
this study). It is now believed that this gene product is required for
activation or processing of nickel. The other hyd genes may also
process the nickel for insertion into hydrogenase or regulate the
expression of all three isoenzymes. Sankar et al. (187, 188), using
maxicells, determined that both hydB (hypE) and hydF (hypD) expression
was rpoN dependent, anaerobically inducible, and nitrate repressible.
Fumarate and TMAO supplementation had no significant effect on
transcription of either gene. This is in correlation with hyp-lac gene


GENETIC REGULATION OF FORMATE HYDROGENLYASE IN ESCHERICHIA COLI
By
JULIE ANNE MAUPIN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991


84
by a typical ribosome binding site (GGA, starting at position 2410; ref.
126). A weak "-10 and -35" o70 promoter consensus sequence is also
indicated in Fig. 9 (positions 2312 to 2317 and 2338 to 2343; ref. 86).
Based on the DNA sequence, the fhlA gene resides between 2,867 and 2,870
kb of the E. coli chromosomal DNA as described by Kohara et al. (125)
and the direction of transcription is clockwise towards cys operon at 59
min (11).
FHL-A protein is a transcriptional activator. The FHL-A protein
has sequence homology with known transcriptional activators like NTR-C
protein of f. coli, NIF-A protein of Klebsiella pneumoniae, and XYL-R
protein of Pseudomonas put ida (Fig. 10; and ref. 58, 103, 157). The
overall homology is about 30% between the four proteins although the
FHL-A protein is considerably larger than the other three. It is 224,
168, and 126 amino acids larger than NTR-C, NIF-A and XYL-R proteins,
respectively. Significant regions of the FHL-A protein were also found
to be homologous with other transcriptional activators of the two-
component regulatory systems including the recently described LEV-R
protein of Bacillus subtilis (53), DCT-D protein of Rhizobium
leguminosarum (181), FLB-D protein of Caulobacter crescentus (176), TYR-
R protein (49) and HYD-G protein of E. coli (224). Homology with this
class of proteins, combined with physiological data, suggest that the
FHL-A protein functions as a transcriptional activator of the o54-
dependent promoters of the FHL pathway (fhlB, fdhF and hyc operons; for
review, see 80, 222).


ebgA Ancestral gene for p-galactosidase
fdhF FDH-H; formate dehydrogenase (linked to H2 evolution)
fdhGHI FDH-N; formate dehydrogenase (linked to N03 reduction)
fhlA Putative DNA binding protein necessary for transcriptional
activation of the fdhF, hyc and fhlB operons
fhlB Putative modulator of the FHL pathway; necessary for FHL,
FDH-H and total HYD activity
flbD Flagellar synthesis; Caulobacter crescentus
fnr Global regulator of anaerobic respiration; homologous to CRP
frdABCD Fumarate reductase
gyrB DNA gyrase
his Histidine biosynthesis and binding-protein-dependent
transport system
hyb Hydrogenase isoenzyme-2
hyc Hydrogenase isoenzyme-3
hydA Total hydrogenase activity
hydFB hypDE\ total hydrogenase activity
hydE hypB\ total hydrogenase activity; Ni suppressible
hydC,D Total hydrogenase activity; nickel transport
hyd-17 0RF5 of the hyc operon, hydrogenase isoenzyme-3
hydG Putative DNA binding protein of dihydrogen metabolism
(uptake)
hyp Total hydrogenase activity, operon includes previously
described hydB, £, and F genes
mal Maltose metabolism and binding-protein-dependent transport
system
molR Molybdate binding-protein-dependent transport system
xi i


LIST OF TABLES
Table page
1 Strains used in this study 31
2 Plasmids used in this study 48
3 Biochemical characterization of strain MC4100 and an
fhlB mutant, strain SE-2011 54
4 Effect of media composition on the expression of
lacZ*) in an fhlB mutant, strain SE-2011 57
5 Plasmid complementation analysis of an fhlB mutant,
strain SE-2011 68
6 Expression of Q(fhlB'-'lacZ+) in different genetic
backgrounds 71
7 Effect of dioxygen on the expression of (fhlA'-'lacZ*) in
an fhlA mutant, strain SE2007 97
8 Effect of media composition on the expression of
Q(fhlA'-'lacZÂ¥) in an fhlA mutant, strain SE-2007 99
9 Expression of Q(fhlA'-'lacZ+) in different genetic
backgrounds 100
10 Anaerobic expression of ^(hyp'-'lacZ+), strains SE-2001 and
SE-2002, in different genetic backgrounds and culture media. 104
11 The effect of molybdenum on expression of Q(fhlB'-'lacZ*),
mutant strain SE-2011, in chlD and molR genetic backgrounds. Ill


56
since the gene is formate inducible (see below) and thus probably plays
a major role in the production of FHL activity. However, defects in the
production of all three HYD isoenzymes and FR can be readily detected in
strain SE-2011.
Formate requirement for expression of the fhlB gene. In order to
better understand the transcriptional control of the FHL pathway,
expression of the fhlB gene was monitored by measuring the levels of p-
galactosidase activity produced by strain SE-2011 from the fhlB
promoter. When cultured under strictly aerobic conditions, strain SE-
2011 produced about 100 U of p-galactosidase activity in all media
tested (Table 4). Upon transfer to anaerobic conditions, the p-
galactosidase activity of the LB culture increased approximately 2.5-
fold after a 4 hr incubation period. Fumarate had no effect on this
anaerobic induction. Nitrate, a high redox potential electron acceptor,
had a repressive effect on anaerobic expression. Both glucose and
formate supplementation elevated fhlB expression anaerobically by about
2-fold and 5-fold, respectively. The glucose enhancement was due to the
endogenous production of formate which was verified (see below) by the
lack of glucose induction in a pfl background, deficient in pyruvate
formatelyase activity. Acidic pH has been shown in the past to increase
FHL activity (17, 78). Formate-dependent induction was reduced by 50%
when the culture medium was buffered at pH 7.0. This parallels the
physiological data which suggests a role for FHL in pH stabilization
during the fermentative growth of E. coli.


113
CCCTGGCTAATGGAACCGGCAACGCAATGTGTGTTTTAGTAGCGAAATCGCGCGCGGTCG 60
CGGAATAATTTTTGTTGGAGCTTAAGGGTGAGAAGGATTTCGGCCTGCATAACAATGTCC 120
T GGCAAAAGTCTT ATT GT GACGGAAAACGAACGCCACGCAAAGCTGACCGCACAAAAGGG 180
GAGTGCTTTTCTGTGCTTAGCGGTTAGAATAGTCTCATGACTATATCTGGAGTTGACCAT 240
Kpnl
GTT AGAGTT ATT AAAAAGT CT GGT ATTCGCCGT AAT CAT GGTACCT GTCGT GAT GGCCAT 300
CATCCTGGGTCTGATTTACGGTCTTGGTGAAGTATTCAACATCTTTTCTGGTGTTGGTAA 360
|£xoIII-l
AAAAGACCAGCCCGGACAAAATCATTGATTCCCTGAATGCCCGCTTAGTCGGGCATTTTC 420
|£xoIII-2
TTTTTCTCAACTTCCTGCTTTTCCTGCCGATATTTTTTCTTATCTACCTCACAAAGGTTA 480
GCAATAACTGCTGGGAAAATTCCGAGTTAGTCGTTATATTGTCGCCTACATAACGTTACA 540
TTAAGGGGTTACCAATGGCTCGTAAATGGTTGAACTTGTTTGCCGGGGCGGCACTCTCTT 600
TCGCTGTTGCTGGCAATGCACTGGCAGATGAAGGGAAAATCACGGTGTTCGCCGCCGCAT 660
CACTGACTAACGCAATGCAGGACATTCTTACGCAGTTTAAAAAAGAGAAAGGCGTGGATG 720
|£xoII1-3
TGGTTTCTTCTTTCGCTTCGTCATCTACTCTCGCCCGTCAGATTGAAGCGGGTGCGCCTG 780
CGGAT CTGTTT ATTT CT GCCGAT CAGAAAT GGAT GGATT AT GCGGTTGAT AAAAAAGCGA 840
TCGATACACGTACGCGTCAGACACTGCTCGGCAATAGCCTGGTCGTTGTAGCACCGAAAG 900
CCAGCGTGCAGAAAGATTTCACCATCGACAGCAAAACCAACTGGACTTCACTGCTGAATG 960
|£xoIII-4
GCGGTCGCCTGGCGGTTGGCGATCCGGAACATGTTCCCGCTGGCATTTATGCAAAAGAAG 1020
CACTGCAAAAACTGGGCGCATGGGATACGCTCTCTCCGAAACTGGCCCCAGCGGAAGATG 1080
TTCGTGGGGCGCTGGCGCTGGTCGAACGTAACGAAGCGCCTCTGGGCATTGTCTACGGTT 1140
CTGACGCAGTTGCCAGCAAAGGGGTAAAAGTGGTTGCCACCTTCCCGGAAGATTCACATA 1200
AAAAAGTGGAATATCCGGTTGCTGTTGTGGAAGGGCATAACAATGCCACAGCGAAACCTT 1260
|£xoIII-5
TTATGATTATCTGAAGGCACCGCAGGCACCCAAATCTTTAAACGTTACGGATTTACAATC 1320
|£xoIII-6
AAGTAATGATACTGACCGATCCAGAATGGCAGGCAGTTTTATTAAGCCTGAAAGTTTCTT 1380
CCCTGGCTGTGCTGTTTAGCCTGCCGTTTGGGATCTTTTTTGCCTGGTTACTGGTGCGTT 1440
GCACGTTTCCGGGCAAAGCTCTGCTCGACAGCGTACTGCATCTACCGCTGGTGTTACCGC 1500
|£xoIII-7
CCGTGGTCGTCGGTTACTTATTATTAGTTTCGATGGGACGGCGCGGATTTATCGGTGAAC 1560
|£xoIII-8
GTCTGTATGACTGGTTTGGTATTACCTTCGCCTTTAGCTGGCGCGGCGCGGTTCTCGCTG 1620
CCGCCGTCATGTCGTTTCCGCTGATGGTGCGGGCAATTCGTCTGGCGCTGGAAGGGGTTG 1680
ATGTCAAACTGGAACAGGCCGCAAGAACACTGGGGGCCGGGCGCTGGCGCGTTTTCTTTA 1740
CTATCACGTTACCGCTGACCTTACCGGGAATTATTGTTGGTACGGTACTGGCTTTTGCTC 1800
Figure 15. Nucleic acid sequence of the mol (chi) operon. Restriction
endonuclease sites for some of the enzymes are highlighted. Exonuclease Ill
generated deletion derivatives, plasmids #1 through 13 are identified.
Translational start sites of 0RF1 and 0RF2 are double underlined (positions 555
to 557 and positions 1326 to 1328, respectively). "Shine-Dalgarno" sequence and
translational start site of the chlD gene are single underlined (positions 2005
to 2009 and positions 2018 to 2020).


88
FHLA EIIGRSEAMYSVLKQVEMVAQSDSTVLIL
NIFA NMVGKSPAMRQIMDIIRQVSRWDTTVLVR
FLBD PMVVRDPAHEQVIKLADQVAPSEASILIT
NTRC DIIAK-PAMQDVFRIIGRLSRSSISVLIN
XYLR G-IGHSPAYKRICETIDKAARGRVSVLLL
DCTD -LIGQTPVMENLRNILRHIADTDVDVLVA
TYRR QIVAVSPKHKHVVEQAQKLAMLSAPLLIT
GETGTGKEl LIARAIHNLSGRNNRRMVKMNCA
GESGTGKEj LIANAIHHNSPRAAAAFVKFNCA
GESGSGKE| VMARYVHGKSRRAKAPFISVNCA
GESGTGKEI LVAHALHRHSPRAKAPFIALNMA
GETGVGKE j VIARSVHLRSERAEQPFVAVNCA
GETGSGKE| VVAQILHQWSHRRKGN FVALNCG
GDTGTGKO| LFAYACHQASPRAGKPYLALNCA
I
FHLA
NIFA
FLBD
NTRC
XYLR
DCTD
TYRR
AM PAG LLESDLFGHERGAFTGASAQRIGRFEL
ALPDNLLESELFGHEKGAFTGAVRQRKGRFEL
AIPENLLESELFGHEKGAFTGAMARRIGKFEE
AIPKDLIESELFGHEKGAFTGANTIRQGRFEQ
AIPPDLIESELFGVDKGAYTGAVNARAGRFER
ALPETVIESELFGHERGAFTGAQKRRTGRIEH
SIPEDAVESELFGH APEGKKGFFEQ
I 1
ADKSSLFLDE
ADGGTLFLDE
ADGGTLLLDE
ADGGTLFLDE
ANGGTIFLDE
ASGGTLFLDE
ANGGSVLLDE
I I
VGDMPLELQPKLLRVLQE
IGESSASFQAKLLRILQE
ISEMDVRLQAKLLRAIQE
IGDMPLDVQTRLLRVLAD
VIELTPRAQATLLRVLQE
IESMPAATQV KM LRVLEM
IGEMS PRMQAKLLRF LND
FHLA QEFERLGSNKIIQTDVRLIAATNRDLKKMVADREFRSDLYYRLNVFPIHLPPLRERPEDIPLLA
NIFA GEMERVGGDETLRVNVRIIAATNRHLEEEVRLGHFREDLYYRLNVMPIALPPLRERQEDIAELA
FLBD REIDRVGGSKPVKVNIRILATSNRDLAQAVKDGTFREDLLYRLNVVNLRLPPLRERPADVISLC
NTRC GQFYRVGGYAPVKVDVRIIAATHQNLEQRVQEGKFREDLFHRLNVIRVHLPPLRERREDIPRLA
XYLR GELERVGGDRTRKVDVRLITATNENLEEAVKMGRFRADLFFRLNVFPVHIPPLRERVEDIPLLV
DCTD REITPLGTNEVRPVNLRVVAAAKIDLGDPAVRGDFREDLYYRLNVVTISIPPLRERRDOIPLLF
TYRR GTFRRVGEDHEVHVDVRVICATQKNLVELVQKGMFREDLYYRLNVLTLNLPPLRDCPQDIMPLT
FHLA KAFT FKIARRLGRNIDSIPAETLRT LSNMEWPGNVRELENVIERAVLLTRG-NVLQL
NIFA HFLVRKIAHSQGRTL-RISDGAIRLLMEYSWPGNVRELENCLERSAVLSES-GLIDR
FLBD EFFVKKYSAANGIEEKPISAEAKRRLIAHRWPGNVRELENAMHRAVLLSAG-PEIEE
NTRC RHFLQVAARELGVEAKLLHPETEAALTRLAWPGNVRQLENTCRWLTVMAAGQEVLIQ
XYLR EHFLRRHHKEYGKKTLGLSDRAMEACLHYQWPGNIRELENALERGVILTES-N--ES
DCTD SHFAARAAERFRRDVPPLSPDVRRHLASHTWPGNVRELSHYAERVVLGVEG-GGAAA
TYRR ELFVARFADEQGVPRPKLAADLNTVLTRYAWPGNVRQLKNAIYRALTQL-DGYEL
Figure 11. Alignment of the predicted "domain D" amino acid sequences
of E. coli FHL-A, K. pneumoniae NIF-A, C. crescentus FLB-D, E. coli NTR-
C, P. putida XYL-R, R. leguminosarum DCT-D, and E. coli TYR-R.
Highlighted residues are identical or functionally similar to the FHL-A
protein. Double underlined regions represent those residues of which
five out of the six aligned amino acids are homologous to FHL-A protein.
Boxed areas are the proposed ATP-binding regions.


BIOGRAPHICAL SKETCH
Julie Anne Maupin was born on July 30, 1963, in Richmond Heights,
Missouri. She received a bachelor's degree in biological sciences in
April 1985 and during that time received a honor society scholarship.
Working in a biochemistry laboratory at the University of Alabama in
Huntsville, she developed an enthusiasm for genetics and molecular
biology. Since 1988, she has been in the Ph.D. program in the
Department of Microbiology and Cell Science at the University of
Florida.
143


76
by strain SE-2011 was enhanced from about 20 units/pg protein to about
330 units/pg protein if the fhlA gene was also present in a multicopy
plasmid (pSE-133-1) (Fig. 8). Immediately after anaerobic conditions
were established, the //? IB-mediated (3-galactosidase activity increased
to about 1,200 units within a generation time. During the second
generation, this activity decreased to about 75% of the observed peak
value. The p-galactosidase activity of the culture reached about 1,000
units and was maintained at that level. On the other hand, strain M9s
[^(fdhF'-' lacZ*")] carries the lac fusion in FDH-H, a known membrane
protein. With the same plasmid, this strain (M9s/pSE-1331) produced p-
galactosidase activity after an initial lag. The differential rate of
synthesis was about 10% of the values of strain SE2011/pSE1331 and
was only about 3-fold higher than strain M9s without the plasmid. The
levels of p-galactosidase activity produced by strains SE-2011 and M9s,
both in the presence and absence of the plasmid pSE-133-1, was not
altered by including Mo or Se to the LB medium, although the FDH-H is a
Mo, Se-containing protein. The amount of formate needed for optimum
expression of Q(fdhF'-'lacZ*)/pSE-133-1 was about 3 mM also which is
similar to that required for strain MJ-9/pSE-133. This rapid induction
of fhlB gene in the presence of multiple copies of fhlA+ gene, which is
in contrast to the fdhF gene, suggests that the FHL-B protein is
cytoplasmic. Its synthesis is apparently not constrained by the
availability of membrane proteins and membrane synthesis.


Regulation of the fhlA Gene 94
Molybdate Metabolism and FHL Activity 110
CONCLUSION 116
REFERENCES 120
BIOGRAPHICAL SKETCH 143
iv


97
Table 7. Effect of dioxygen on the expression of Q(fhlA'-'lacZ*) in
an fhlA mutant, strain SE-2007
Conditions
Innoculum
size
Incubation
time
p-Galactos i dase Activity3
Microaerobic
16 hr
1,800
Aerobic-1
1%
2 hr
990
Aerobic-2
1%
1 hr
290
Anaerobic
10%
4 hr
1,800
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
All cultures were grown at 37C in Luria Broth.
An overnight stationary (1 ml in 13 x 100 mm metal-cap tube) LB culture of
strain SE-2007 served as the inoculum for this experiment. This culture
is referred above as the microaerobic culture. During the first part, 0.1
ml of this culture was inoculated into 10 ml LB (125 ml flask) and shaken
at 250 rpm. After 2 hr of growth, 0.3 ml of this aerobic culture was
transferred to 30 ml of fresh LB (1 L flask) and grown in the shaker for
1 hr. This culture was used to inoculate LB at 10% (v/v) and grown under
anaerobic conditions. p-Galactosidase activities of all the cultures at
the time of transfer to fresh medium (except the anaerobic culture, which
was harvested after 4 hr growth) are presented in the Table.


59
PROTEIN (jig)
Figure 3. Differential rate of synthesis of p-galactosidase activity by
lacZ*) strain SE-2011 grown in LB medium with different
supplements. LBG, LBF, and LBGF represent LB-glucose, LB-formate, and
LB-glucose-formate media, respectively. p-Galactosidase and protein
activities are expressed as units per ml and pg per ml, respectively.


Table 9. Expression of {fhlA'-' lacZ+) in different
genetic backgrounds
Strain
Relevant genotype
pGal actos i dase activity3
SE-2007
<^{fhlA,-lacZ+)
1,800
MJ101
${fhlA'-'lacZf) rpoN
2,100
MJ102
lacZ+) narL
1,200
MJ103
[fhlA'-' lacZ*) fnr
1,600
MJ107
Q(fhlA'-'lacZ*) cya
.Q
O
O
CO
MJ-108
QifhlA'-'lacZ+) molR
1,400
MJ-109
*{fhlA'-'lacZ+) pfl
2,300
SE-2007
HfhlA'-'lacZ+)
650/680
SE1762
*(fhlA'-'lacZ+) chlD
830/800
All cultures were grown for 4 hr anaerobically at 37C in Luria Broth
unless otherwise indicated.
aExpresssed as nanomoles of o-nitrophenol produced per minute per
milligram of protein.
bPoor cell growth.
independent experiment in which cells were grown in LB/and LB
supplemented with 1 mM Mo.


GENETIC REGULATION OF FORMATE HYDROGENLYASE IN ESCHERICHIA COLI
By
JULIE ANNE MAUPIN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991

ACKNOWLEDGEMENTS
I would like to sincerely thank my advising professor Dr. K. T.
Shanmugam. His continual encouragement, guidance and enthusiasm for
science have definitely influenced me for a lifetime. I would like to
thank the K. T. S. laboratory group including both old and new. I would
also like to extend my thanks to Drs. Richard Boyce, Francis Davis Jr.,
John Gander, and Lonnie Ingram for their helpful questions and counsel
while serving on my committee. Additionally, I would like to thank Drs.
A. Bock and B. Bachmann for providing several strains and plasmids
necessary for this study. Finally, special thanks are extended to my
family and fianc for their willingness to listen to my ups and downs
throughout the course of this work.

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS
TABLE OF CONTENTS Hi
LIST OF FIGURES v
LIST OF TABLES vii
ABBREVIATIONS viii
LIST OF GENE SYMBOLS xi
ABSTRACT xiv
INTRODUCTION 1
LITERATURE REVIEW 7
Anaerobiosis 11
Nitrate 16
Formate 18
Molybdenum 21
Nickel 25
Selenium 27
MATERIALS AND METHODS 30
Materials 30
Bacterial Strains and Media 30
Isolation of Mutants 30
Enzyme Activities and their Respective Culture Conditions 36
Genetic and Molecular Biological Experiments 41
RESULTS AND DISCUSSION 53
Physiological Properties of an fhlB Mutant, Strain SE-2011 ... .53
Genetic Characteristics of Strain SE-2011, Q(fhlB'lacZ*) ... .64
Trans-acting Factors of the fhlB Operon 70
Analysis of the fhlA Gene 79

Regulation of the fhlA Gene 94
Molybdate Metabolism and FHL Activity 110
CONCLUSION 116
REFERENCES 120
BIOGRAPHICAL SKETCH 143
iv

Figure
LIST OF FIGURES
page
1 Model of the genetic regulation of formate hydrogenlyase in
E. coli 4
2 Mixed-acid fermentation of E. coli 9
3 Differential rate of synthesis of p-galactosidase activity by
[fhlB'-'lacZ+) strain SE-2011 grown in LB medium with
different supplements 59
4 Effect of formate on the induction of p-galactosidase
activity by QifhlB-lacZ*) strain SE-2011 and a pfl
derivative, strain MJ-9 61
5 Effect of nitrate supplementation on fhlB gene expression. .62
6 Differential rate of induction of the Q(fhlB'-'lacZ+)
fusion in strain SE-2011 in the presence and absence of
plasmid pSE-133 73
7 Effect of formate concentration on the levels of 0-galacto-
sidase activity produced by a Q(fhlB'-'lacZ*) pfl double
mutant, strain MJ-9, in the presence of plasmids pSE-133 and
pSE-133-2 (fhlA::Jn5) 74
8 Differential rate of synthesis of <\>(fdhF'-'lacZ+) t strain
M9s, and $(fhlB'-'lacZ+), strain SE-2011, in LB medium
supplemented with 3 mM formate in the presence and absence
of the fhlA+ gene in a multicopy plasmid (pSE-133-1) 77
9 Nucleic acid and predicted protein sequences of the
partial hypC gene and complete hydB, hydF and fhlA genes . .80
10Alignment of the predicted seguences of E. coli FHL-A,
E. coli NTR-C, K. pneumoniae NIF-A, and P. put ida XYL-R
proteins 85
v

11 Alignment of the predicted "domain D" amino acid sequences
of E. coli FHL-A, K. pneumoniae NIF-A, S. typhimurium FLB-D,
E. coli NTR-C, P. putida XYL-R, R. leguminosarum DCT-D, and
E. coli TYR-R 88
12 Alignment of potential DNA-binding motifs in the FHL-A,
HYD-G, NTR-C, XYL-R and NIF-A proteins 90
13 Localization of the promoter lac fusions in strains SE-2007
[Q(fhlA'-'lacZ*)], SE-2001 [*{hyp'-'lacZ+)1], and SE-2002
[Q{hyp'lacZ+)2] 96
14 Differential rate of synthesis of Q(fhlAlacZ*), strain
SE-2007, and (hypX'-' lacZ+)li strain SE-2001, in LB
medium supplemented with 3 mM formate in the presence and
absence of the fhlA+ gene in a multicopy plasmid (pSE-133). 108
15 Nucleic acid sequence of the mol {chi) operon 113
VI

LIST OF TABLES
Table page
1 Strains used in this study 31
2 Plasmids used in this study 48
3 Biochemical characterization of strain MC4100 and an
fhlB mutant, strain SE-2011 54
4 Effect of media composition on the expression of
lacZ*) in an fhlB mutant, strain SE-2011 57
5 Plasmid complementation analysis of an fhlB mutant,
strain SE-2011 68
6 Expression of Q(fhlB'-'lacZ+) in different genetic
backgrounds 71
7 Effect of dioxygen on the expression of (fhlA'-'lacZ*) in
an fhlA mutant, strain SE2007 97
8 Effect of media composition on the expression of
Q(fhlA'-'lacZÂ¥) in an fhlA mutant, strain SE-2007 99
9 Expression of Q(fhlA'-'lacZ+) in different genetic
backgrounds 100
10 Anaerobic expression of ^(hyp'-'lacZ+), strains SE-2001 and
SE-2002, in different genetic backgrounds and culture media. 104
11 The effect of molybdenum on expression of Q(fhlB'-'lacZ*),
mutant strain SE-2011, in chlD and molR genetic backgrounds. Ill

LIST OF ABBREVIATIONS
A Absorbance
Ac-CoA Acetyl coenzyme A
ATP Adenosine triphosphate
BV Benzyl viologen
CAT Chloramphenicol acetyl transferase
CRP cAMP repressor protein
CTAB Hexadecyltrimethyl ammonium bromide
DMS Dimethyl sulfide
DMSO Dimethyl sulfoxide
FDH-H Formate dehydrogenase (linked to H2
evolution)
FDH-N Formate dehydrogenase (linked to N03
reduction)
FHL Formate hydrogenlyase
FR Fumarate reductase
HUP Hydrogen uptake activity
HYD Hydrogenase
HYD-1 Hydrogenase isoenzyme-1
HYD-2 Hydrogenase isoenzyme-2
HYD-3 Hydrogenase isoenzyme-3
LB Luria Broth
vi i i

LBG Luria Broth + glucose
LBGF Luria Broth + glucose + formate
LBF Luria Broth + formate
LBM Luria Broth + maltose
LBN Luria Broth + nitrate
LBNF Luria Broth + nitrate + formate
MES 2-(N-Morpholino)ethanesulfonic acid
Mo Molybdenum
MOPS 3-(N-Morpholino)propanesulfonic acid
MPT Molybdopterin
MGD Molybdopterin guanine dinucleotide
NAD+, NADH Nicotinamide adenine dinucleotide and its
reduced form, respectively
OAA Oxaloacetate
ONPG Ortho-nitrophenyl-p-D-galactopyranoside
ORF Open reading frame
PDH Pyruvate dehydrogenase
PEP Phosphoenolpyruvate
PFL Pyruvate formatelyase
PIPES Piperazine-N,N'-bis[2-ethanesulfonic
acid]; 1,4-piperazinediethanesulfonic acid
PMF Proton motive force
SDS Sodium dodecyl sulfate
TES N-Tris-(hydroxymethyl)methyl-2-
aminoethanesulfonic acid
TMA Trimethyl amine
ix

TMAO Trimethyl amine-N-oxide
Tris Tris-(hydroxymethyl)-ami nomethane
X-gal 5-Bromo-4-chloro-3-indolyl-p-D-
galactopyranoside
x

LIST OF GENE SYMBOLS
All the genes listed below are from Escherichia coli unless otherwise
indicated.
Gene Alternate gene symbols; phenotype affected
symbol
ant See hyc; anaerobic electron transport
arcA Aerobic regulatory control, putative DNA binding
protein of Arc modulon
arcB Aerobic regulatory control, histidine-protein-
kinase of Arc modulon
chi A Synthesis of the pterin component of MPT
chlB "Association factor-FA"; synthesis of functional MPT
chlC See narGHJI
chlD Peripheral protein of molybdate binding-protein-dependent
transport system
chlE Synthesis of the pterin component of MPT
chlF See fdhGHI
chlG Molybdate-restorable nitrate reductase activity
chlJ Integral membrane protein of molybdate binding-protein-
dependent transport
cyd Cytochrome d\ high-affinity oxidase
cyo Cytochrome o; low-affinity oxidase
dctD C-4 dicarboxylate transport; Rhizobium leguminosarum
dmsABC DMSO and TMAO reductase activity
xi

ebgA Ancestral gene for p-galactosidase
fdhF FDH-H; formate dehydrogenase (linked to H2 evolution)
fdhGHI FDH-N; formate dehydrogenase (linked to N03 reduction)
fhlA Putative DNA binding protein necessary for transcriptional
activation of the fdhF, hyc and fhlB operons
fhlB Putative modulator of the FHL pathway; necessary for FHL,
FDH-H and total HYD activity
flbD Flagellar synthesis; Caulobacter crescentus
fnr Global regulator of anaerobic respiration; homologous to CRP
frdABCD Fumarate reductase
gyrB DNA gyrase
his Histidine biosynthesis and binding-protein-dependent
transport system
hyb Hydrogenase isoenzyme-2
hyc Hydrogenase isoenzyme-3
hydA Total hydrogenase activity
hydFB hypDE\ total hydrogenase activity
hydE hypB\ total hydrogenase activity; Ni suppressible
hydC,D Total hydrogenase activity; nickel transport
hyd-17 0RF5 of the hyc operon, hydrogenase isoenzyme-3
hydG Putative DNA binding protein of dihydrogen metabolism
(uptake)
hyp Total hydrogenase activity, operon includes previously
described hydB, £, and F genes
mal Maltose metabolism and binding-protein-dependent transport
system
molR Molybdate binding-protein-dependent transport system
xi i

narL
Putative DNA binding protein of nitrate regulation
narX
Putative membrane-bound histidine-protein-kinase of
nitrate regulation
narQ
Second proposed histidine-protein-kinase of nitrate
regulation
narGHJI
Nitrate reductase
nifA
Positive activator for nitrogen fixation; Klebsiella
pneumoniae
ntrC
glnG, nitrogen metabolism
pfl
Pyruvate formatelyase
pgi
oxrC, phosphoglucose isomerase
tyrR
Aromatic amino acid biosynthesis and transport
xylR
Degradative pathway of aromatic hydrocarbons; Pseudomonas
put ida

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
GENETIC REGULATION OF FORMATE HYDROGENLYASE IN ESCHERICHIA COLI
By
Julie Anne Maupin
December, 1991
Chairman: Dr. K. T. Shanmugam
Major Department: Microbiology and Cell Science
A new gene (fhlB) whose product regulates the production of
formate hydrogenlyase (FHL) has been identified in Escherichia coli.
Biochemical analysis of a mutant, strain SE-2011 [$>(//IB'-' lacZ*)],
revealed that this mutant lacks formate dehydrogenase activity
associated with FHL (FDH-H) and hydrogenase activity. As a consequence,
dihydrogen production and uptake were undetectable in strain SE-2011.
Expression of the fhlB gene (measured as p-galactosidase activity) was
increased 2- to 3-fold by anaerobic conditions, and enhanced by formate,
but only under anaerobic conditions. Maximum expression of (fhlB'~
'lacZ+) required rpoN, fhlA, chlD, and molR gene products. The
concentration of formate required for maximum expression of the fhlB
gene was reduced in the presence of fhlA gene in a multicopy plasmid
from about 15 mM to 3 mM. The fhlB gene in molR or chlD genetic
xiv

background still retained formate inducibi1ity; however, molybdate was
required at high concentrations for expression to wild type levels. DNA
sequence analysis of fhlA gene revealed that the FHL-A protein is
homologous to other known transcriptional activators. It is proposed
that formate, in association with the FHL-A protein, initiates
transcription of the fhlB, hyc, hyp and fdhF operons. Analysis of
mutant strain SE-2007 [${fhlAlacZ*)] indicated that the transcription
of the fhlA gene, located at the 3'-end of the hyp operon, is
constitutive. This is in contrast to the hyp operon transcription which
has dual controls: formate-dependent and FNR-dependent. The DNA
sequence of the mol operon (essential for molybdate transport, FHL
activity and fhlB expression) was also determined to investigate the
role of molybdate metabolism in the regulation of FHL in E. coli. These
studies revealed that the fermentative dihydrogen production is
regulated by a complex series of reactions involving the availability of
formate, Mo and absence of 02. It is postulated that the FHL-B protein
is one of the modulators of this control system.
xv

INTRODUCTION
The production of dihydrogen by the members of the
Enterobacteriaceae family has been known since 1901 (166). Although
much has been learned since that time, many avenues of research remain
to be explored. It is now understood that the formate hydrogenlyase
complex is composed of FDH-H, HYD-3, and associated electron transport
proteins (10, 27, 72, 173, 193, 215, 243). This complex catalyzes the
oxidation of formate to H2 and C02 (2, 166) and is found only in
anaerobically growing cells. The reaction catalyzed by the FHL complex
operates at a standard free energy (AG0') of zero and does not appear to
be involved in cellular energy production (2). It is presumed that the
FHL system plays an important role in later stages of the
microorganism's fermentative growth when organic acids such as formate
accumulate and lower the pH of the culture medium. The cell apparently
synthesizes the FHL complex in order to modulate the external pH, thus
facilitating growth (77).
The FHL pathway is an attractive research field due to the
potential of harnessing dihydrogen production as an alternative energy
source. Current events, including the Persian Gulf War and concern over
the greenhouse effect, have aroused the public's attention. Although
lobbyists continue to solicit Congress for the oil industry, the
1

2
American population is more aware of this crisis and open to
environmentally and economically sound options. Research in these
fields must continue in order for alternative fuels to become more
competitive. Unfortunately, historical events, i.e. the "Hindenberg",
have curbed the use of dihydrogen as an energy source. When properly
controlled, dihydrogen is a clean burning fuel. The end product being
H20, instead of CO, C02, S02 and nuclear waste (the current waste
products of energy production in our country).
Additionally, the question of how an organism responds to various
environmental stimuli such as dioxygen and regulates the metabolism by
activating or repressing specific genes remains to be fully elucidated.
By studying the FHL system of Escherichia coli, one can not only
integrate regulatory similarities from this system to other pathways,
but also study modes of control which are unique to FHL in order to gain
a clear understanding of gene expression. Associating this with the
physiological role of anaerobic H2 and C02 production is also of
interest. The metabolic fate of the end products of FHL are poorly
understood.
Following the advent of DNA sequencing techniques and computer
programs designed to organize the sequences generated, studies on
protein homology have enabled the researcher to identify apparent
function from the primary structure. It is now understood that there
are two categories of proteins involved in signal transduction: sensor
and receiver. Generally, the sensor is a membrane-bound, histidine-

3
protein-kinase. Appropriate environmental stimulation results in sensor
autophosphorylation cascading to phosphorylation of the receiver
protein. Once in an active form, the receiver is a positive regulator
of specific modulon transcription (163, 183, 222).
Much remains to be understood about how the "appropriate
environmental stimulation" is transmitted to the sensor protein. It has
been proposed that the FHL system is regulated by five environmental
signals which include anaerobiosis, intracellular formate, pH,
molybdate, and nickel. Although the genes encoding the structural
components of the FHL complex [fdhF (FDH-H) and hyc (HYD-3) operons] are
located approximately 33 minutes apart in the E. coli chromosome, their
transcription appears to be coordinated (11, 24, 25, 171).
In order to decipher the mode by which these signals are
transmitted to the transcription of the formate modulon, a series of
promoter lac fusion strains were constructed and characterized for
anaerobic induction and dihydrogen related enzyme activities.
Mutational analysis revealed a unique gene, later denoted as the fhlB
gene, encoding a product essential for FHL activity including FDH-H and
total hydrogenase activity. However, FHL-B protein was not necessary
for expression of the fdhF gene and is therefore currently assumed
modulate transcription of the FHL pathway (Fig. 1). Transcription of
the fhlB gene was repressed by high-redox potential electron acceptors
(dioxygen and nitrate) and required formate for maximum anaerobic
induction. Further analysis revealed that rpoN (encoding the o54

4
(fhlX)?
h y
fhIA
y i
fi- ii y
I URS URS T
-i 'transport proteins r-
HYD3J ^ |_FDH-H
FHL
FORMATE H2 + CO*
Figure 1. Model of genetic regulation of formate hydrogenlyase in E.
coli.

5
subunit of RNA polymerase), fhlA, chlD and molR gene products were
necessary for optimum fhlB gene transcription (Fig. 1).
Subsequent investigation of the fhlB gene transcription
demonstrated that high cytoplasmic concentrations of FHL-A protein
(using multicopy plasmid pSE-133) decreased but did not eliminate the
formate requirement. Because of these observations, the DNA sequence of
the fhlA gene was determined to identify the characteristics of the gene
and its product. The FHL-A protein was found to have sequence homology
with putative o54-dependent transcriptional activators (receivers) of
the two-component regulatory system. However, the amino terminus of the
FHL-A protein was unique and did not contain the conserved secondary
structure or aspartate and lysine residues which have been shown to be
phosphorylated by the respective histidine-protein-kinase (sensor).
This would suggest that phosphorylation is not required for active FHL-A
protein, and a sensor protein may not be involved. Interestingly,
transcription of the fhlA gene was shown to be from the weak
"-35 and -10" fhlA promoter (constitutive) and not from the upstream
54
FNR- or o -dependent promoters of the hyp operon. It is therefore
possible that the FHL-A protein is synthesized in an inactive form and
activated in the presence of formate (Fig. 1).
During investigation of the molybdate requirement for FHL, it was
determined that the fhlB gene in molR or chlD genetic backgrounds still
retained formate inducibility in the presence of multiple copies of the
fhlA+ gene (exogenous molybdate was required for expression to wild type

6
levels). This indicates that FHL is regulated by a dual regulatory
system, one involving molybdate and a second formate-dependent pathway
(Fig. 1). The results presented show that fermentative dihydrogen
production is regulated by a complex series of reactions involving the
availability of formate, molybdate and the absence of oxygen.

LITERATURE REVIEW
Facultative anaerobes, such as Escherichia coli, have evolved
respiratory networks organized such that the most energetically
favorable electron transport pathway is utilized for the specific redox
state of the environment. E. coli is capable of using a variety of
electron acceptors which include in order of decreasing potential:
l/202/H20 (E0'= +0.82 V), N03"/N02" (E0'= +0.42 V), N02"/NH4+
(E0'= +0.36 V), DMSO/DMS (EQ' = +0.16 V), TMAO/TMA (E0'= +0.13 V),
fumarate/succinate (E0'= +0.03 V), pyruvate/lactate (E0'= -0.19 V),
acetaldehyde/ethanol (E0*= -0.2 V), H+/H2 (E0' = -0.42 V; ref. 16, 228).
Pathway preference is determined by the difference between the standard
oxidation-reduction potential of the initial electron-donor (NADH) and
terminal acceptor system (AE0') available in the growth medium. The
standard free energy change (AG0) of a reaction is given by
AG0I= -nfAE0'
where n is the number of electrons transferred and F is Faraday's
constant. Therefore the greater the AE0, the more free energy can be
harnessed by the organism for biosynthetic purposes.
This establishes a hierarchy with aerobic respiration being the
most energetically favorable for the cell's survival. Thus, other
7

8
electron transport pathways are not synthesized in the presence of
dioxygen. Furthermore when nitrate is present in an anaerobic
environment, the nitrate respiratory pathway is induced and alternate
respiratory systems are repressed (for review, see 82, 102, 139, 175).
Presumably, a complex interconnected regulation involving transcription,
translation, protein processing, and allosteric effectors is
conceivable.
Respiration involves the generation of a proton motive force
(PMF), consisting of a pH gradient (ApH) and electrochemical gradient
(Aip), by electron transport (for review, see 84, 160). In general,
respiratory components are organized into "modules" or pathways. These
include a substrate dehydrogenase, which transfers electrons to a
quinone pool, and ultimately a membrane-bound reductase; thus coupling
substrate oxidation to the reduction of an electron acceptor (139). A
fermenting cell, on the other hand, produces endogenous electron
acceptors and derives most of its energy from substrate-level
phosphorylation (42). Although it is generally believed that the FHL
system is a fermentative process discrete from anaerobic respiration,
the precise function of the FHL pathway remains unclear.
E. coli is a mixed-acid fermentor producing acetate, lactate,
formate, ethanol, succinate, H2 and C02 (Fig. 2). The ratio of these
major end products is regulated (conceivably by redox-mediated
modulation) to balance the reducing equivalents generated during
glycolysis (3, 206). All of these fermentation products are derived

9
2[H]
GLUCOSE
ADP
ATP
PEP
LACTATE"
2[H]
I
12
ADP
ATP
PYRUVATE -
2[H]
V
10
ACETYL-CoA
si
CO,
FORMATE16
2[H]
I \
.Lp
h* CoA
ACETALYDEHYDE ACETYL-P
V- adp
2[H]H 11 9 [
ETHANOL
ATP
ACETATE
- OAA
2[H]
MALATE
sl
H* -A 7
A *
FUMARATE
*
2[H]
SUCCINATE
Figure 2. Mixed-acid fermentation of E. coli. 1, Phosphoenolypyruvate
carboxylase; 2, malate dehydrogenase; 3, fumarase; 4, fumarate
reductase; 5, pyruvate formatelyase; 6, formate hydrogenlyase complex
(formate dehydrogenase-H, hydrogenase isoenzyme-3, and associated
electron transport proteins); 7, hydrogenase isoenzyme-2; 8,
phosphotransacetylase; 9, acetate kinase; 10, acetaldehyde
dehydrogenase; 11, alcohol dehydrogenase; 12, lactate dehydrogenase.
Redrawn with modifications from Alam and Clark (3).

10
from pyruvate. It has been proposed that PDH is inhibited by the high
[NADH]/[NAD+] ratio reached upon shift to anaerobic conditions (83).
Therefore anaerobically active PFL, encoded by the pfl operon, plays a
central role in glucose metabolism (122, 170, 191). The PFL complex
catalyzes the nonoxidative cleavage of pyruvate leading to transfer of
"acetyl" group to coenzyme A (Ac-CoA) and formate (75). Consequently,
the C02 and reduced NADH produced aerobically by PDH is replaced
anaerobically by formate. This is critical since the cell must maintain
an acceptable ratio of [NADH]/[NAD+] (42). The FHL catalyzes the
oxidation of formate to H2 and C02. This minimizes the accumulation of
formic acid which would otherwise lower the pH (77). The metabolic
fates of the gaseous products, H2 and C02, are poorly understood. It
is hypothesized that FHL is the major anaerobic enzyme producing C02 for
the synthesis of oxaloacetate (OAA) from phosphoenol pyruvate (PEP; ref.
42). OAA can be further metabolized to succinate or 2-oxoglutarate, all
three of these metabolites being essential for biosynthetic reactions
such as amino acid production. E. coli can utilize the H2 produced from
formate oxidation by FHL to reduce fumarate to succinate (HUP; ref.
148). This energy yielding pathway is HYD-2 dependent (12, 151, 193).
This would link FHL to the membrane-bound fumarate reductase and thus a
respiratory process which provides the cell with 1 mole of ATP per mole
of fumarate reduced, presumably by the generation of PMF (116).
Therefore, FHL appears to be linked to both fermentative and respiratory
pathways.

11
Formate hydrogenlyase is composed of FDH-H, HYD-3, and redox
carriers linking the two enzymes (27, 77, 173, 193). The fdhF gene
(92.4 min) encodes the 80 kDa selenopolypeptide constituent of FDH-H
(11, 243). Additionally, the FDH-H protein requires molybdenum
(molybdopterin-guanylate) for activity (33, 73, 112). The hyc operon
(59 min) consists of 8 ORFs and is presumed to encode the remaining FHL
components. Five of these ORFs code for electron carriers (previously
termed ant and hyd-17; ref. 11, 27, 171, 242). The 0RF5 (hyd-17) shows
significant sequence homology with the large subunit from other Ni/Fe
hydrogenases and is the presumptive structural gene for HYD-3.
Numerous mutants have been isolated with defects in H2 metabolism
(Fhl-), the majority of which have been deficient in all three
hydrogenase isoenzymes (35, 37, 76, 107, 128, 135, 167, 171, 231).
Through the characterization of mutant strains, DNA sequence analysis
and mapping of the genes affected, FHL regulation can be more clearly
understood. It is proposed that the FHL system is regulated by signals
which include anaerobiosis, nitrate, intracellular formate, low pH,
molybdate, nickel, and selenium. How these signals are transmitted to
the transcription, translation and post-translational modification of
the components of the FHL complex remains poorly understood.
Anaerobiosis
The presence or absence of terminal electron acceptors, such as
dioxygen or nitrate, play a major role in regulating genes involved in
respiration and fermentation. It was estimated, by using 2-dimensional

12
protein electrophoresis, that 18 or more proteins were induced by
anaerobiosis in E. coli (205). Later studies using a physiological
approach with lac gene fusions, estimated that 50 genes were induced
when dioxygen and alternative electron acceptors were absent, 20% of
these were H2 repressive, and only a small number of mutants failed to
grow microaerobically on glucose minimal medium (41).
The FHL pathway has been shown to be induced anaerobically by a
variety of methods. Initially, FHL induction was monitored by an
increase in specific activity of FHL and the enzymes of the pathway (10,
57, 78, 127, 203, 235, 236). Later, transcription of the fdhF and hyc
(hyd-17) operons (structural genes of FHL), measured as 0-galactosidase
activity using gene fusion strains, was found to be anaerobically
inducible (171). These studies demonstrate that anaerobically growing
organisms require the synthesis of additional proteins to generate
energy and maintain redox balance. Unfortunately, little is known about
how the organism senses the onset of anaerobiosis and activates or
represses specific pathways.
Many earlier studies have suggested that anaerobic electron
transport systems are subject to a "dual control" in which an
unidentified effector molecule somehow senses the "redox" status of the
environment and activates FNR protein, a pleiotropic transcriptional
activator and repressor. In combination with this global regulatory
mechanism, specific transcriptional effectors responded to terminal
electron acceptors such as nitrate or fumarate (36, 47, 197, 198). With

13
the identification and characterization of the Arc system and other
forms of respiratory control, current research forms a much more complex
picture of regulation (105, 106, 212).
Available evidence suggests that this aerobic-anaerobic switch may
involve several genes including arcA, arcB, fnr and pgi (involved in
DNA-supercoiling); mutants deficient in any one of these genes have
pleiotropic phenotypes. The Arc modulon is a two-component sensor-
regulator system (105, 106). Through DNA sequence analysis, ARC-B is
presumed to be a membrane-bound, histidine-protein kinase.
Autophosphorylation of ARC-B occurs in response to dioxygen-limitation
(possibly redox control). The signal is then transmitted to ARC-A by
phosphorylation to produce a transcriptional activator of the cytochrome
d (a high-affinity oxidase) gene (cyd). ARC-A is also a repressor of
"aerobic" enzyme synthesis (i.e. succinate dehydrogenase and cytochrome
o; ref. 68, 105). Cytochrome o, having a lower affinity for dioxygen,
is only synthesized aerobically; where as cytochrome d is stimulated
under microaerobic conditions as the terminal electron carrier (for
review, see 175).
The FNR protein, likewise, acts as both a transcriptional
activator and repressor (for review, see 211, 212). Mutations in the
fnr gene were originally isolated as strains deficient in nitrate
reductase and fumarate reductase (133). It is now known that several
other anaerobically inducible enzymes require FNR for transcription and
include nitrite reductase (both cyt552 and NADH-1inked; ref. 38, 45,

14
159), DMSO:TMAO reductase (216, 232), PFL (190, 191), FDH-N (19, 25);
anaerobic glycerol-3-phosphate dehydrogenase (130), fumarase B (18,
237), aspartase, and asparaginase II (109). The FNR protein represses
its own synthesis (autoregulation; ref. 209) as well as cytochrome o
(low-affinity oxidase; ref. 50, 68) and NADH dehydrogenase II (213).
Recent evidence is conflicting on FNR regulation at the cyd promoter.
By monitoring cyd expression at varying levels of dissolved dioxygen
concentrations, Fu et al. (68) suggest that FNR, a weaker
transcriptional activator at this operon, outcompetes ARC-A for operator
binding as the culture shifts from microaerobic to anaerobic conditions.
The FNR protein has been shown through sequence analysis to be
homologous to cAMP receptor protein (CRP) in the helix-turn-helix and
nucleotide binding domains (199). However, the FNR amino-terminus
differs. It is cysteine rich and three of the four cysteine residues
are proposed to be involved in redox-sensitive iron-binding (210). The
addition of ferrozine, an iron specific chelator, to the growth medium
reduced the level of transcription of the frd operon (encoding fumarate
reductase). Since this operon is positively regulated by FNR, iron in
association with FNR may be involved in this regulation. In support of
this possibility, Green et al. (79) purified FNR in the presence of
externally added iron and demonstrated specific in vitro binding of the
pure protein to a synthetic (postulated) FNR-binding site by both DNasel
and methylation-protection (DMS) footprinting. Although direct evidence
is still lacking, parallels can be drawn with the thoroughly

15
investigated, metal-dependent regulation of FUR (iron uptake) and MER-R
(mercury resistance) systems (for review, see 90). It is possible that
molybdenum and nickel (necessary for transcriptional repression of chlD
and hydC genes, respectively) be also involve a metal-protein complex.
Formate hydrogenlyase synthesis is independent of the ARC system,
and FNR-dependence appears to be indirect (44, 105, 171). The
transcription of the pfl gene which is essential for the production of
formate is partially FNR dependent (190, 191). The gene coding for
nickel transport (hydC) is also FNR dependent (239). The hyp operon,
whose products presumably process the nickel into forms suitable for
hydrogenase, appears to be FNR regulated when the intracellular formate
concentration is limiting (this study).
DNA supercoiling has been suggested to be essential for
transcription from several promoters including the FNR-independent
promoters of the FHL pathway. Early studies have shown that mutants
lacking the DNA gyrase activity are also impaired in anaerobic growth
(241). Additionally, FHL activity is absent and plasmid supercoiling is
altered in a glucose-grown pgi (oxrC) mutant. Phosphoglucose isomerase,
a glycolytic enzyme encoded by the pgi (oxrC) gene, is necessary for the
synthesis of FDH-H, peptidase T, tripeptide permease, HYD-1 and HYD-3 in
Salmonella typhimurium (107, 108). Recently, Hsieh et al. (98) have
compared changes in [ATP]/[ADP] ratios and negative supercoiling of both
chromosomal and plasmid DNA upon shifting f. coli from aerobic to
anaerobic conditions. They monitored the effect of dioxygen tension on

16
supercoiling activity in wild type and gyrB (DNA gyrase) mutants. It
was concluded that the respiratory state of the organism influenced DNA
supercoiling. Furthermore, this was mediated by DNA gyrase since,
changes in supercoiling paralleled the [ATP]/[ADP] ratios. This is
analogous to in vitro studies which demonstrated that the [ATP]/[ADP]
ratio influences gyrase-mediated DNA supercoiling (233). Therefore, the
energy status of the organism may be an environmental signal affecting
DNA supercoiling. In general, the state of chromosomal supercoiling is
presumed to be a mechanism of transcriptional control; however,
verification of this hypothesis has been difficult.
Nitrate
Anaerobically, nitrate supplementation induces the nitrate-formate
respiratory system and represses other respiratory and fermentative
pathways of lower redox potential. The narGHJI (nitrate reductase; ref.
36, 62, 137, 216) and fdhGHI (FDH-N; ref. 19) operons, both required for
nitrate respiration, are induced. Whereas, the frdABCD operon (fumarate
reductase; ref. 104, 115), the dmsABC operon (DMSO and TMAO reductase;
ref. 51, 104), the pfl gene (190), the fdhF gene and the hyc operon (25,
171) are repressed in the presence of the high redox potential acceptor,
nitrate.
Current evidence suggests that a two-component regulatory system
mediates transcriptional control of the narGHJI, fdhGHI, frdABCD, and
pfl operons. The NAR-X protein (and possibly NAR-Q) displays sequence
homology to membrane-bound histidine-protein-kinases (sensors) and NAR-L

17
to other known DNA-binding proteins (receivers; ref. 81, 164, 220). The
NAR-Q protein was proposed by Egan and Stewart (60) as a second protein
kinase which could also activate NAR-L via phosphorylation at aspartate
residue 59; however, this role has not been confirmed. Mutations in the
narX gene confer variable phenotypes. In-frame deletions are
phenotypically indistinguishable from the wild type (59), while
narX::Jn5 and point mutants relieve (to varying levels) nitrate
inhibition at the frd operon (60, 118). Kalman and Gunsalus (118) have
sequenced three such point mutants which no longer require nitrate for
repression and have localized these single-amino acid changes to an 11-
residue domain. Molybdate is required for frd operon repression in one
*
out of the three narX strains tested, suggesting that NAR-X may have an
additional role in molybdenum sensing. The narL gene function is less
ambiguous, since all mutants studied to date render frd operon
expression nitrate insensitive (104, 117).
When cells are grown in buffered, rich medium with glucose, pfl
operon expression is repressed approximately 2.5-fold in the presence of
nitrate. The repression is directly meditated by NAR-L and not relieved
by the addition of formate (190). The physiological significance of
this partial NAR-L repression is unclear since formate is the substrate
for nitrate respiration. The NAR-L protein may serve as a modulator of
formate levels at the site of pfl transcription to prevent formic acid
accumulation during nitrate respiration. Transcription of the hyc (hyd-
17) and fdhF genes are repressed in the presence of nitrate (171);

18
therefore, FHL activity is also reduced. However, this control does not
appear to be mediated by the NAR-X, NAR-L cascade (218). High
concentrations of formate relieve both nitrate and fumarate repression
of the FHL structural genes (171). Interestingly, mutations in nor/.,
narK (hypothetical nitrate transport gene) and narGHJI partially
relieved nitrate inhibition at the level of hyc and fdhF operon
expression when tested in the absence of formate (218). Likewise,
strain WL24, deficient in the FDH-N selenopeptide encoded by the fdhGHI
operon, is derepressed for the synthesis of FDH-H when grown
anaerobically in the presence of nitrate (192). These results suggest
that the nitrate effect at the fdhF and hyc level may be a consequence
of formate (an obligate inducer) being channeled to nitrate respiration.
Formate
Irrespective of whether high redox potential electron acceptors
(dioxygen or nitrate) are present in the environment, glucose is
actively transported into the cytoplasm by the phosphotransferase system
(PTS) and catabolized to pyruvate (for review, see 75). Therefore, the
pivotal metabolic step signalling anaerobiosis could potentially be
pyruvate degradation by PFL to formate which occurs in the absence of
dioxygen (42). Pyruvate dehydrogenase (PDH) and pyruvate formatelyase
(PFL) are tightly controlled by dioxygen both transcriptionally and
allosterically (123, 124, 205). Anaerobiosis represses and inhibits PDH
while inducing PFL synthesis and catalytic activity (83, 170). Pyruvate
formatelyase constitutes up to 3% of the cytoplasmic protein in

19
anaerobically growing cells (122). Lactate dehydrogenase (LDH),
responsible for the oxidation of pyruvate to lactic acid, is presumed to
be only fully active at high concentrations of pyruvate (17).
The pathway of formate oxidation is determined by the presence or
absence of nitrate. Anaerobically, in the absence of nitrate, both
formate and low pH enhance FHL activity (17, 46, 78). Additionally,
formate and dihydrogen are known inducers of hydrogenase synthesis (128,
135, 171). Formate is considered an obligate inducer for expression of
the fdhF and hyc {hyd-17) operons. The DNA sequence of both operons has
been determined (27, 145, 243), and the structural genes appear to have
comparable regulatory patterns in various media and genetic backgrounds
tested, as measured by the amount of p-galactosidase activity produced
by the appropriate lac fusion mutants (25, 171). DNA sequence analysis
suggests a common upstream regulatory sequence (URS) positioned between
bases 101/142 {fdhF) and 53/79 {hyc) relative to the transcription
start site. The URS exhibits several characteristics comparable to
eukaryotic enhancer elements (121). The fdhF URS consists of two tandem
conserved hexanucleotide sequences (GTCACG; ref. 22). Deletion analysis
of the fdhF upstream region show that the URS is a cis-acting DNA
element essential for formate induction and dioxygen/nitrate repression
(22). Construction of chimeric promoter regions, exchanging the
Klebsiella pneumoniae nif upstream activating sequence (UAS for nitrogen
fixation genes) with the fdhF URS and vice versa, established that the
cis-acting element mediated complete regulatory control of the fdhF gene

20
and not the spacer region (23). The hyc operon, on the other hand, is
divergently transcribed with the hyp operon. The 210 bp intragenic
region contains 3 central hexanucleotide repeats (GTCGAC) which are
comparable to the fdhF URS (145).
Further analysis of both promoters revealed sequence homology to
the "-24 and -12" promoter consensus (TGGCAC-N5-TTGC) which is
recognized by the a54 subunit of RNA polymerase (24, 227). This subunit
is encoded by the constitutive rpoN (ntrA or glnF) gene and is required
for expression of a number of operons whose physiological roles are
apparently diverse although many are involved in nitrogen metabolism
(for review, see 131). The rpoN gene product was originally identified
as a trans-acting factor required for glutamine synthetase transcription
in enteric bacteria and is now known to encode a unique sigma factor
(69, 132), distinct both structurally and functionally from the major
o70 subunit of RNA polymerase. Based on DNA sequence analysis, no
significant amino acid homology between the o54 subunit and the major
family of sigma subunits was observed (for review, see 89). However, a
helix-turn-helix motif can be predicted in the C-terminal end of both
sigma factors (152). Mutagenesis coupled with DNA footprinting has
shown that the second helix is probably involved in promoter recognition
(48, 70, 204). All o54-dependent promoters, which have been analyzed,
are transcriptionally controlled by activators (homologous to the
receiver proteins of the two-component systems) which commonly bind to
enhancer-like upstream elements. The NTR-C and NIF-A proteins,

21
transcriptional activators which have been most thoroughly investigated,
are essential for catalysis of isomerization from closed to open
promoter complexes. This would suggest that the fdhF and hyc operons
require a common trans-acting, DNA-binding protein for transcription.
Sankar et al. (186) reported the presence of a putative regulatory
element encoded by the fhlA gene which was required for transcription at
both promoters. This was subsequently verified by Schlensog et al.
(196). The fhlA mutant isolated was phenotypically deficient in Fhl
activity (FDH-H and HYD-3) which would be expected for a positive
regulator of the FHL modulon. Transduction and plasmid complementation
analysis revealed that fhlA was linked to the hydEFB gene cluster (hyp
operon) and adjacent to the hydB gene. "Maxicell" experiments
established the apparent molecular weight of FHL-A protein as 78 kDa and
suggested transcription occurred both aerobically and anaerobically
(186). This present study includes DNA sequence analysis of the fhlA
gene and predicted amino acid sequence of FHL-A, now known to be
homologous to DNA-binding proteins found in two-component regulatory
systems. By studying regulation of the fhlB operon, it was shown that
both formate and FHL-A were necessary for its transcription. Hypo
thetically, formate could activate the FHL-A protein or an unidentified
protein which positively interacts with FHL-A during transcription.
Molybdenum
Five redox enzymes of E. coli are known to contain molybdenum (Mo)
as MPT: FDH-H, FDH-N, nitrate reductase, TMAO/DMSO reductase, and

22
biotin-sulfoxide reductase (55, 92, 111, 140, 141, 202, 219; for
review, see 217). Molybdopterin is a complex composed of a nonprotein
organic moiety (6-alkyl-pterin) with a Mo atom (111, 113). Initially,
Pateman et al. (169) linked anaerobic chlorate-resistance to nitrate
respiration in Aspergillus nidulans by demonstrating that the nitrate-
respiration defective mutant is unable to reduce chlorate to toxic
chlorite. The chlorate-resistance (chi) genes exhibited pleiotropic
phenotypes and were later identified as essential for molybdenum
transport, regulation and formation of functional molybdopterin
containing enzymes (for review, see 94). Complementation analysis and
deduced sequence homology to other periplasmic binding and transport
systems estimated at least 11 chi genes mapping at 5 loci which are
necessary for MPT biosynthesis in E. coli (110, 178).
The chlA operon, consisting of three complementation groups (18
min), and the chlE operon, composed of two (18 min), are presumed to be
necessary for synthesis of the organic portion of the MPT (11, 113, 178,
219). Mutations in either one of these operons give rise to defects in
pterin biosynthesis (114). Baker and Boxer (14) constructed
merodiploids containing chlA+ / (chlA'- lacZ*) to study the
transcription of the chlA locus. Various chi mutations (chlA, chlB,
chlD, chlG and chlE) were introduced into the merodiploid strains.
These results suggested that the chlA operon is anaerobically inducible
and repressed in the presence of MPT. These investigators concluded

23
that chlA repression is probably mediated by a complex of MPT and a MPT-
binding protein which has been reported previously (6, 189).
The constitutively synthesized chlB gene (86 min) product has been
purified (Mr = 35 kDa) and is denoted as "association factor-FA" which
is essential for synthesis of functional MPT (143, 180). A mutation in
this gene inhibits insertion of the MPT into the apoprotein; therefore,
association factor-FA may have a direct role in mediating the terminal
step of apoprotein maturation (71, 146). Johnson et al. (112) recently
developed a more sensitive fluorescence technique to analyze cell
extracts for MPT and MGD derivatives. By comparing chlB mutant extracts
to wild type, it was concluded the chlB gene was required for the
formation of MGD, a step occurring late in the MPT maturation process.
The chlC (27 min) and chlF (32 min) operons code for nitrate reductase
and FDH-N respectively. Since neither are necessary for MPT
biosynthesis, they have been renamed narGHJI (chlC) and fdnGHI (chlF)
(11, 19, 219). Mutations in both the chlG (0 min) and chlD operons (17
min) are suppressed by Mo supplementation of the growth medium (11, 207,
219). Phenotypically, the chlG mutants lack nitrate reductase activity,
even though detectable levels of the protein were produced in the
original isolates, and retain wild type levels of Fhl activity (219).
It is currently postulated that the CHL-G proteinfunctions to
incorporate Mo into molybdoenzymes and Mo-binding proteins (94).
Mutations in the chlD operon can be phenotypically distinguished
from the chlG gene. Dihydrogen production is lacking in chlD mutants

24
due to a deficiency in both FDH-H and HYD-3 activities (33, 73, 76,
196). The partial sequence of the chlD operon discloses significant
homology to binding-protein-dependent transport systems (110).
Generally, this type of transport is a unidirectional process. It is
now known that this process requires ATP for energy instead of acetyl-
phosphate (20, 95, 97, 100), and the substrate does not undergo
modification during transport (4). In the systems which have been
analyzed, the number and location of proteins involved in the transport
process are conserved (for review, see 5, 52). These proteins include:
(i) a high-affinity, substrate-binding protein located in the
periplasmic space which is released upon cold osmotic shock (i.e. HISJ,
MAL-E), (ii) two integral membrane proteins present in lower
concentration than the binding protein (i.e. HIS-Q, HIS-M, MAL-F, MAL-
G), and (iii) another membrane-associated protein, presumed to be
peripheral, which contains a nucleotide-binding domain with a conserved
amino acid sequence (i.e. HIS-P, MAL-K). The central domain of the
complete CHL-D sequence revealed extensive homology to peripheral
transport proteins (110). The partial CHL-J sequence suggested that
this protein is an integral membrane component of Mo transport (110).
Both the chlD gene and the molR gene, recently described by Lee et
al. (136) are essential for fdhF and hyc (ant, hyd-17) operon expression
as measured by 0-galactosidase activity (134, 196). This implies that
transcription of these FHL operons either directly or indirectly
requires Mo or a Mo-derivative. Interestingly, both molR and chlD genes

25
are located in the same operon as determined by transduction and plasmid
complementation analysis (Shanmugam, unpublished results; this study).
However, MOL-R is expressed constitutively (134) while CHL-D synthesis
is Mo repressible (155) thus suggesting at least two promoters for this
operon.
Nickel
E. coli synthesizes three distinct nickel (Ni) containing
hydrogenase isoenzymes (12, 193). The HYD-1 protein and an
enzymatically active fragment of HYD-2 have been purified to homogeneity
(1, 13, 66, 168, 194). Biochemical characterization of these proteins
revealed that HYD-1 and HYD-2 contain nonheme iron, inorganic sulfur and
nickel. The HYD-3 protein, responsible for dihydrogen evolution, is
electrophoretically labile and has remained elusive to purification
(193). DNA sequence analysis of the large subunit of HYD-3 (hyc 0RF5),
however, displays significant homology to the "nickel-amino acid"
consensus sequence of other NiFe-hydrogenases (27, 195, 214). The large
subunits of all sequenced NiFe-hydrogenases contain the consensus R-X-C-
G-X-C-Xg-H in the amino-terminus and D-P-C-X2-C-X2-H at the carboxy-
terminus (177). Apparently the conserved sites at both termini are
somehow involved in liganding nickel at the active site (61, 87).
Several genes required for total hydrogenase activity (thus Fhl
activity) have been characterized and are presumed to be involved in
nickel transport, nickel processing, or hydrogenase regulation. These
include hydA (59 min), hydB or hypE (59 min), hydC (77.6 min), hydD

26
(77.6 min), hydE or hypB (59 min), and hydF or hypD (59 min; ref. 35,
119, 128, 135, 185, 187, 188, 231, 238). Although the hyp operon has
been sequenced, the DNA did not display any detectable homology to the
DNA available in Genbank data base and thus possible function could not
be deduced (144). Mutations in both the hydC and hydE genes are
suppressed by high concentrations of nickel in the growth medium (231,
238). This would suggest that both are essential for transport of the
divalent metal ion. Transcription of the FNR-controlled hydC is fully
repressed by approximately 0.2 mM NiC12 which is correlated to the in
dependent restoration of hydrogenase activity (239). Hypothetically, a
nickel-protein complex could function as a transcriptional repressor at
the hydC gene operator. The hydE or hypB gene, now known to be a
component of the hyp operon, does not appear to be nickel repressive at
either of its two promoters (FNR or o54-dependent), as measured by RNA
transcript levels and pgal actos i dase activity of hyp gene fusions (144,
this study). It is now believed that this gene product is required for
activation or processing of nickel. The other hyd genes may also
process the nickel for insertion into hydrogenase or regulate the
expression of all three isoenzymes. Sankar et al. (187, 188), using
maxicells, determined that both hydB (hypE) and hydF (hypD) expression
was rpoN dependent, anaerobically inducible, and nitrate repressible.
Fumarate and TMAO supplementation had no significant effect on
transcription of either gene. This is in correlation with hyp-lac gene

27
fusion expression and transcript levels under the same conditions (144;
this study).
Recent studies have established that nickel is not essential for
transcription of the electrophoretically stable HYD-1 and HYD-2
isoenzymes, but necessary for hydrogenase activity. Lutz et al. (144)
reported that mutations in hypBCDE did not alter hydrogenase
transcription as monitored by immunoblotting analysis with anti-HYD-1
and HYD-2 antibodies; however, the electrophoretic mobility was altered
in a 10% SDS-polyacrylamide gel. Potentially the lack of nickel
processing and insertion could lead to a modified hydrogenase
conformation and proteolytic susceptibility. Menon et al. (151)
obtained similar results for HYD-1 in a hydE (hypB) mutant. Although
HYD-1 transcription was comparable to wild type in these mutants in the
absence of Ni, activity was absent. Additionally, the hydE gene product
appeared essential for membrane localization of HYD-1, and this
requirement was nickel repressible. Labile HYD-3 is probably regulated
by a similar post-translational mechanism requiring nickel incorporation
for Fhl activity.
Selenium
A number of enzymes from both prokaryotic and eukaryotic
organisms, including FDH-H, require selenium in the form of
selenocysteine for activity (172; for review, see 26). DNA sequence
analysis of the fdhF gene [coding for the 701 amino acid FDH-H subunit
(Mf=79 kDa)] established that a UGA "nonsense" codon at amino acid

28
position 140 with an associated unique stem loop structure is essential
for selenocysteinyl-tRNA incorporation during translation (15, 64, 65,
243, 244). By constructing a plasmid in which the first 39 amino acids
of FDH-H were fused to p-galactosidase, it was determined that selenium
was not required for anaerobic transcription of the fdhF gene (243).
Selenium regulation of Fhl activity is evidently at the translational
level of the fdhF gene.
In summary, the FHL system is presumed to be regulated by a number
of elements including anaerobiosis, nitrate, formate, low pH, molybdate,
nickel, and selenium. The absence of dioxygen induces the transcription
of fdhF, hyc, and other operons whose products are required for FHL
activity. Additionally, enzymes necessary for dihydrogen production are
often irreversibly inactivated by the presence of dioxygen thus enabling
tighter metabolic control. Nitrate repression of FHL appears to be
mediated by the NAR-X and NAR-L, two-component regulatory system at the
level of pfl gene transcription. Therefore, it is possible that the
nitrate repressive effect on the transcription of FHL structural
components is indirectly a result of the absence of the obligate
inducer, formate as well as low pH. Currently it is unknown whether
molybdenum is directly or indirectly required for Fhl transcription.
Mutations in both molR and chlD genes abolish expression of fdhF and hyc
operons and are reversed by high molybdate. Nickel is essential for
HYD-3 activity at the post-translational level; whereas, selenium is
required for translation of the selenopolypeptide, FDH-H.

29
In this study, the mechanism by which formate and molybdenum
regulate the FHL pathway was investigated. This includes isolation of a
specific mutant, strain SE-2011 lacZ*)], physiological
characterization of this fhlB mutant, and trans-acting factors
regulating the fhlB gene. These trans-acting factors were found to be
the FHL-A protein of the formate-dependent pathway and an
uncharacterized component of the molybdate-dependent pathway.

MATERIALS AND METHODS
Materials
Biochemicals were purchased from Sigma Chemical Co. Analytical-
grade inorganic and organic chemicals were from Fisher Scientific.
Bacterial Strains and Media
The bacterial strains are derivatives of E. coli K12 and are
listed in Table 1. Basal minimal, dihydrogen/fumarate and
glycerol/fumarate media and LB were prepared as described previously
(135). Cultures were grown at 37C in LB which was supplemented with
glucose (0.3%) or sodium formate (0.1% to 0.5%), as needed. Ampicillin
(100 pg/ml), kanamycin (50 pg/ml), streptomycin (100 pg/ml),
tetracycline (15 pg/ml), chloramphenicol (5 pg/ml) or X-gal (20-40
pg/ml) were added as needed.
Isolation of Mutants
Strain MC4100, grown in LB+maltose (0.3%; LBM) medium was
mutagenized with XpZocMu53 and ApMu507, as described by Bremer et al.
(29). Kanamycin resistant mutants were transferred by replica plating
techniques to LB + X-Gal medium (156) and incubated under aerobic or
anaerobic conditions. Mutants which are Lac+ only under anaerobic
growth conditions were identified and inoculated into 1 ml of LBG medium
in 12 x 75 mm tubes. These tubes were sealed with serum stoppers and
30

31
Table 1. Bacterial strains used in this study
Strain
Relevant Genotype or Phenotype
Source or Reference
E. coli
BW545
h[lacU)169, rpsL
G. Walker (242)
CSH26
ara, A{lac-pro), thi
Laboratory
collection
JRG780
trpA9761, frdAll, trpR72, gal-25,
rpsL195
CGSC #5916
JRG861a
gal, trpA9761, iclR, trpR, rpsL, fnr
J. Guest
LCB898
thr-1, leuB6, pfl-1, thi-1, lacYl,
rpsL175, tonA21
CGSC #6161
LS853
trpA9605, his-85, cya-2, trpR55
CGSC #5381
M2508
Hfr, relAl, spoTl, metBl, melA7
CGSC #4926
M9s
MC4100, 4>{fdhF'-'lacZ+)
A. Bock (171)
MC4100
araD139, 5(argF-lacll)205, ptsF25,
relAl, rpsL150, deoCl, flb5301
CGSC #6152
MJ-2
[fhlB'-lacZ+), hydF102, cysalnlO
PI transduction
(SE-2011 x SE-67-1)
MJ-3
MC4100, (fhlB'-' lacZ+)
PI transduction
(MC4100 x SE-2011)
MJ-4
BW545, *{fhlB'-'lacZ+)
PI transduction
(BW545 x SE-2011)
MJ-5
Q[fhlB'-'lacZ+), rpoN::Tni0
PI transduction
(SE-2011 x YMC18)
MJ-6
[fhlB'-' lacZ+), fnr, zcj-5::JnlO
PI transduction
(SE-2011 x SE1188)
MJ-7
HfhlB'-'lacZ+), narL215::Tr)10
PI transduction
(MJ-4 x RK5278)

32
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
MJ-8
lacZ+), cya-2, zif-4::JnlO
PI transduction
(SE2011 x SE1162)
MJ-9
*{fhlB'-'lacZ+), pfl-1, zba-6: :TnJ0
PI transduction
(SE2011 x SE1265)
MJ-18
MJ-19, Hfr PO(fhlB)
Conjugation
(MJ-19 x TT627)
MJ-19
CSH26, *{fhlB'-'lacZ+)
PI transduction
(CSH26 x SE2011)
MJ-20
Q(fhlB'-'lacZ*), fhlA::Tni0
PI transduction
(SE2011 x SE1174)
MJ-21
SE1000, metBl, melA7,
thr+, arg+, leu+, F~
Conjugation
(SE-1000 x M2508)
MJ-40
^(fhlB'-'lacZ+)t molR
PI transduction
(SE2011 x SE1704)
MJ-50
SE-1000, PI transduction
(SE-1000 x SE2011)
MJ101
(fhlA'-' lacZ+), rpoN::lr)10
PI transduction
(SE-2007 x YMC18)
MJ-102
4>(fhlA,-'lacZ+), nor/,215: :TnJ0
PI transduction
(SE-2007 x RK5278)
MJ-103
Q>(fhlA'-' lacZ+), /nr, zcj-5::TnI0
PI transduction
(SE-2007 x SE-1188)
MJ107
(fhlA'-'lacZ+), cya-2, zi/-4::TnJ0
PI transduction
(SE-2007 x SE1162)
MJ-108
Q(fhlA'-' lacZ+), moZ/?
PI transduction
(SE-2007 x SE1704)
MJ-109
(fhlA'-' lacZ+), pfl-1, zba-6: :TnJZ?
PI transduction
(SE-2007 x SE1265)

33
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
PC0287
thr-20, leu-32, proA35, argF58,
argI60, lacYl, gal-6, rpsL125,
tonA48, tsx-70, supE44
CGSC #5404
RK5278
narL215::lnlO
V. Stewart
SE-1000
cysC-43, srl-3000::lnl0, thr-1,
leu-6, thi-1, lacY-1, galK2, ara-14,
xyl-5, mtl-1, proA2, his-4, argE3,
rpsL31, tsx-33, supE44
Laborotory
collection
SE-67-1
hydFI02, cys::Tnl0
PI transduction
(SE67 x SE1300)
SE-1100
BW545, 4>{tnolR'-' lacZ*)
Laboratory
collection (136)
SE-1162
LS853, zif-4::JnlO
Laboratory
collection
SE-1174
fhlA::JnlO
Laboratory
collection (186)
SE-1188
JRG861a, zcj-5::lnlO
Laboratory
collection
SE1265
LCB898, zba-6::Tni0
Laboratory
collection
SE1300
BW545, cys::TnT0
Laboratory
collection
SE1651
4>{hyp'-' lacZ+) 1, fnr, zcj-5::JnlO
PI transduction
(SE2001 x SE-1188)
SE1652
4>(hyp'-' lacZ+)2, fnr, zcj-5: :Tni0
PI transduction
(SE-2002 x SE-1188)
SE1654
4>{hyp'-' lacZ*) 1, narL2l5::lr\10
PI transduction
(SE2001 x RK5278)

34
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
SE1655
(hyp'-' lacZ+)2, nor/,215: :Tni0
PI transduction
(SE-2002 x RK5278)
SE1657
(/?yp'-'lacZ+)\i rpoNulnlO
PI transduction
(SE-2001 x YMC18)
SE1658
Q(hyp'- lacZ*) 1, rpoN::lr\10
PI transduction
(SE-2001 x YMC18)
SE1659
[hyp'-' lacZ+)2i rpoN: :lnlO
PI transduction
(SE-2002 x YMC18)
SE1660
4>(/jyp'-'lacZ+)2, rpoN::JnlO
PI transduction
(SE-2002 x YMC18)
SE-1704
moZ/?::Tn5, zgg::lr\10
Laboratory
collection
SE1714
QtfhlB'-'lacZ*), chlD::lnlO
PI transduction
(SE-2011 x VJS720)
SE1760
${hyp'-'lacZ*) 1, chlD::lr\10
PI transduction
(SE-2001 x VJS720)
SE1761
(hyp'-'lacZ+)2, chlD::lnlO
PI transduction
(SE-2002 x VJS720)
SE1762
PI transduction
(SE-2007 x VJS720)
SE-2001
MC4100, (hyp'-'lacZ+)l
This study
SE-2002
MC4100, This study
SE-2007
MC4100, QifhlA'-'lacZ*)
This study
SE-2009
MC4100, (hydC'-'lacZ+)
This study
SE2011
MC4100, Q{fhlB'lacZ*)
This study
VJS720
chlDalnlO
V. Stewart

35
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
YMC18
endA, thi, hsdR, h(lacU)169,
rpoN:JnlO
B. Magasanik
Salmonella typhimurium
TT627 strAl, pyrC7/F'tsll4 zz/::TniO
J. Roth (40)

36
the gas phase was replaced with dinitrogen. After 16 hr of incubation
at 37C, dihydrogen in the gas phase of the culture tubes was determined
using a gas chromatograph (Varian; Model 920) fitted with a 50 nm
molecular sieve column. From a total of 68 mutants, 13 were found to be
defective in dihydrogen production (Fhl_) and were analyzed further.
Enzyme Activities and their Respective Culture Conditions
P-qalactosidase activities and culture conditions. For anaerobic
induction of p-galactosidase activity in mutant strains SE-2001, SE-2002
and SE-2011, 120 ml of medium in a 160 ml "Wheaton" bottle was
inoculated (1% V/V) with a 1.5 hr old aerobic culture, grown at 37C, in
a shaker, at 250 rpm. However, with strain SE-2007 or derivatives
thereof, alternate procedures were used to maximally aerate the culture
at low cell density before starting the experiment. A 2 hr old aerobic
culture (37C; 250 rpm) was transferred to fresh LB medium (1% V/V) and
grown again in the shaker for 1 hr. This culture was used to inoculate
the experimental medium at 10% (V/V) and then grown under anaerobic
conditions in a "Wheaton" bottle. The bottles were closed with rubber
stoppers and secured with aluminum seals. The gas phase was replaced
with argon. Samples were removed at different time periods with a
syringe and needle and growth of the culture and p-galactosidase
activity of the cells were determined. In another set of experiments,
the aerobic cultures were used to inoculate (1% V/V) the appropriate
medium in 13 x 100 mm screw cap tubes filled to the top. Cells from

37
these cultures were harvested after 4 hr of incubation at 37C
(standing) and used for enzyme assays.
The amount of p-galactosidase present in cells was determined as
described by Miller (156), after permeabilization with SDS and
chloroform. The specific activity of the enzyme is expressed as
nanomoles ortho-nitrophenol produced per min per mg cell protein. The
differential rate of synthesis of p-galactosidase activity was
calculated as units of activity per pg cell protein produced by the
culture and represents the increase in the amount of p-galactosidase
activity produced by the culture in relation to the increase in total
cell protein.
Dihydrogen related metabolic activities and culture conditions.
Whole cells were utilized for all biochemical determinations (135).
Cells were inoculated (5% V/V) from overnight LB-grown cultures into
fresh LB6 medium in 250 ml screw cap bottles or 20 ml screw cap tubes
(16 x 150 mm) filled to the top. Cultures were grown anaerobically for
4 hr at 37C. Cells were collected by centrifugation (3,000 x g for 10
min) at 4C, washed with half volume of phosphate buffer (0.1 M sodium
phosphate, pH 7.0; 1 mM glutathione; 0.1 mg/ml chloramphenicol). The
cell pellet was resuspended in 1.0 ml buffer and diluted to 1.75 mg cell
protein per ml, as determined in a Spectronic 710 spectrophotometer
(Baush and Lomb) in which 1 A420nm unit equalled 350 pg cell protein
per ml. Samples were maintained under N2 at 4C and assayed
immediately.

38
Total hydrogenase activity, measured as tritium exchange (7, 138),
was determined using 50 pg cell protein adjusted to 0.2 ml volume in
sodium phosphate buffer (0.1M; pH 7.0). Assays were carried out in 12 x
75 mm thick walled test tubes, sealed with 11 x 17 mm serum stoppers.
The gas phase was replaced with helium. The reactions were initiated
with 0.7 ml of dihydrogen and 0.55 pCi tritium gas (11.2 mCi/mmol; New
England Nuclear Corp.) and terminated after 1 hr incubation at room
temperature. After removing the stopper, the reaction mixture was
agitated with a vortex mixer and allowed to stand for 10 min. Tritium
in a 50 pi sample in 2.5 ml Scintiverse-E scintillation fluid was
determined, and total hydrogenase activity was calculated as nanomoles
of tritiated water produced per min per mg cell protein.
Hydrogen uptake (HUP) was measured both as the ability to reduce
benzyl viologen (BV) (96, 167) and fumarate with dihydrogen as electron
donor (148). The reduction of BV was monitored in 13 x 100 mm test
tubes with "subaseal" stoppers. The assay mixture (0.1 M sodium
phosphate buffer, pH 7.0; 4 mM BV) was degassed, the gas phase replaced
with dihydrogen and reduced with sodium diothionite until a faint purple
color appeared. The reaction was initiated by the addition of whole
cells to a final volume of 2.5 ml. The rate of BV reduction was
correlated to AA550nm using a DW2C spectrophotometer (SLM Instruments;
Urbana, Illinois). The HUPBV activity was expressed as nanomoles BV
reduced per min per mg cell protein. Fumarate reduction was measured as
the rate of dihydrogen consumption from the gas phase using a gas

39
chromatograph as described above. Assay mixture consisted of 0.1 M
phosphate buffer (pH 7.0) and 50 mM fumarate brought to a final volume
of 1.0 ml with cells. "Wheaton" vials (10 ml) with serum stoppers and
aluminum seals were used to establish anaerobic conditions (10% H2/90%
N2 gas phase). The HUPfumarate activity was calculated as nanomoles of
H2 consumed per min per mg cell protein.
Formate-dependent reduction of BV (FDH-H, FDH activity associated
with FHL), was assayed in an assay mixture containing 0.33 M sodium
phosphate buffer (pH 7.0), 6.5 mM BV, 40 mM formate and cells in a final
volume of 4.0 ml (135). Test tubes (13 x 100 mm) with "subaseal"
stoppers were used to monitor the reaction in a N2 atmosphere. Assay
mixture was reduced with sodium dithionite until a faint purple color
developed. Reaction was initiated by adding whole cells and the
AAgsonm was correlated to BV reduction. The FDH-H activity was
calculated as nanomoles BV reduced per min per mg of cell protein.
Formate hydrogenlyase activity was measured in 70 mM sodium
phosphate buffer (pH 6.5) containing 0.1 M formate (final volume of 1.0
ml with cells; 96). Wheaton" vials (10 ml) with serum stoppers and
aluminum seals were used to monitor the production of dihydrogen from
formate in a dinitrogen atmosphere. Dihydrogen evolution was quantified
using a gas chromatograph. The FHL activity was calculated as nanomoles
of H2 produced per min per mg of cell protein.
Fumarate reductase activity was assayed in 92.5 mM sodium
phosphate buffer (pH 7.0), 30 mM fumarate, and 0.35 mM BV under a N2

40
atmosphere. Activity was measured as fumarate-dependent oxidation of
BVred. Assay mixture was reduced with sodium dithionite to 2.0
absorbance units at 550 nm, whole cells were added to a final volume of
5.0 ml, and oxidation of reduced BV was monitored as AA550nm. Activity
was expressed as nanomoles BVred oxidized per min per mg cell protein
(208).
Chloramphenicol acetyltransferase (CAT) assay and culture
conditions. Plasmid pSV208 (fdhF'cat) was generously provided by Dr.
A. Bock (196). Cell extract was prepared according to Brosius and
Lupski (30) with modifications. Transformants were inoculated into 1.0
ml LB + ampicillin (50 pg/ml) medium and grown to stationary phase.
This was used as an inoculum for 20 ml (5% V/V) of the same medium
supplemented with glucose (0.3%), formate (0.2%), and trace metals
(FeS04.7H20, 0.01 mg/ml; NaMo04.2H20, 0.01 mg/ml; NaSe03.5H20, 0.263
ng/ml). The cultures were grown anaerobically (16 x 150 mm screw cap
tubes filled to top) to approximately 5 x 108 CFU/ml (4 hr; 37C).
Cells were harvested by centrifugation at 5,000 rpm for 10 min at 4C,
washed with 20 ml of assay buffer (50 mM Tris-HCl, pH 7.8; 30 pM
dithiothreitol), and resuspended in 1.0 ml of same buffer. This was
transferred to 1.5 ml plastic centrifuge tubes and placed at -70C for 1
hr. Cells were thawed at 37C and then disrupted by sonication (one 20
seconds pulse at full power; Heat Systems sonifier with microprobe)
after dilution to 2.0 ml volume in conical-bottomed glass test tubes in
an ice-water bath. Cellular debris were removed by centrifuging in

41
"Eppendorf" micro-centrifuge for 5 min at 4C. Supernatant was
collected and kept on ice for immediate assay using a modified procedure
described by Shaw (200). The reaction mixture was freshly prepared by
dissolving 4 mg of 5,5' dithiobis-2-nitrobenzoic acid (DTNB) in 1.0 ml
Tris-HCl (pH 7.8), adding 0.2 ml of 5 mM acetyl-CoA, and then making the
total volume up to 10 ml. After measuring the rate of change of
absorbance at 412 nm with 900 ul of reaction mixture and 80 pi of
extract, the reaction was started with 20 pi of chloramphenicol (Cm; 5
mM in 70% ethanol) added to a final concentration of 0.1 mM. The
difference in the rate of change at A412nm with and without Cm was
calculated. Protein concentration of the extract was determined using
Coomassie brilliant blue (28). The CAT activity was expressed as
nanomoles of free 5-thio-2-nitrobenzoate produced per min per mg cell
protein.
Genetic and Molecular Biological Experiments
Bacteriophage (PI and lambda) preparation by plate lysis. Host
strain was grown to stationary phase in LB medium for PI infection.
Cells were sedimented by centrifugation (3,500 x g) at 25C and
resuspended in an equal volume of PI adsorption medium (5 mM CaCl2.2H20;
10 mM MgCl2.6H20). Bacteriophage PI (105 to 106 PFU) was added to 0.2
ml of host cells and incubated for 5 min at 25C. Then 3 ml of LCTG
soft agar (5g/L yeast extract; 10 g/L each of NaCl and trypticase
peptone; 2.5 mM CaCl2 2H20; 25 mg/ml thymine; 60 mM glucose; 0.6%

42
agar) at 50C was added to the mixture and after mixing was overlayed on
LB+thymine (25 pg/ml) plates. After 9 hr of incubation at 42C, the
overlay from the plates with confluent lysis was harvested after
addition of 2.5 ml Pl-diluent (10 g/L trypticase peptone; 10 mM
MgCl2.6H20). Chloroform (0.5 ml) was added to the lysate and mixed with
a glass pipet. This was centrifuged (12,100 x g) for 15 min at 4C.
The supernatant was again extracted with choloroform, and the final
supernatant was stored at 4C with 0.1 ml chloroform. Two consecutive
series of PI infection were done to ensure enrichment of host strain
mutation.
Procedures implemented for replication of bacteriophage A were
comparable to PI with some critical exceptions. Strain LE392 (supF,
supE) was used as the host strain and was grown in LBM medium. Cells
were pelleted and resuspended in an equal volume of 10 mM MgS04.7H20.
Water-Thy agar (0.6% agar; 0.1 mg/ml thymine) was used as the soft agar
overlay, and plates were incubated at 37C. Lambda diluent (10 mM Tris-
HC1, pH 7.5; 10 mM MgS04.7H20; 50 mM NaCl; 0.1% gelatin) was used to
titer and harvest phage.
Transduction using bacteriophage PI Cm cZrlOO. Transduction
experiments were carried out according to Miller (156) with
modifications. Two milliliters of a "mid-log phase" culture of
recipient cells were centrifuged (3,500 x g) for 5 min at 25C and
resuspended in 1.0 ml of PI adsorption medium. Then 0.2 ml of the
resuspension was infected at a M.0.1. of 1 to 10 with the appropriate

43
phage preparation. After 30 min at 25C, 1 ml of PI diluent was added
and the bacteria-phage mixture was vortexed. This was then centrifuged
(3,500 x g) for 5 min at 25C, resuspended in 0.1 ml of LB and incubated
for 1 hr at 25C before plating on selective medium for transductants
(25C). Tranduction of donor mutation was confirmed prior to and after
curing recipients off bacteriophage at 42C.
Conjugation. The F' complementation analysis was performed
according to Miller (156) using F'1431 (57 to 60 min), F126 (17 to 30
min), F' 116 (60 to 65 min), F' 128 (6 to 8 min; lacZ::lr\10), F 112 (90 to
98 min) and F1104 (0 to 7 min) provided by Dr. B. Bachmann. The Hfr-
mediated transfer of chromosomal DNA was accomplished by aerobically
(250 rpm) growing the donor and recipient strains to approximately 5 x
o
10 CFU/ml at 37C. Mating was initiated by mixing 1.0 ml of donor with
1.0 ml of recipient in 3.0 ml of fresh LBG (125 ml flask; 50 rpm).
Immediately upon removal, samples were vortexed, diluted in minimal
medium and centrifuged (3,500 x g) for 5 min at 25C. Exconjugants were
then selected on appropriate media.
Transformation by CaClg-heat shock method. Routine transformation
was performed by two methods, depending on the amount of competent cells
required. Both were a modification of the calcium chloride-heat shock
method described by Mandel and Higa (149) which was later established
for transforming episomal elements (43). The first method was basic and
expedient. An overnight LB culture of the desired host strain was
inoculated into fresh medium in 13 x 100 mm tubes (0.02 ml into 2.0 ml)

44
and incubated for 2 hr standing at 37C. Cells were sedimented (3,500 x
g; 5 min; 25C) and resuspended in 0.4 ml of 0.1 M CaCl2.2H20. DNA
(about 50 ng) was added to 0.2 ml of cell suspension and incubated on
ice for 20 min. This was transferred to 42C for 2 min and then
returned to ice for 10 min. Fresh LB medium (1.0 ml) was added, and
cells were incubated at 37C for 1 to 2 hr and then plated on applicable
selection medium. If more cells were needed, the overnight culture was
inoculated into 10 ml LB medium and aerobically grown at 30C for 1.5 to
2 hr. This was transferred to 37C for 30 min and then harvested (3,500
x g; 5 min; 25C). After washing with 0.1 M NaCl, the cells were
resuspended in equal volume of 0.1 M CaCl2.2H20 and incubated for 20 min
at 25C. This was then pelleted and resuspended in CaCl2 solution at
approximately one-fifth the original volume. Similar procedures were
then followed to that described above.
For high efficiency transformation, competent cells were prepared
by a modified procedure and stored at -70C (Sankar, personal
communication). An overnight culture was used to inoculate 50 ml of
fresh LB medium. This was grown to early-log phase (2 x 108 CFU/ml)
aerobically at 37C. The cells were centrifuged (3,000 x g; 5 min;
4C), washed in 0.1 M cold MgCl2.6H20, resuspended in equal volume of
0.1 M cold CaCl2.2H20, and incubated on ice 20 min. Treated cells were
then pelleted (3,000 x g; 5 min; 4C) and resuspended in one-tenth the
starting volume with 0.1 M CaCl2 containing 15% glycerol. This was
aliquoted and stored at -70C for later use. Cells were thawed slowly

45
on ice and 0.1 ml was transferred to a tube for transformation. DNA
(approximately 50 ng) was added to the competent cells. This was
incubated on ice for 20 min, transferred to 37C for 5 min, and returned
to ice for 2 min. Transformants were preincubated in SOC medium (20 g/L
trypticase peptone; 5 g/L bacto-yeast extract; 10 mM NaCl; 2.5 mM KC1;
10 mM MgCl2.6H20; 10 mM MgS04.7H20; 20 mM glucose) with aeration at 37C
for 1 hr prior to plating on selection medium.
Transposon Tn5 mutagenesis of cloned genes in plasmid DNA. The
two transposon Tn5 derivatives of plasmid pSE-133 were constructed as
described before (188). Plasmid pSE-133 which carries the hydB+ and
fhlA+ genes was described previously (187). Strain MBM7014 (supF) was
utilized as the host strain for plasmid DNA (pSE-133) Tn5 mutagenesis.
A transformant was aerobically grown (250 rpm) in LBM medium (20 ml) to
mid-log phase (3 x 108 CFU/ml) at 37C, the culture was centrifuged
(3,500 x g; 25C), and the pellet was resuspended in 1.0 ml of 10 mM
MgS04.7H20. Cells were infected with NK421 (Tn5) at a M.0.1. of 10 for
30 min at 25C. The infected cells were then vortexed, centrifuged
(3,500 x g; 25C), washed with 5.0 ml of LB medium, and resuspended in
final volume of 10 ml. This was subcultured (2.0 ml inoculum) in 10 ml
fresh LBG medium supplemented with 10 mM sodium citrate and aerobically
grown (250 rpm) at 37C for 30 min and then shifted to 30C for 1 hr.
Kanamycin (50 pg/rol) and ampicillin (100 pg/ml) were added after the hr.
Then the culture was grown to stationary phase (18 hr), and plasmid DNA
was extracted by the alkaline lysis method described below (150).

46
Plasmids carrying the transposon Tn5 in the gene of interest were
selected as Kmr Ampr transformants and then screened for lack of
complementation in the appropriate mutant. Location of the transposon
Tn5 was determined by analyzing "restriction endonuclease" digests of
the DNA. The relevant genotype of plasmid pSE-133-1 is hydB::Jn5, fhlA+
and pSE133-2 carries the transposon in the fhlA gene (hydB+,
fhlA::Tn5).
Plasmid and chromosomal DNA preparations. Small and large scale
plasmid isolation were carried out following standard alkaline lysis
protocol with some modifications (150). The cesium chloride gradient
consisted of (final concentration) 1 g/ml cesium chloride and 0.625
mg/ml ethidium bromide. This was centrifuged at 50,000 rpm (160,000 x
g), for 18 hr, at 18C. Plasmid band was recovered with syringe fitted
with a 22G x 1" needle, and ethidium bromide was removed with water
saturated 1-butanol. Extracted plasmid solution was diluted with 2
volumes of deionized water and then ethanol precipitated at -20C for 18
hr. After centrifugation (12,100 x g; 30 min; 4C), the pellet was
washed with 70% cold ethanol and vacuum dried. This was resuspended in
a small volume of sterile H20.
Chromosomal DNA was prepared according to Ausubel et al. (9) with
modifications. A culture of the E. coli strain of interest (1 liter)
was grown to mid-log phase (3 x 108 CFU/ml). The cells were harvested
by centrifugation at 2,300 x g, for 10 min at 4C. The pellet was
resuspended in 95 ml of TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0).

47
Sodium dodecyl sulfate and proteinase K were added to a final
concentration of 0.5% and 0.1 mg/ml, respectively. This was incubated
for 1 hr at 37C. The NaCl concentration of the sample was then
adjusted to 0.7 M, mixed thoroughly and CTAB/NaCl solution (10%
hexadecyl trimethyl ammonium bromide in 0.7 M NaCl) was added to a final
concentration of 1% CTAB. This was heated to 65C for 10 min, extracted
with equal volume of chloroform: isoamyl alcohol (24:1) and extracted
with phenol/chloroform/isoamyl alcohol (25:24:1). The DNA was
precipitated with isopropanol and washed with 70% ethanol. The pellet
was vacuum-dried, resuspended in TE buffer (7.5 ml) and centrifuged in a
cesium chloride gradient (1 mg/ml cesium chloride; 0.625 mg/ml ethidium
bromide) using similar parameters to the large scale plasmid
preparation. Chromosomal DNA was removed from the gradient using a
syringe fitted with a 16G x needle and then extracted and
precipitated as with plasmid DNA. Concentration was determined by
agarose gel electrophoresis with known DNA standards and
fluorometrically with Hoechst 33258 dye (using the TK0 100-dedicated
Mini-Fluorometer; Hoeffer Scientific). Concentration as well as the
purity of the DNA was determined from the absorption spectrum between
the wavelengths 200 nm and 300 nm.
DNA sequence determination. DNA sequence was determined using the
Sanger dideoxy method with double stranded plasmid DNA (85, 184). The
plasmids used are listed in Table 2. Plasmids pSE-130, pSE-132 and pSE-
133 used in the fhlA gene sequencing experiments were described

48
Table 2. Plasmids used in this study
Plasmid
Relevant Genotype or Phenotype
Reference
pRBH
Cmr, 14 kb CZoI-flomHI fragment with
the complete hyd-17 gene cluster from
MC4100 in pACYC184
A. Bock (27)
pSE-111
Apr, 14.7 kb SauSM fragment with the
complete hyd [hyp) and partial hyc
operon in pBR322
Laboratory
collection
(185)
pSE-125
Tcr, 2.8 kb Sail fragment from pSE-22 with
partial hypC and fhlA genes and complete
hypDF [hydFB) genes in pBR322
Laboratory
collection
(187)
pSE-125-1
Tcr, 0.8 kb Sall-Clal fragment from
pSE-125 with partial hypCD [hydXF) genes
in pBR322
This study
pSE-128
Apr, 6.5 kb SoZI-Sou3Al fragment from
pSE-22 with partial hypC [hydX) gene
and complete hypDF [hydFB) and fhlA
genes in pBR322
Laboratory
collection
(185)
pSE-130
Apr, 4.7 kb Pst I fragment with partial
hypA and fhlA genes and complete hypBCDF
(hydFXFB) genes in pBR322
Laboratory
collection
(186)
pSE-132
Apr, 2.0 kb Kpnl-Sall fragment from Laboratory
pSE-125 with partial hypD [hydF) and fhlA collection
genes and complete hypF [hydB) gene in pUC19
(187)
pSE-133
Apr, 3.8 kb CZoI fragment with partial
hydF gene and complete hydB and fhlA genes
Laboratory
collection
(187)
pSE-133-1
As pSE-133, hydB::lr\5
This study
pSE-133-2
As pSE-133, fhlA::Tr\5
This study
pSE-137
Apr, 1.0 kb Kpnl-Sall fragment from
with partial hypCD [hydXF) genes in pUC19
Laboratory
collection
(138)
pSE-190
Apr, 1.3 kb SoZI-Pstl fragment from
pSE-111 with partial fhlA gene in pUC19
Laboratory
collection
(188)

49
Table 2.
continued
Plasmid
Relevant Genotype or Phenotype
Reference
pSE1009
Apr, 2.9 kb Kpnl-EcoRV fragment from
pSE-1007, complementing molR and chlD
mutants, in pUC19
Laboratory
collection (136)
pSE-1009
Exo #1-13
Apr, ExoIII deletions from Kpnl, in pUC19
This study
pSE-1004
Apr, 4.7 kb Pvull-Clal fragment from
pSE-1001, complementing molR and chlD
mutants, in pBR322
Laboratory
collection (136)
pSE1213
Apr, 6.5 kb Sau3M fragment with
partial hyb operon in pBR-322
Laboratory
collection
33pBR
Tcr, 6.2 kb Pstl-EcoRI fragment with
complete hyb operon
A. E. Przybyla
pSV208
Apr, Cmr, EcoRl-BamHl promoter fragment
from pBN208 [Apr, 240 bp upstream of fdhF] into pKK232-8
A. Bock (196)

50
previously (187, 188). Plasmid pSE-190 carries a 1.0 kb Sall-Pstl
internal fragment of fhlA gene from previously described plasmid pSE-128
(187) in vector plasmid pUC19. Sequencing of the hydX'FB {hypC'DE)
genes, upstream of the fhlA gene, was accomplished using plasmid pSE-
137, a Sall-Kpnl fragment containing the partial hydX (hypC) and hydF
(hypD) genes in vector plasmid pUC19, pSE-132 which was described
previously, and plasmid pSE125-1 which was constructed as a 2.38 kb
Clal deletion of previously described plasmid pSE-125 (185). The mol
operon was sequenced using previously described plasmid pSE-1009 (134,
136) and exonuclease Ill-generated deletion derivatives from the Kprtl
(the 5'-end of the open reading frames) towards the fcoRV site (88).
Procedures from Promega "Erase-a-base system" technical manual were
followed in these experiments. Plasmid pSE1004 was also used to
establish the fcoRV to Clal DNA sequence. Both strands of chromosomal
DNA present in these plasmids were sequenced using appropriate primers.
New oligonucleotide primers were synthesized, as needed, based on the
partial DNA sequence of the genes, by the DNA synthesis core laboratory,
Interdisciplinary Center for Biotechnology Research, and by Dr. F.C.
Davis, Department of Microbiology and Cell Science, University of
Florida. Commercially available sequencing primers were obtained from
US Biochemical Corporation, Pharmacia-LKB or New England BioLabs. DNA
sequence was determined using T7 DNA polymerase (Sequenase), obtained
from either US Biochemical Corporation or Pharmacia-LKB, and ^S-dATP
was supplied by DuPont-New England Nuclear. The protocols supplied by

51
the manufacturers were followed. The DNA sequence was manipulated and
homology with other known sequences in the Genbank and EMBL library was
determined using the computer programs provided by Genetics Computer
Group, University of Wisconsin (56, 234) and Genepro (Hoeffer
Scientific).
DNA sequencing gels (both 21 x 50 cm and 38 x 50 cm) were poured
and run according to the procedures supplied by BioRad Laboratories with
modifications. A 6% polyacrylamide gel (acrylamide:bis 19:1) in 1 X TBE
(0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) with 8 M urea
was used to separate the reaction products. For increasing the length
of DNA sequence determined, a variety of successful techniques were
implemented. These included the use of wedge spacers provided by BioRad
laboratories, "double loading", and the addition of sodium acetate
(final concentration of approximately 0.1 M) to the bottom chamber in
order to form an electrolyte gradient (201).
Southern transfer and hybridization. Restriction endonuclease
digested chromosomal DNA was separated by electrophoresis in a vertical
1.0% agarose (Sigma type 1: low electroendosmosis grade) gel. The DNA
was stained with ethidium bromide and photographed for calculating the
fragment size of the hybridizing fragments. The DNA was depurinated
(0.25 M HC1 for 10 min) and denatured (0.5 N NaOH; 1 M NaCl for 30 min),
and then the gel was neutralized (0.5 M Tris-HCl pH 7.4; 3 M NaCl for 30
min). Southern transfer to Zeta-Probe membranes (Bio-Rad Laboratories)
was implemented using standard blotting techniques with 10 X SSC (1.5 M

52
NaCl; 0.15 M trisodium citrate) buffer (150). Membranes were dried
under vacuum at 80C for 3 hr and stored dry between two pieces of
"Whatman" 3MM filter paper in plastic bags until used for hybridization.
DNA probes were labeled by random primed incorporation of digoxigenin-
labeled deoxyuridine-triphosphate (Dig-dUTP), hybridized under stringent
conditions to the transferred DNA, and immunologically detected using an
alkaline phosphatase linked antibody-conjugate. Procedures provided
with the Boehringer-Mannheim Biochemicals "Genius"-kit were performed to
carry out these experiments.
For localization of the lac fusion in strain SE2007, an internal
fhlA gene fragment (1.3 kb Sall-Pstl) from plasmid pSE-190 was labeled
and used to probe chromosomal DNA from strains SE-2007 and MC4100
(parent) digested with either a single or two restriction endonucleases
(Sail and Bgll, Sail and fcoRI, Sail and Clal, or Clal). Similarly lac
fusion strain SE-2001 (strains MC-4100 and SE-2009 were used as
controls) chromosomal DNA was digested with endonucleases Kpnl and SoZI,
Clal and Bgll, Bgll, or SoZI and EcoRI and probed with a 0.7 kb SoZI-
Kpnl fragment from plasmid pSE-137 (carrying the partial hypCD genes).
Further localization of the lac fusion was done on both mutant strains,
SE-2001 and SE-2002, by digesting chromosomal DNA with Pst I and then
using 4.0 kb and 4.6 kb Pstl fragments from plasmid pSE-111 as probes.

RESULTS AND DISCUSSION
Physiological Properties of an fhlB Mutant, Strain SE-2011
Biochemical characteristics of an fhlB mutant, strain SE-2011.
Using ApZocMu53 mutagenesis (29), strain SE-2011 was isolated as a lac
operon fusion derivative of strain MC4100 which produced p-galactosidase
activity anaerobically and was deficient in fermentative dihydrogen
production (Fhl-). Upon detailed biochemical analysis, this strain was
found to be affected in the production of hydrogenase, formate
dehydrogenase-H, and fumarate reductase activities (Table 3). The lack
of tritium exchange activity in strain SE-2011 shows that all three HYD
isoenzymes are absent in this strain. As a consequence of this defect,
both FHL, which requires active HYD-3, and hydrogen uptake, mediated by
HYD-2, activities were not detectable in this strain. The FDH-H
activity in strain SE-2011 was less than 10% of the levels observed in
the parent, strain MC4100. On the basis of this property, strain SE-
2011 can be distinguished from all known hyd mutants, which produced
FDH-H activity. Similarly, the SE-2011 phenotype can be readily
distinguished from FDH-H mutants using the hydrogen uptake
characteristics of the other strains. In experiments which are similar
to the ones described in Table 3, strain M9s, a known fdhF mutant (171),
produced 75 to 85% of hydrogen uptake activity of the parent, strain
53

54
Table 3. Biochemical characteristics of strain MC4100 and an fhlB
mutant, strain SE-2011
Specific activity
Enzyme MC4100 SE-2011
(parent) <\>(fhlB'-' lacZ*)
Hydrogenase3
J Q 3
( H2-exchange)
1,200
44
Hydrogen uptake
(H2 to BV)b
700
29
(H2 to fumarate)0
290
UD9
Formate hydrogenlyased
170
UD
Formate dehydrogenase-He
490
38
Fumarate reductasef
600
100
o 3
Expressed as nanomoles of H20 produced per minute per milligram of cell
protein.
Expressed as nanomoles of BV reduced per minute per milligram of cell
protein.
Expressed as nanomoles of H2 consumed per minute per milligram of cell
protein.
Expressed as nanomoles of H2 produced per minute per milligram of cell
rotein.
Expressed as nanomoles of BV reduced per minute per milligram of cell
protein.
Expressed as nanomoles of BVred oxidized per minute per milligram of cell
rotein.
UD, Undetectable

55
MC4100, measured either as BV or fumarate reduction. The hydrogenase
activity of this strain, measured as tritium exchange was close to 100%
of the parent. Strain M9s produced elevated levels of FDH-N (6.6-fold)
and lower levels of fumarate reductase (28%) as compared with its
parent, strain MC4100. These values which are comparable to the
phenotype described by Pecher et al. (171) are quite distinct from the
properties of strain SE-2011 (Table 3).
Strain SE-2011 is normal for nitrate respiration which requires
active FDH-N, but has not been tested for the recently described third
FDH isoenzyme which is presumed to be a major component of formate
oxidase, expressed both aerobically and anaerobically (192). The level
of fumarate reductase activity in strain SE-2011 was also lower (less
that 20% of the parent value). This deficiency is probably the reason
for the growth characteristics of strain SE-2011. The aerobic and
anaerobic growth of this mutant was comparable to that of the parent,
strain MC4100, in LB supplemented with different sugars. However,
strain SE-2011 failed to grow in glucose-minimal medium and to produce
succinate as a fermentation product. Since succinate is a necessary
precursor for biosynthesis, this would account for the poor growth of
the organism in defined medium. As will be discussed later, the same
phenotype was observed when this mutation was transduced into strain
MC4100 or other lac deletion mutants of E. coli (strains CSH26 and
BW545), indicating that the pleiotropic effect is due to a single gene
defect in these genetic backgrounds. The altered gene is termed fhlB

56
since the gene is formate inducible (see below) and thus probably plays
a major role in the production of FHL activity. However, defects in the
production of all three HYD isoenzymes and FR can be readily detected in
strain SE-2011.
Formate requirement for expression of the fhlB gene. In order to
better understand the transcriptional control of the FHL pathway,
expression of the fhlB gene was monitored by measuring the levels of p-
galactosidase activity produced by strain SE-2011 from the fhlB
promoter. When cultured under strictly aerobic conditions, strain SE-
2011 produced about 100 U of p-galactosidase activity in all media
tested (Table 4). Upon transfer to anaerobic conditions, the p-
galactosidase activity of the LB culture increased approximately 2.5-
fold after a 4 hr incubation period. Fumarate had no effect on this
anaerobic induction. Nitrate, a high redox potential electron acceptor,
had a repressive effect on anaerobic expression. Both glucose and
formate supplementation elevated fhlB expression anaerobically by about
2-fold and 5-fold, respectively. The glucose enhancement was due to the
endogenous production of formate which was verified (see below) by the
lack of glucose induction in a pfl background, deficient in pyruvate
formatelyase activity. Acidic pH has been shown in the past to increase
FHL activity (17, 78). Formate-dependent induction was reduced by 50%
when the culture medium was buffered at pH 7.0. This parallels the
physiological data which suggests a role for FHL in pH stabilization
during the fermentative growth of E. coli.

57
Table 4. The effect of media composition on the expression of
Q(fhlB'-lacZ+) in an fhlB mutant, strain SE-2011
Medium composition
3-Gal actosi dase
activity3
Aerobic
Anaerobic
Luria Broth
110
280
+ Nitrate (1.0%)
90
120
+ Fumarate (0.5%)
110
250
+ Glucose (0.3%)
92
580
+ Formate (0.1%)
170
1,300
+ Formate and Glucose
190
1,400
+ Formate and bufferb
NDC
700
Cells were grown as described in the "Methods" section.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
b0.1 M Phosphate buffer at pH 7.0
CND, not determined

58
Upon detailed analysis of fhlB gene expression, monitored at
specific time intervals, it was found that there was an exponential
increase in p-galactosidase activity which paralleled the growth. The
specific activity of the culture reached maximum value during early
stationary phase and remained constant over an additional 8 hr of
incubation (data not shown). In LB medium, maximum activity observed
was about 250 U (Fig. 3). In this medium, the increase in specific
activity of the enzyme was coupled to growth and the differential rate
of induction was about 1.0. In LB medium supplemented with glucose, the
differential rate of p-galactosidase production increased exponentially
during growth, probably due to continued production of formate by the
growing culture (since the amount of formate produced by the culture is
proportional to the cell density). The maximum activity reached was
about 600 units during the early stationary phase of growth when the
cell density was about 100 pg protein/ml. With the addition of formate
to the medium, the growth rate and final cell yield decreased, although
the differential rate of synthesis of p-galactosidase was enhanced to as
high as 130 units/pg cell protein. In this medium, the maximum activity
produced by the culture increased to about 1,200 units. In LB medium
supplemented with both glucose and formate, the differential rate of
induction of p-galactosidase activity was similar to the values obtained
with LB-formate cultures but the total amount of the enzyme produced by
strain SE-2011 was slightly higher (about 1,400 units). The final cell
yield of the culture in the latter two media was comparable. These

59
PROTEIN (jig)
Figure 3. Differential rate of synthesis of p-galactosidase activity by
lacZ*) strain SE-2011 grown in LB medium with different
supplements. LBG, LBF, and LBGF represent LB-glucose, LB-formate, and
LB-glucose-formate media, respectively. p-Galactosidase and protein
activities are expressed as units per ml and pg per ml, respectively.

60
experiments clearly show that the transcription of the fhlB operon is
dependent on formate, either produced internally or added externally to
the medium.
The amount of p-galactosidase activity produced by the Q(fhlB-
,lacZ+) strain increased linearly with increasing formate concentration
up to about 5 mM (Fig. 4). The activity continued to increase at a
lower rate until the maximum was reached at about 15 mM formate in the
medium. For these experiments, strain SE-2011 was grown under anaerobic
conditions, in LB-formate medium, and the cells were harvested after 4
hr for enzyme assays. Similar results were also obtained with strain
MJ-9 [(fhlB'-' lacZ*), pfl] which lacks the ability to produce formate
internally due to a loss of pyruvate formatelyase activity. At 15 mM
formate, strain MJ-9 produced only about 60% of the p-galactosidase
activity observed in the pfl+ parent strain. At higher formate
concentrations (about 30 mM), the specific activity of p-galactosidase
detected in strain MJ-9 was comparable to the pfl+ strain. These
results suggest that the internally produced formate plays a significant
role in the transcription of the fhlB operon.
Nitrate repression of fhlB gene expression. Further analysis of
the repressive effect of nitrate on fhlB operon expression, was carried
out by monitoring the p-galactosidase activity and nitrite produced by
strain SE-2011 over a 6 hr growth period in an anaerobic environment.
The fhlB mutant was grown in LB, LB-formate, LB-nitrate and LB-formate-
nitrate medium (LB, LBF, LBN and LBFN respectively; Fig. 5). Nitrite

61
FORMATE (mM)
Figure 4. Effect of formate on the induction of p-galactosidase activity
by <\>(fhlB-lacZ+) strain SE-2011 and a pfl derivative, strain MJ-9.
Specific activity represents the maximum value observed at each formate
concentration.

62
Figure 5. Effect of nitrate supplementation on fhlB gene expression.
Cultures were grown in LB, LB-formate, LB-nitrate, and LB-nitrate-
formate media (LB, LBF, LBN and LBNF, respectively). A. Nitrate
respiration (as measured by nitrite produced) in fhlB mutant strain SE-
2011 grown in LB and LBNF. B. Rate of induction of (3gal actos i dase
activity by 4>(fhlB'-'lacZÂ¥) strain SE-2011.

63
production was measured in the cultures supplemented with nitrate to
estimate the rate of nitrate respiration. Results of these experiments
indicate that significant levels of nitrate respiration are not evident
until 2 hr after initiation of anaerobiosis and growth (Fig. 5A). After
this initial lag, nitrite accumulated in the medium throughout the
remaining time of the experiment. In the absence of exogenous formate,
nitrate repression of fhlB gene transcription (as measured by p-
galactosidase activity) paralleled the initiation of nitrate respiration
(LBN; Fig. 5B). Formate supplementation (73 mM) partially suppressed
this repressive effect, and a narL mutation did not affect the fhlB
operon regulation. This is comparable to the regulatory patterns
observed for other genes of the FHL pathway (hyd-17 and fdhF genes;
171). These results suggest that both formate and anaerobiosis are
necessary for maximal induction of fhlB operon transcription. Although
nitrate and neutral pH repress this expression, this effect can be
overcome by formate.

64
Genetic Characteristics of Strain SE-2011, QtfhlB'-' lacZ*)
F1 complementation analysis. Initial complementation studies were
performed using F'143-1, an episomal element which carried DNA from 57
to 61 min in the E. coli chromosome. This was done since the previously
described hyd and fhlA genes, which demonstrated pleiotropic phenotypes
for dihydrogen metabolism, mapped to the 58 to 59 min region (185, 186,
187, 188). Mutant strain MJ-1, a cys::Tni0 derivative of SE-2011, was
used as a recipient for F'1431 transfer. Exconjugants were selected
for Cys+ phenotype and then screened for FHL activity. Approximately 20%
of the Cys+ clones were Fhl+. Among those complemented for dihydrogen
production, HUP activity was not restored (as measured by H2 to BV). It
is possible that the 20% complementation for FHL activity was because
the entire region necessary for restoration of strain SE-2011 to
parental phenotype was not completely transferred or that deletions may
have occurred in the plasmid during growth with uracil, a needed
nutrient. Therefore, strain MJ-50 [(fhlB'-'lacZ+) cys, srl] was used
to select a F* element carrying the entire region between cys+ and srl+
genes. A Fhl+ exconjugant (5% of the clones tested) was isolated and
used as a F*1431 donor strain to select for Cys+ in recipient strain
MJ-1, 4>(fhlB'-'lacZÂ¥) cys. Of the exconjugants analyzed, 100% were Fhl +
and Hup+ which suggested that the mutation mapped in this region.
PI transduction. Similar results were obtained with PI
transduction experiments using strain BW545 as the donor. Of the 72

65
Cys+ clones tested, 12.5% were restored for FHL activity. One lysogen
from this experiment tested positive for HUP activity but the clone
cured of the lysogenic phage was deficient in both FHL and HUP
activities.
Hfr mediated conjugation analysis of the fhlB gene. Because of
these inconclusive results the approximate map location of the altered
gene in the E. coli chromosome was determined by Hfr-mediated
conjugation analysis. For these experiments, an Hfr-derivative of
strain SE-2011 in which the origin of DNA transfer is the fhlB gene was
constructed, using lac homology, as described before (40, 136). In a 30
min conjugation period, strain MJ-18, transferred argl gene (96.6 min;
11) and not frdA gene (94.4 min; 11) indicating that the fhlB gene was
located between these two genes. Both thr and leu were also transferred
at high frequency during this 30 min duration. Since the orientation of
the lac operon with respect to the origin of transfer is known (40),
results of these experiments were also used to determine the direction
of transcription of the fhlB gene. According to the results, it
appeared that the fhlB gene was transcribed in a clockwise direction,
towards argl, thr and leu.
The accuracy of utilizing this procedure in certain lac fusion
strains is currently under investigation (Shanmugam, personal
communication). Therefore after reevaluating the physiological data and
mapping results of SE-2011, it appeared probable that the localization
of fhlB gene to the 96.6 min position was due to homology of the

66
chromosomal region (i.e. melAB at 93 min) to the episomal lac DNA
sequences used in the construction of the Hfr (PO-fhlB). Comparable
mapping complications were encountered in another study with strain SE-
1100, a (molR'-' lacZ+) fusion. The molR gene was originally mapped to
66 min using similar Hfr procedures. Phage Pl-mediated transduction
experiments later mapped the molR gene at 17 min in the E. coli
chromosome. Interestingly, a second ancestral gene for p-galactosidase,
the ebgA gene, which is homologous to the lacZ gene was found to map in
this region (68 min; ref. 8, 11, 32, 225, 226, 230).
Stability of the $(fhlB'-' lacZ^_) mutation. The pleiotropic nature
of the fhlB mutation led to the question of whether there was one (or
multiple) chromosomal mutation(s) leading to the observed phenotype in
strain SE-2011. Initially, the same biochemical characteristics as well
as operon expression (p-galactosidase activity) were observed when the
mutation from strain SE-2011 was transduced into various E. coli
strains, including strains MC4100, BW545 and CSH26. Even upon several
years of maintaining the mutant strains at -70C in 20% glycerol, the
original characteristics of strain SE-2011 remained stable. However, in
recent experiments, the majority of transductants which were selected
for Lac+ (Xgal+) and Kmr were no longer analogous to the fhlB mutation
previously described. Only about 1 to 5% of the transductants were
Fhl". Dihydrogen uptake activity of these strains was comparable to
wild type levels of activity. Many of these mutants were also altered
in fhlB operon expression. Basal level anaerobic expression, in the

67
absence of formate was increased 2- to 3-fold. The significance of
these results remains to be determined.
Plasmid complementation. Previously described plasmid pSE-133,
which carries the partial hydF and complete hydB, fhlA genes (186),
restored FHL activity of strain SE-2011 to low levels (data not shown).
As later experiments would show, this effect is due to a physiological
effect of the FHL-A protein, a transcriptional activator for the FHL
system.
It is now known that the two divergently transcribed operons in
the 58 to 59 min region (the hyc and hyp operons) span approximately
15.5 kb of DNA. Plasmid pRBH, which carries the complete hyc operon on
low copy vector pACYC184 restored the Fhl+ activity of strain SE-2011 to
parental levels. Although the H2 to BV activity was increased, this
activity was attributable to HYD-3 (Table 5). Additionally, the p-
galactosidase activity produced by strain SE-2011/pRBH was 2-fold lower
than strain SE-2011 (LB-formate; 10 mM). Lutz et al. (144) have
recently reported that the first gene of the hyc operon is a putative
repressor and this is probably responsible for this decrease in fhlB
expression.
Interestingly, multicopy plasmid pSE-1213 which carries the
partial hyb operon (encoding HYD-2) and complements hyb mutants restored
strain SE-2011 to parental levels of HUP and low levels of FHL activity
(10% of wild type levels). Multicopy plasmid 33pBR which carries the
complete hyb operon complemented strain SE-2011 for both FHL and HUP

68
Table 5. Plasmid complementation analysis of fhlB mutant,
strain SE-2011
Specific activity
Strain/plasmid
FHLa
HUPb
(H2 to BV) (H2
HUPC
to fumarate)
BW545
100%
694
44
SE-2011
UDd
UD
UD
SE-2011/pRBH
100%
+e
NDf
SE2011/pSE1213
10%
1,017
63
SE201l/33pBR
75%
616
91
Expressed as % wild type levels of H2 produced.
Expressed as nanomoles of BV reduced per minute per milligram of cell
protein.
^Expressed as nanomoles of H2 consumed per minute per milligram of cell
rotein.
UD, Undetectable.
eMainly HYD-3 activity.
fND, Not determined.

69
activities to 75% and 100% of wild type levels, respectively. This
suggests that the mutation is more complex than anticipated. Further
analysis is currently being done to determine the extent of the hyb
operon on the Sau3M fragment which is carried by plasmid pSE1213.
Also, the exact location of the fhlB mutation in strain SE-2011 is under
investigation using polymerase chain reaction (PCR) procedures combined
with ${fhlB'-'lacZ+) cloning methods.

70
Trans-Ac ting Factors of the fhlB Operon
Genetic regulation of the fhlB operon. Expression of the enzymes in
dihydrogen metabolism requires the products of several genes and these
include hyd, fnr, fhlA, rpoN, molR and chlD gene products (25, 35, 135,
136, 171, 185, 186, 187, 188, 196, 223, 224, 231, 238, 242). In order to
study the role of these gene products on the expression of 4>(//>Zfi'-
1 ZacZ4-), appropriate double mutant strains were constructed in which one
of the putative regulatory genes is defective. Analysis of these double
mutant strains revealed that the fhlA, rpoN, chlD and molR gene products
are needed for the anaerobic formate-dependent induction of Q(fhlB'-
ZocZ+) (Table 6). The differential rate of expression of p-galactosidase
activity in the double mutants, strains MJ-5, MJ-20 and MJ-40 (rpoN, fhlA
and molR, respectively) was unity indicating that the induction and cell
growth are coupled and the enhancing effect of formate was absent. In an
fnr mutant (strain MJ-6), although the differential rate of expression was
comparable to strain SE-2011, the maximum activity was reduced by about
35%. The reduction in the amount of p-galactosidase activity produced by
the fhlB, fnr double mutant could be a consequence of lower cell yield of
the culture since the production of p-galactosidase activity by (fhlB'~
'lacZ+) required growth of the organism. HydF and cya mutations had no
apparent effect on the expression of (fhlB'-'lacZ*) operon with and
without formate supplementation. Introducing narL::Tni0 (217) into strain

71
Table 6. Expression of Q(fhlB'-'lacZ*) in different genetic backgrounds
Strain
Relevant
Genotype
0-Galactosidase
Activity3
Differential
Rate of Inductionb
(U/pg Protein)
Maximum
Activity
SE-2011
(fhlB-lacZ+)
130
1,300
MJ-5
QifhlB-lacZ*), rpoN
1
110
MJ-20
QifhlB-lacZ*), fhlA
1
150
MJ-2
Q^fhlB-lacZ*), hydF
NDC
1,200
MJ-6
QifhlB-lacZ*), fnr
110
840
MJ-8
QifhlB-lacZ*), cya
160d
1,300
MJ-40
^{fhlB-lacZ^), molR
1
130
SE1714
QtfhlB-lacZ*), chlD
ND
160
Expressed as nmoles ONP produced per min per mg protein. All cultures
were grown anaerobically at 37C in LB + formate medium except strain MJ-6
which was grown in LB medium with formate and glucose to enhance cell
vield.
The differential rate of induction of 0-galactosidase activity was
calculated as the amount of enzyme activity produced by the culture in
relation to the increase in total cell protein.
^ND, Not Determined.
dPoor cell growth.

72
SE2011 did not alter the amount of p-galactosidase activity produced by
the culture both in the presence and absence of nitrate (data not shown).
Effect of multiple copies of the fhlA gene on fhlB transcription.
The effect of increasing the copy number of the fhlA gene on the
expression of (fhlB'-'lacZ+) was investigated using plasmid pSE-133
which carries the complete fhlA+ and hydB+ genes (186, 187). In the
presence of plasmid pSE-133, the differential rate of production of p-
galactosidase activity by (fhlB'-' lacZ+) was about 700 units / pg cell
protein in LB-formate medium (Fig. 6). This value is greater than 5
times the rate of about 130 units / pg cell protein for strain SE-2011
cultured in the same medium. The maximum activity produced by strain
SE201l/pSE-133 is also greater than 2 times the values obtained with
strain SE-2011 and this increase was detected immediately after
establishing anaerobic conditions. The maximum activity observed in
strain SE-2011/pSE133, grown in LB medium without formate
supplementation, was also increased to about 1,100 units of p-
galactosidase activity. This level of activity is comparable to the
values obtained with strain SE-2011 grown in LB medium with 30 mM
formate (about 1,000 units) although the differential rate of induction
observed with strain SE-2011/pSE133, in LB medium, was lower.
Inserting transposon Tn5 into the fhlA gene in the plasmid (pSE-133-2;
Fig. 7) abolished this enhancing effect of plasmid pSE-133 while a
hydB::lr\5 mutation in the plasmid (pSE-133-1) had no effect indicating
that the plasmid-mediated increase is due to the fhlA gene. Transferring

73
PROTEIN (jig)
Figure 6. Differential rate of induction of the lacZ*) fusion
in strain SE-2011 in the presence and absence of plasmid pSE-133. LB, LB
medium; LBF, LB medium supplemented with 30 mM formate. p-Galactosidase
and protein activities are expressed as units per ml and pg per ml,
respectively.

74
FORMATE (mM)
Figure 7. Effect of formate concentration on the levels of p-
galactosidase activity produced by a ^{fhlB'-'lacZf) pfl double mutant,
strain MJ-9, in the presence of plasmids pSE-133 and pSE133-2
[fhlA::Tn5). Cultures were grown for 4 hr in LB medium with appropriate
concentrations of formate under anaerobic conditions before the assay.

75
an F1 element carrying fhlA+ gene (F143-1) into strain SE-2011, did not
significantly alter the rate or the level of p-galactosidase activity.
These results suggest that increasing the copy number of the fhlA+ gene
either decreased the concentration of formate required for transcription
of lac2+) or eliminated the need for formate.
Formate is required for FHL-A activation of the fhlB operon. In
order to distinguish between the two possibilities, plasmid pSE-133 was
transferred to strain MJ-9 [Q(fhlB'-'lacZ+), pfl] and the amount of p-
galactosidase activity produced by the culture was determined after
culturing the cells in either LB or LB-formate medium. The strain MJ-
9/pSE-133 produced about 100 units of p-galactosidase activity when
grown in LB medium and about 1,300 units when grown in LB-formate medium
(Fig. 7). The minimum amount of formate needed for this transcription
was about 3 mM. At formate concentrations higher than 3 mM, the amount
of p-galactosidase activity produced by strain MJ9/pSE133 increased
slowly reaching a maximum value of about 1,500 units at about 30 mM.
The amount of p-galactosidase activity produced by strain MJ-9/pSE-133-2
(fhlA::Tn5) was actually lower than strain MJ-9 itself and under the
conditions used in these experiments, this activity never exceeded 300
units. These results show that in the presence of multiple copies of
fhlA gene, the concentration of formate required for optimum expression
of fhlB gene is considerably reduced but is still a needed inducer.
The FHL-B protein is cytoplasmic. With 3 mM formate in the
medium, the differential rate of synthesis of p-galactosidase activity

76
by strain SE-2011 was enhanced from about 20 units/pg protein to about
330 units/pg protein if the fhlA gene was also present in a multicopy
plasmid (pSE-133-1) (Fig. 8). Immediately after anaerobic conditions
were established, the //? IB-mediated (3-galactosidase activity increased
to about 1,200 units within a generation time. During the second
generation, this activity decreased to about 75% of the observed peak
value. The p-galactosidase activity of the culture reached about 1,000
units and was maintained at that level. On the other hand, strain M9s
[^(fdhF'-' lacZ*")] carries the lac fusion in FDH-H, a known membrane
protein. With the same plasmid, this strain (M9s/pSE-1331) produced p-
galactosidase activity after an initial lag. The differential rate of
synthesis was about 10% of the values of strain SE2011/pSE1331 and
was only about 3-fold higher than strain M9s without the plasmid. The
levels of p-galactosidase activity produced by strains SE-2011 and M9s,
both in the presence and absence of the plasmid pSE-133-1, was not
altered by including Mo or Se to the LB medium, although the FDH-H is a
Mo, Se-containing protein. The amount of formate needed for optimum
expression of Q(fdhF'-'lacZ*)/pSE-133-1 was about 3 mM also which is
similar to that required for strain MJ-9/pSE-133. This rapid induction
of fhlB gene in the presence of multiple copies of fhlA+ gene, which is
in contrast to the fdhF gene, suggests that the FHL-B protein is
cytoplasmic. Its synthesis is apparently not constrained by the
availability of membrane proteins and membrane synthesis.

77
PROTEIN (fig)
Figure 8. Differential rate of synthesis of and lacZ*), strain SE-2011, in LB medium supplemented with 3 mM
formate in the presence and absence of the fhlA+ gene in a multicopy
plasmid (pSE-133-1). p-Galactosidase and protein activities are
expressed as units per ml and pg per ml, respectively.

78
Expression of QjfdhF'-'cat) in an fhlB mutant, strain SE2011.
The FDH-H activity was undetectable in strain SE2011. In order to
determine whether this effect is at the transcriptional or post-
transcriptional level, a plasmid carrying a 'CAT' gene fusion in the
fdhF gene was transformed into strain SE-2011 and the effect of the fhlB
mutation on fdhF gene mediated CAT activity was determined. Cells were
grown anaerobically (4 hr) in LB medium supplemented with glucose and
formate for maximal expression. Due to the multicopy nature of the
expression vector, trace metals (Se, Mo, and Fe) were included to insure
saturation. The fdhF transcription measured as CAT activity in strain
SE-2011 [QtfhlB'-'lacZ*)] was comparable to parent strain MC-4100 at 140
and 110, units respectively. Whereas, in strain SE-2007 [Q(fhlA'-
'lacZ+)] expression was reduced over 5-fold, to about 20 units
confirming the need for FHL-A protein. This suggests that FHL-B protein
is not required for transcription of fdhF gene but is required for the
production of active FDH-H (Table 3).

79
Analysis of the fhlA Gene
Primary structure of the fhlA gene. Previous experiments
identified the fhlA gene product as a putative regulatory element of
both fdhF and hyd-17 genes (185, 187, 196). The results presented above
(Table 6, Fig. 6-8) also show that the FHL-A protein is a needed
regulatory element for the fhlB operon. Because of these observations,
the DNA sequence of the fhlA gene was determined to identify the
characteristics of the gene and its product. The fhlA gene (coding
region position 2421 to 4497; Fig. 9) codes for a protein of 692 amino
acids with an anhydrous molecular weight of 78,467 Da which is
comparable to the apparent molecular weight of 78,000 Da obtained by
other experiments (144, 185). This protein did not contain any
significant hydrophobic region indicating that the primary location of
this protein is the cytoplasm. Eight base pairs from the end of
translational stop codon (position 4497), the coding region is followed
by an inverted repeat (underlined in Fig. 9; positions 4508 to 4517 and
4524 to 4533) which can produce a 10 base pairs stem and a 6 bases
(positions 4518 to 4523) loop structure. This region is followed by a
stretch of 6 thymine residues at a distance of 14 bases (positions 4548
to 4553). Five more thymine residues can be found 9 bases from the
first set of thymines (positions 4563 to 4567). This segment of DNA, in
appropriate configuration may function as a p-independent transcription
termination site. The 5'-end of the putative coding region is preceded

80
Sal
GTCGACGTCTGCGGCATTCAGCGCGATGTCGATTTAACGTTAGTCGGCAGCTGCGATGAA 60
VDVCGIQRDVDLTLVGSCDE
AACGGTCAGCCGCGCGTGGGCCAGTGGGTACTGGTACACGTTGGCTTTGCCATGAGCGTA 120
NGQPRVGQWVLVHVGFAMSV
ATTAATGAAGCCGAAGCACGCGACACTCTCGACGCCTTACAAAACATGTTTGACGTTGAG 180
INEAEARDTLDALQNMFDVE
CCGGATGTCGGCGCGCTGTTGTATGGCGAGGAAAAATAATGCGTTTTGTTGATGAATATC 240
PDVGALLYGEEK ***
MRFVDEYR
GCGCGCCGGAACAGGTGATGCAGTTAATTGAGCATCTGCGCGAACGTGCTTCACATCTCT 300
APEQVMQLIEHLRERASHLS
CTTACACCGCCGAACGCCCTCTGCGGATTATGGAAGTGTGTGGCGGTCATACCCACGCTA 360
YTAERPLRIMEVCGGHTHAI
TCTTTAAATTCGGCCTCGACCAGTTACTGCCGGAAAACGTTGAGTTTATCCACGGTCCGG 420
FKFGLDQLLPENVEFIHGPG
GGTGCCCGGTGTGCGTACTGCCGATGGGTAGAATCGACACCTGCGTGGAGATTGCCAGCC 480
CPVCVLPMGRIDTCVEIASH
ATCCGGAAGTCATCTTCTGTACCTTTGGCGACGCGATGCGCGTGCCGGGGAAACAGGGAT 540
PEVIFCTFGDAMRVPGKQGS
CGCTGTTGCAGGCAAAAGCACGCGGTGCCGATGTGCGCATCGTTTACTCGCCGATGGATG 600
LLQAKARGADVRIVYSPMDA
CGTTGAAACTGGCGCAGGAGAATCCAACCCGCAAAGTGGTGTTCTTCGGCTTAGGTTTTG 660
LKLAQENPTRKVVFFGLGFE
AAACCACTATGCCGACCACCGCTATCACTCTGCAACAGGCGAAAGCGCGTGATGTGCAGA 720
TTMPTTAITLQQAKARDVQN
ATTTTTACTTCTTCTGCCAGCACATTACGCTTATCCCGACGTTGCGCAGTTTGCTGGAAC 780
FYFFCQHITLIPTLRSLLEQ
Clal
AGCCGGATAACGGTATCGATGCGTTCCTCGCGCCGGGTCACGTCAGTATGGTTATCGGCA 840
PDNGIDAFLAPGHVSMVIGT
CCGACGCCTATAATTTTATCGCCAGCGATTTTCATCGTCCGCTGGTGGTTGCTGGATTCG 900
DAYNFIASDFHRPLVVAGFE
AACCCCTTGATCTACTACAAGGCGTGGTCATGCTGGTGCAGCAGAAAATAGCGGCCCACA 960
PLDLLQGVVMLVQQKIAAHS
Kpnl
GCAAGGTAGAGAATCAGTATCGTCGAGTGGTACCGGATGCCGGTAACCTGCTGGCGCAAC 1020
KVENQYRRVVPDAGNLLAQQ
Figure 9. Nucleic acid and predicted amino acid sequences of the
partial hypC gene and complete hydB, hydF and fhlA genes. The
termination codons are indicated by three asterisks. The "Shine-
Dalgarno" sequences and the weak "-35 and -10" region of the fhlA gene
are double underlined. Restriction sites for some of the enzymes are
highlighted. The inverted triangle between positions 4,404 and 4,405
represents the position of transposon Tn5 in plasmid pSE133-2, as
determined by DNA sequence analysis.

81
AGGCGATTGCCGATGTGTTCT6T6TCAACGGCGACAGCGAATGGCGCGGCTTAGGCGTGA 1080
AIADVFCVNGDSEWRGLGVI
TTGAATCTTCTGGCGTGCACCTGACGCCGGATTATCAACGATTCGATGCCGAAGCACATT 1140
ESSGVHLTPDYQRFDAEAHF
TCCGCCCGGCACCGCAGCAGGTCTGCGATGACCCGCGCGCGCGTTGTGGTGAGGTATTAA 1200
RPAPQQVCDDPRARCGEVLT
CGGGCAAATGTAAGCCGCATCAATGCCCGCTGTTTGGTAACACCTGTAATCCTCAAACCG 1260
GKCKPHQCPLFGNTCNPQTA
CGTTTGGTGCGCTGATGGTTTCCTCCGAAGGAGCGTGCGCCGCGTGGTATCAGTATCGTC 1320
FGALMVSSEGACAAWYQYRQ
AGCAGGAGAGTGAAGCGTGAATAATATCCAACTCGCCCACGGTAGCGGCGGCCAGGCGAT 1380
Q E S E A *** M
GCAGCAATTAATCAACAGCCTGTTTATGGAAGCCTTTGCCAACCCGTGGCTGGCAGAGCA 1440
QQLINSLFMEAFANPWLAEQ
GGAAGATCAGGCACGTCTTGATCTGGCGCAGCTGGTAGCGGAAGGCGACCGTCTGGCGTT 1500
EDQARLDLAQLVAEGDRLAF
CTCCACCGACAGTTACGTTATTGACCCGCTGTTCTTCCCTGGCGGTAATATCGGCAAGCT 1560
STDSYVIDPLFFPGGNIGKL
GGCGATTTGCGGCACAGCCAATGACGTTGCGGTCAGTGGCGCTATTCCGCGCTATCTCTC 1620
AICGTANDVAVSGAI PRYLS
CTGTGGCTTTATCCTCGAAGAAGGATTGCCGATGGAGACACTGAAAGCCGTAGTGACCAG 1680
CGFI LEEGLPMETLKAVVTS
CATGGCAGAAACCGCCCGCGCGGCAGGCATTGCCATCGTTACTGGCGATACTAAAGTGGT 1740
MAETARAAGIAIVTGDTKVV
GCAGCGCGGCGCGGTAGATAAACTGTTTATCAACACCGCTGGCATGGGCGCAATTCCGGC 1800
QRGAVDKLFINTAGMGAIPA
GAATATTCACTGGGGCGCACAGACGCTAACCGCAGGCGATGTATTGCTGGTGAGCGGTAC 1860
NIHWGAQTLTAGDVLLVSGT
ACTCGGCGACCACGGGGCGACTATCCTTAACCTGCGTGAGCAGCTGGGGCTGGATGGCGA 1920
LGDHGATI LNLREQLGLDGE
ACTGGTCAGCGACTGCGCGGTGCTGACGCCGCTTATTCAGACGCTGCGTGACATTCCCGG 1980
LVSDCAVLTPLIQTLRDIPG
CGTGAAAGCGCTGCGTGATGCCACCCGTGGTGGTGTAAACGCGGTGGTTCATGAGTTCGC 2040
VKALRDATRGGVNAVVHEFA
GGCAGCCTGCGGTTGTGGTATTGAACTTTCAGAAGCGGCACTGCCTGTTAAACCTGCCGT 2100
AACGCGI ELSEAALPVKPAV
GCGTGGCGTTTGCGAATTGCTGGGACTGGACGCCCTGAACTTTGCCAACGAAGGCAAACT 2160
RGVCELLGLDALNFANEGKL
AGTAATAGCTGTTGAACGCAACGCGGCAGAGCAAGTGCTGGCAGCGTTACATTCCCATCC 2220
VIAVERNAAEQVLAALHSHP
ACTGGGGAAAGACGCGGCGCTGATTGGTGAAGTGGTGGAACGTAAAGGTGTTCGTCTTGC 2280
LGKDAALIGEVVERKGVRLA
CGGTCTGTATGGCGTGAAACGAACCCTCGATTTACCACACGCCGAACCGCTTCCGCGTAT 2340
GLYGVKRTLDLPHAEPLPRI
Figure 9continued

82
MGCTAATAAAATTCTAAATCTCCTATAGTTAGTCAATGACCTTTTGCACCGCTTTGCGG 2400
0 ******
TGCTTTCCTGGAAGAACAAAATGTCATATACACCGATGAGTGATCTCGGACAACAAGGGT 2460
MSYTPMSDLGQQGL
TGTTCGACATCACTCGGACACTATTGCAGCAGCCCGATCTGGCCTCGCTGTGTGAGGCTC 2520
FDITRTLLQQPDLASLCEAL
TTTCGCAACTGGTAAAGCGTTCTGCGCTCGCCGACAACGCGGCTATTGTGTTGTGGCAAG 2580
SQLVKRSALADNAAIVLWQA
CGCAGACTCAACGTGCGTCTTATTACGCGTCGCGTGAAAAAGACACCCCCATTAAATATG 2640
QTQRASYYASREKDTPI KYE
AAGACGAAACTGTTCTGGCACACGGTCCGGTACGCAGCATTTTGTCGCGCCCTGATACGC 2700
DETVLAHGPVRSI LSRPDTL
TGCATTGCAGTTACGAAGAATTTTGTGAAACCTGGCCGCAGCTGGACGCAGGTGGGCTAT 2760
HCSYEEFCETWPQLDAGGLY
ACCCAAAATTTGGTCACTATTGCCTGATGCCACTGGCGGCGGAAGGGCATATTTTTGGTG 2820
PKFGHYCLMPLAAEGHIFGG
GCTGTGAATTTATTCGTTATGACGATCGCCCCTGGAGCGAAAAAGAGTTCAATCGTCTGC 2880
CEFIRYDDRPWSEKEFNRLQ
Hpal
AAACATTTACGCAGATCGTTTCTGTCGTCACCGAACAAATCCAGAGCCGCGTCGTTAACA 2940
TFTQIVSVVTEQIQSRVVNN
Sail
ATGTCGACTATGAGTTGTTATGCCGGGAACGCGATAACTTCCGCATCCTGGTCGCCATCA 3000
VDYELLCRERDNFRILVAIT
CCAACGCGGTGCTTTCCCGCCTGGATATGGACGAACTGGTCAGCGAAGTCGCCAAAGAAA 3060
NAVLSRLDMDELVSEVAKEI
TCCATTACTATTTCGACATTGACGATATCAGTATCGTCTTACGCAGCCACCGTAAAAACA 3120
HYYFDIDDISIVLRSHRKNK
AACTCAACATCTACTCCACTCACTATCTTGATAAACAGCATCCCGCCCACGAACAGAGCG 3180
LNIYSTHYLDKQHPAHEQSE
AAGTCGATGAAGCCGGAACCCTCACCGAACGCGTGTTCAAAAGTAAAGAGATGCTGCTGA 3240
VDEAGTLTERVFKSKEMLLI
TCAATCTCCACGAGCGGGACGATTTAGCCCCCTATGAACGCATGTTGTTCGACACCTGGG 3300
NLHERDDLAPYERMLFDTWG
GCAACCAGATTCAAACCTTGTGCCTGTTACCGCTGATGTCTGGCGACACCATGCTGGGCG 3360
NQIQTLCLLPLMSGDTMLGV
TGCTGAAACTGGCGCAATGCGAAGAGAAAGTGTTTACCACTACCAATCTGAATTTACTGC 3420
LKLAQCEEKVFTTTNLNLLR
GCCAGATTGCCGAACGTGTGGCAATCGCTGTCGATAACGCCCTCGCCTATCAGGAAATCC 3480
QIAERVAIAVDNALAYQEIH
ATCGTCTGAAAGAACGGCTGGTTGATGAAAACCTCGCCCTGACCGAGCAGCTCAACAATG 3540
RLKERLVDENLALTEQLNNV
TTGATAGTGAATTTGGCGAGATTATTGGCCGCAGCGAAGCCATGTACAGCGTGCTTAAAC 3600
DSEFGEI IGRSEAMYSVLKQ
Figure 9--continued.

83
AAGTTGAAATGGTGGCGCAAAGTGACAGTACCGTGCTGATCCTCGGTGAAACTGGCACGG 3660
VEMVAQSDSTVLI LGETGTG
GTAAAGAGCTGATTGCCCGTGCGATCCATAATCTCAGTGGGCGTAATAATCGCCGCATGG 3720
KELIARAIHNLSGRNNRRMV
TCAAAATGAACTGCGCGGCGATGCCTGCCGGATTGCTGGAAAGCGATCTGTTTGGTCATG 3780
KMNCAAMPAGLLESDLFGHE
AGCGTGGGGCTTTTACCGGTGCCAGCGCCCAGCGTATCGGTCGTTTTGAACTGGCGGATA 3840
RGAFTGASAQRIGRFELADK
AAAGCTCCCTGTTCCTCGACGAAGTGGGCGATATGCCACTGGAGTTACAGCCGAAGTTGC 3900
SSLFLDEVGDMPLELQPKLL
TGCGTGT ATTGCAGGAACAGGAGTTTGAACGTCTCGGCAGCAACAAAATCATTCAGACGG 3960
RVLQEQEFERLGSNKI IQTD
ACGTGCGTCTAATCGCCGCGACTAACCGCGATCTGAAAAAAATGGTCGCCGACCGTGAGT 4020
VRLIAATNRDLKKMVADREF
TCCGTAGCGATCTCTATTACCGCCTGAACGTATTCCCGATTCACCTGCCGCCACTACGCG 4080
RSDLYYRLNVFPIHLPPLRE
AGCGTCCGGAAGATATTCCGCTGCTGGCGAAAGCCTTTACCTTCAAAATTGCCCGTCGTC 4140
RPEDIPLLAKAFTFKIARRL
TGGGGCGCAATATCGACAGCATTCCTGCCGAGACGCTGCGCACCTTGAGCAACATGGAGT 4200
GRNIDSIPAETLRTLSNMEW
GGCCGGGTAACGTACGCGAACTGGAAAACGTCATTGAGCGCGCGGTATTGCTAACACGCG 4260
PGNVRELENVI ERAVLLTRG
Pst I
GTAACGTGCTGCAGCTGTCATTGCCAGATATTGTTTTACCGGAACCTGAAACGCCGCCTG 4320
NVLQLSLPDIVLPEPETPPA
CCGCAACGGTTGTCGCCCTGGAGGGCGAAGATGAATATCAGTTGATTGTGCGCGTGCTGA 4380
ATVVALEGEDEYQLIVRVLK
Tn5

AAGAAACCAACGGCGTGGTTGCCGGGCCTAAAGGCGCTGCGCAACGTCTGGGGCTGAAAC 4440
ETNGVVAGPKGAAQRLGLKR
GCACGACCCTGCTGTCACGGATGAAGCGGCTGGGAATTGATAAATCGGCATTGATTTAAC 4500
TTLLSRMKRLGIDKSALI ***
TGCAAATTGCCGGACAGATCTGCCTGTCCGGCATACTATTCATGAGGTTTTTTCGGACGA 4560
Clal
TATTTTTCCGGCAGTTCTGGCACCGGACGCTTGTCATCGAT 4601
Figure 9continued.

84
by a typical ribosome binding site (GGA, starting at position 2410; ref.
126). A weak "-10 and -35" o70 promoter consensus sequence is also
indicated in Fig. 9 (positions 2312 to 2317 and 2338 to 2343; ref. 86).
Based on the DNA sequence, the fhlA gene resides between 2,867 and 2,870
kb of the E. coli chromosomal DNA as described by Kohara et al. (125)
and the direction of transcription is clockwise towards cys operon at 59
min (11).
FHL-A protein is a transcriptional activator. The FHL-A protein
has sequence homology with known transcriptional activators like NTR-C
protein of f. coli, NIF-A protein of Klebsiella pneumoniae, and XYL-R
protein of Pseudomonas put ida (Fig. 10; and ref. 58, 103, 157). The
overall homology is about 30% between the four proteins although the
FHL-A protein is considerably larger than the other three. It is 224,
168, and 126 amino acids larger than NTR-C, NIF-A and XYL-R proteins,
respectively. Significant regions of the FHL-A protein were also found
to be homologous with other transcriptional activators of the two-
component regulatory systems including the recently described LEV-R
protein of Bacillus subtilis (53), DCT-D protein of Rhizobium
leguminosarum (181), FLB-D protein of Caulobacter crescentus (176), TYR-
R protein (49) and HYD-G protein of E. coli (224). Homology with this
class of proteins, combined with physiological data, suggest that the
FHL-A protein functions as a transcriptional activator of the o54-
dependent promoters of the FHL pathway (fhlB, fdhF and hyc operons; for
review, see 80, 222).

85
FHL-A MSYTPMSDLGQQGLFDITRTLLQQPDLASLCEALSQLVKRSALADNAAIVLWQAQTQRAS 60
FHL-A YYASREKDTPIKYEDETVLAHGPVRSILSRPDTLHCSYEEFCETWPQLDAGGLYPKFGHY 120
FHL-A CLMPLAAEGHIFGGCEFIRYDDRPWSEKEFNRLQTFTQIVSWTEQIQSRWNNVDYELL 180
NIF-A MIHKSDSDTTV 11
XYL-R MSLTYKPKMQHEDMQDLSSQIRFVAAEGKIWLG 33
FHL-A CRERDNFRILVAITNAVLSRLDMDELVSEVAKEIHYYFDIDDISIVLRSHRKNKLNIYST 240
NIF-A RRFDLSQQFTAMQRISWLSRATEASKTLQEVLSVLHNDAFMQHGMICLYDSQQEILSIE 71
XYL-R EQRMLVMQLSTLASFRREIISLIGVERAKGFFLRLGYQSGLMDAELARKLRPAMREEEVF 93
FHL-A HYLDKQHPAHEQSEVDEAGTLTERVFKSKEMLLINLHERDDLAPYERM-LFDTWGNQIQT 299
NTR-C MQRGIVWWDDDSSIRWVLERALAGAGLTCTTFENGAEVLEA-LASKTPDVLLS 53
NIF-A ALQQTEDQTLPGSTQIRYRPGEGLVGTVLAQGQSLVLPRVADDQRFLDRL-SLYDYDLPF 130
XYL-R LAGPQLYALKGMVKVRLLTMDIAIRDGRFNVEAEWIDSFEVDICRTELGL-MNEPVCWTV 152
FHL-A LCLLPLMSGDTMLGVLKLAQCEEKVFTTTN-LNLLRQIA ERVAIA-VDNALAYQEIH 354
NTR-C DIRMPGMDGLALLKQIKQRHPMLPVIIMTA-HSDLDAAVSAYQQGAFDYLPKPFDIDEAV 112
NIF-A IAVPLMGPHSRPIGVLAAHAMARQEERLPA-CTRFLETV ANLIAQ-TIRLMILPTSA 185
XYL-R LGYASGYGSAFMGRRIIFQETSCRGCGDDKCLIVGKTA EEWGDVSSFEAYFKSDPI- 208
FHL-A RLKERLVDENLALTEQLN-NVDSEFGEIIGRSEAMYSVLKQVEMVAQSDSTVLILGET 411
NTR-C ALVERAISHYQEQQQPRNVQLNGPTTDIIAKP-AMQDVFRIIGRLSRSSISVLINGES 169
NIF-A AQAPQQSPRIERPRACTP-SRGFGLENMVGKSPAMRQIMDIIRQVSRWDTTVLVRGES 242
XYL-R -VDERYELQTQVANLRNRLK-QYDGQYYG-IGHSPAYKRICETIDKAARGRVSVLLLGET 265
FHL-A GTGKELIARAIHNLSGRNNRRMVKMNCAAMPAGLLESDLFGHERGAFTGASAQRIGRFEL 471
NTR-C GTGKELVAHALHRHSPRAKAPFIALNMAAIPKDLIESELFGHEKGAFTGANTIRQGRFEQ 229
NIF-A GTGKELIANAIHHNSPRAAAAFVKFNCAALPDNLLESELFGHEKGAFTGAVRQRKGRFEL 302
XYL-R GVGKEVIARSVHLRSERAEQPFVAVNCAAIPPDLIESELFGVDKGAYTGAVNARAGRFER 325
FHL-A ADKSSLFLDEVGDMPLELQPKLLRVLQEQEFERLGSNKIIQTDVRLIAATNRDLKKMVAD 531
NTR-C ADGGTLFLDEIGDMPLDVQTRLLRVLADGQFYRVGGYAPVKVDVRIIAATHQNLEQRVQE 289
NIF-A ADGGTLFLDEIGESSASFQAKLLRILQEGEMERVGGDETLRVNVRIIAATNRHLEEEVRL 362
XYL-R ANGGTIFLDEVIELTPRAQATLLRVLQEGELERVGGDRTRKVDVRLITATNENLEEAVKM 385
FHL-A REFRSDLYYRLNVFPIHLPPLRERPEDIPLLAKAFTFKIARRLGRNIDSIPAETLRTLSN 591
NTR-C GKFREDLFHRLNVIRVHLPPLRERREDIPRLARHFLQVAARELGVEAKLLHPETEAALTR 349
NIF-A GHFREDLYYRLNVMPIALPPLRERQEDIAELAHFLVRKIAHSQGRTL-RISDGAIRLLME 421
XYL-R GRFRADLFFRLNVFPVHIPPLRERVEDIPLLVEHFLRRHHKEYGKKTLGLSDRAMEACLH 445
FHL-A MEWPGNVRELENVIERAVLLTRG-NVLQLSLPDIVLPE-PETPPAATWALE-GEDEY 646
NTR-C LAWPGNVRQLENTCRWLTVMAAGQEVLIQDLPGELFES-TVAESTSQMQPDSWATLLA 406
NIF-A YSWPGNVRELENCLERSAVLSES-GLIDRDVILFNHRDNPPKALASSGPAED-G 473
XYL-R YQWPGNIRELENALERGVILTES-NESINVESLFPG-LATATEGDRLSSE-GRLEEES 500
FHL-A QLIVRVLKETNGWAG PKGAAQRLGLKRTTLLSRMKRLG 685
NTR-C QWADRALRSGHQNLLSEAQPELERTLLTTALRHTQGHRUEAARLLGWGRNTLTKKLKELG 466
NIF-A -WLDNSLDERQRLIAALEKAGWV OMAAKL LGMTFRD VA Y R10IMD 518
XYL-R GDSWFRQIIDQGVSLEDLEAGLMRTAMDRCGQN1SQAAKLLGLTRPAMAYRLKKLD 556
FHL-A IDKSALI* 692
NTR-C ME* 468
NIF-A ITMPRL* 524
XYL-R PSLSVKAMGR* 566
Figure 10. Alignment of the predicted sequences of E. coli FHL-A, E.
coli NTR-C, K. pneumoniae NIF-A, and P. putida XYL-R proteins.
Identical and functionally similar amino acids are highlighted.
Underlined regions represent those residues of which two out of the
three aligined amino acids are homologous to FHL-A protein. The helix-
turn-helix motif is double underlined.

86
Based on the sequence homology with these proteins, the FHL-A
protein can be divided into 4 regions which are interspersed with
segments of variable length. Region I is unique to FHL-A and includes
the first 353 amino acids. Similarly, NIF-A, FLB-D, LEV-R and TYR-R are
composed of a unique region at the amino-terminus. This singular region
has been thoroughly analyzed in the NIF-A protein which is synthesized
in an active form (21, 31, 63). In K. pneumoniae, the amino-terminus of
the NIF-A protein is presumed to be the site of direct interaction with
NIF-L (58). The NIFL/NIF-A complex is inactive in the presence of 02
and fixed nitrogen (93, 153).
The N-terminal domain of NTR-C and DCT-D, however, demonstrate
significant homology to other pleiotropic control proteins which have
been shown or are presumed to be phosphorylated by a sensor at an
exposed aspartate residue (91, 101, 120, 161, 162, 240). The 5.
typhimurium CHE-Y protein is included in this class of phosphorylated
regulators. The three-dimensional structure of purified CHE-Y protein
has been resolved to 0.27 nm. It was found to be composed of a central
hydrophobic core of five parallel p-sheets surrounded by five a-helices
(221). Comparison of amino acid sequence suggested that this N-terminal
secondary structure is conserved among the phosphorylated receivers.
Stock et al. (222) reported that the highly conserved aspartate and
lysine residues of these proteins are clustered at the carboxy-terminal
end of the p-sheets 1,3 and 5. Furthermore, these conserved residues
are the active site of the transcriptional regulator. Based on the

87
sequence, the FHL-A protein does not have this domain and thus may not
undergo phosphorylation. The FHL-A region I probably determines the
specific regulatory function for this protein in response to FHL
inducers (i.e. formate and anaerobiosis).
The next region of about 24 amino acids, (region II, 356-374 amino
acids) aligns with the proposed "interdomain linker" of both NTR-C and
NIF-A proteins (58). The high content of glutamine (Q), serine (S),
glutamic acid (E), arginine (R) and proline (P) residues, 55% in NTR-C
and 74% in NIF-A, suggests a coil or turn conformation in the protein
structure. This could hypothetically link the region in the N-terminus
to the central ATP-binding region (discussed below). In FHL-A protein,
the extent of these residues is apparently random (26%) suggesting an
alternative secondary structure in this region.
The region III is about 240 amino acids (positions 380 to 619) and
is highly conserved among transcriptional activators of the two-
component regulatory systems which are proposed to interact at o54-
dependent promoters (80). Alignment with FHL-A protein is presented in
decreasing order of homology: NIF-A (53%), FLB-D (48%), NTR-C (47%),
XYL-R (46%), DCT-D (43%), and TYR-R (39%; Fig. 11). This region is
analogous to the "domain D" of the NIF-A and NTR-C proteins (58). The
boxed residues are potential ATP-binding sites (53, 67, 229). Current
evidence suggests that the activator protein binds to the o54-RNA
polymerase holoenzyme via this domain and catalyzes the ATP-dependent
isomerization of closed to open transcriptional complex (174). Non-

88
FHLA EIIGRSEAMYSVLKQVEMVAQSDSTVLIL
NIFA NMVGKSPAMRQIMDIIRQVSRWDTTVLVR
FLBD PMVVRDPAHEQVIKLADQVAPSEASILIT
NTRC DIIAK-PAMQDVFRIIGRLSRSSISVLIN
XYLR G-IGHSPAYKRICETIDKAARGRVSVLLL
DCTD -LIGQTPVMENLRNILRHIADTDVDVLVA
TYRR QIVAVSPKHKHVVEQAQKLAMLSAPLLIT
GETGTGKEl LIARAIHNLSGRNNRRMVKMNCA
GESGTGKEj LIANAIHHNSPRAAAAFVKFNCA
GESGSGKE| VMARYVHGKSRRAKAPFISVNCA
GESGTGKEI LVAHALHRHSPRAKAPFIALNMA
GETGVGKE j VIARSVHLRSERAEQPFVAVNCA
GETGSGKE| VVAQILHQWSHRRKGN FVALNCG
GDTGTGKO| LFAYACHQASPRAGKPYLALNCA
I
FHLA
NIFA
FLBD
NTRC
XYLR
DCTD
TYRR
AM PAG LLESDLFGHERGAFTGASAQRIGRFEL
ALPDNLLESELFGHEKGAFTGAVRQRKGRFEL
AIPENLLESELFGHEKGAFTGAMARRIGKFEE
AIPKDLIESELFGHEKGAFTGANTIRQGRFEQ
AIPPDLIESELFGVDKGAYTGAVNARAGRFER
ALPETVIESELFGHERGAFTGAQKRRTGRIEH
SIPEDAVESELFGH APEGKKGFFEQ
I 1
ADKSSLFLDE
ADGGTLFLDE
ADGGTLLLDE
ADGGTLFLDE
ANGGTIFLDE
ASGGTLFLDE
ANGGSVLLDE
I I
VGDMPLELQPKLLRVLQE
IGESSASFQAKLLRILQE
ISEMDVRLQAKLLRAIQE
IGDMPLDVQTRLLRVLAD
VIELTPRAQATLLRVLQE
IESMPAATQV KM LRVLEM
IGEMS PRMQAKLLRF LND
FHLA QEFERLGSNKIIQTDVRLIAATNRDLKKMVADREFRSDLYYRLNVFPIHLPPLRERPEDIPLLA
NIFA GEMERVGGDETLRVNVRIIAATNRHLEEEVRLGHFREDLYYRLNVMPIALPPLRERQEDIAELA
FLBD REIDRVGGSKPVKVNIRILATSNRDLAQAVKDGTFREDLLYRLNVVNLRLPPLRERPADVISLC
NTRC GQFYRVGGYAPVKVDVRIIAATHQNLEQRVQEGKFREDLFHRLNVIRVHLPPLRERREDIPRLA
XYLR GELERVGGDRTRKVDVRLITATNENLEEAVKMGRFRADLFFRLNVFPVHIPPLRERVEDIPLLV
DCTD REITPLGTNEVRPVNLRVVAAAKIDLGDPAVRGDFREDLYYRLNVVTISIPPLRERRDOIPLLF
TYRR GTFRRVGEDHEVHVDVRVICATQKNLVELVQKGMFREDLYYRLNVLTLNLPPLRDCPQDIMPLT
FHLA KAFT FKIARRLGRNIDSIPAETLRT LSNMEWPGNVRELENVIERAVLLTRG-NVLQL
NIFA HFLVRKIAHSQGRTL-RISDGAIRLLMEYSWPGNVRELENCLERSAVLSES-GLIDR
FLBD EFFVKKYSAANGIEEKPISAEAKRRLIAHRWPGNVRELENAMHRAVLLSAG-PEIEE
NTRC RHFLQVAARELGVEAKLLHPETEAALTRLAWPGNVRQLENTCRWLTVMAAGQEVLIQ
XYLR EHFLRRHHKEYGKKTLGLSDRAMEACLHYQWPGNIRELENALERGVILTES-N--ES
DCTD SHFAARAAERFRRDVPPLSPDVRRHLASHTWPGNVRELSHYAERVVLGVEG-GGAAA
TYRR ELFVARFADEQGVPRPKLAADLNTVLTRYAWPGNVRQLKNAIYRALTQL-DGYEL
Figure 11. Alignment of the predicted "domain D" amino acid sequences
of E. coli FHL-A, K. pneumoniae NIF-A, C. crescentus FLB-D, E. coli NTR-
C, P. putida XYL-R, R. leguminosarum DCT-D, and E. coli TYR-R.
Highlighted residues are identical or functionally similar to the FHL-A
protein. Double underlined regions represent those residues of which
five out of the six aligned amino acids are homologous to FHL-A protein.
Boxed areas are the proposed ATP-binding regions.

89
polar mutations in "domain D" of NTR-C abolish the positive (not
negative) control of the protein (147). Deletions in NIF-A suggest that
only this central domain is necessary for transcriptional activation at
the nif promoters (99). However, the other NIF-A domains are probably
essential for specific regulation. Due to the high degree of homology,
the central region of the FHL-A protein probably serves a similar
function.
The final region IV (amino acids 663 to 682) of 20 amino acids is
the suggested DNA binding domain (helix-turn-helix; ref. 39, 165) of the
receiver proteins (58, 222). Both NTR-C and NIF-A have been shown to
bind to upstream promoter sites by DNA footprinting (158, 179). This
final segment of FHL-A protein is 50%, 45%, 40% and 30% homologous with
the HYD-G, NTR-C, XYL-R and NIF-A proteins respectively (Fig. 12).
Critical amino acids, Ala-5, Gly-9, Leu-15 and hydrophobic amino acids
at positions 4, 8 and 10 (165) can be detected starting at position 663
(PKGAA QRLGL KRTTL LSRMK). This segment of the protein is crucial for
the biological activity of the FHL-A protein. Inserting transposon Tn5
between the amino acids 662 and 663 (between the DNA bases G and G at
positions 4404 and 4405 in plasmid pSE-133-2; Fig. 7) completely
abolished the formate dependent expression of lacZ*). Thus,
the helix-turn-helix motif is necessary in vivo for specific activation
of genes with FHL-A binding sites, potentially the UAS described by
Birkmann and Bock (23). The R. meliloti NIF-A studies suggest that the
C-terminal domain can be deleted without abolishing in vivo activation;

90
i
4 5 8 9 10
15 18 20
FHL-A
663
HYD-G
417
NTR-C
444
XYL-R
534
NIF-A
496
Pro-Lys-Gly-Ala-Ala-Gln-Arg-Leu-Gly-Leu-Lys-Arg-Thr-Thr-Leu-Leu-Ser-Arg-Met-LyB
Lys-Thr-Glu-Ala-Ala-Arg-Gln-Leu-Gly-Ile-Thr-Arg-Lys-Thr-Leu-Leu-Ala-LyB-Leu-Ser
Lys-Thr-Glu-Ala-Ala-Arg-Leu-I.eu-Gly-Trp-Gly-Arg-Asn-Thr-Leu-Thr-Arg-Lys-Leu-I.yB
Ile-Ser-Gln-Ala-Ala-Arg-Leu-Leu-Gly-Leu-Thr-Arg-Pro-Ala-Met-Ala-Tyr-Arg-Leu-Lys
Gln-Ala-Lys-Ala-Ala-Arg-Leu-Leu-Gly-Met-Thr-Pro-Arg-Gln-Val-Ala-Tyr-Arg-Ile-Gln
Figure 12. Alignment of potential DNA-binding motifs in the FHL-A, HYD-
G, NTR-C, XYL-R and NIF-A proteins. Identical and functionally similar
amino acids are highlighted. Critical amino acids for helix-turn-helix
formation are indicated.

91
however, binding site specificity may be determined by this region,
since R. meliloti nifH promoter activation was reduced 2-fold in E. coli
and 10-fold in R. meliloti (99). Likewise, mutations in the helix-turn-
helix region eliminate NTR-C repressive function in which binding to a
specific operator sequence is required (147).
Primary structure of the hydX'FB [hypC DE) genes upstream of the
fhlA gene. Amino acid sequence data of the FHL-A protein suggests that
a typical "sensor" protein (histidine-protein-kinase) is probably not
required for FHL-A activity. However, the possibility of a nearby
region encoding a sensor protein can not be ruled out. For example, the
K. pneumoniae nifLA operon encodes both the activator (NIF-A) and its
repressor (NIF-L). Sankar and Shanmugam (187, 188) described two genes,
hydF and hydB, which were upstream of the fhlA gene. Based on genetic
analysis, the 44 kDa and 32 kDa polypeptides coded by the hydF and hydB
genes, respectively, appeared to interact with the fhlA gene product.
Partial hydF gene products were capable of restoring total hydrogenase
activity but only in the presence of multiple copies of fhlA gene (188).
There appeared to be a direct correlation between the size of the
overexpressed, partial HYD-F protein and the level of hydrogenase
activity produced by the hydF mutants, strains SE-65 and SE-67.
Additionally, the hydB gene appeared to substitute for the hydF gene in
specific genetic backgrounds (SE-203 hydF, x~). This suggested that
hydF, hydB, fhlA, and an unidentified gene product could potentially
interact in the production of FHL activity.

92
Therefore, the upstream DNA was sequenced in order to identify the
characteristics of the hydF and hydB genes and their products. The
sequence of a partial gene was also determined since it was present in
the plasmid used for these experiments. Sequence analysis revealed that
the hyd genes were transcribed in the same orientation as the fhlA gene
(Fig. 9). The first open reading frame (bases 1 to 216) encodes the
partial hypC product (3' end). The 72 amino acid residues identified
have an anhydrous molecular weight of 7,848 Da. The second open reading
frame, positions 219 to 1337, comprises the hydF gene. The gene codes
for a protein of 373 amino acids with an anhydrous molecular weight of
41,363 Da which is comparable to the apparent molecular weight of
44,000 Da obtained by "maxicell" experiments (188). Positions 1379 to
2344 constitute the hydB gene which codes for a 322 amino acids product
of an anhydrous molecular weight of 33,712 Da which again is comparable
to the apparent molecular weight obtained by Sankar and Shanmugam (187).
A typical "-10 and -35" or "-12 and -24" promoter sequence was not
evident (between positions 1 to 2311); however, "Shine-Dalgarno"
consensus sequences (positions 208 to 212 and 1367 to 1371) were located
in front of both complete genes (hydF and hydB) sequenced. A 38 base
pair intergenic region was located between the hydF and hydB genes. An
inverted repeat (underlined in Fig. 9; positions 2387 to 2393 and
positions 2397 to 2403) that can produce a 7-base-pairs stem and a 3-
base (2394 to 2396) loop structure followed the hydB open reading frame.
This region is not followed by a thymine residue stretch of significant

93
length, but the 17 base pairs region between this secondary structure
and the start codon of the fhlA gene is 65% AT rich. Whether this is
physiologically important has not been determined. The hydB gene
product was extremely hydrophobic with a hydrophilic region in the
amino-terminus. No significant homology of any hyd {hyp) genes
sequenced could be found with known sequences in the GenBank or EMBL
data bases and thus the potential mechanism by which these proteins
participate in H2 metabolism cannot be deduced.

94
Regulation of the fhlA Gene
Localization of an fhlA gene fusion. In order to study the
regulation of fhlA gene, mutant strain SE-2007 was isolated as a lac
operon fusion derivative of strain MC4100, using AplacMu53 (29). Upon
detailed analysis, this strain was found to be defective in the
production of dihydrogen. Plasmid complementation studies suggested
that the fusion was located in the fhlA gene. The FHL activity of
strain SE-2007 was restored by plasmids pSE-133 and pSE-133-1
[hydB::Tn5) but not by pSE133-2 [fhlA::Tn5). Construction of a Hfr
PO(fhlA) suggested the fusion mapped between the srl (58 min) and cys
(59 min) genes; however, chromosomal transfer implied that direction of
transcription was in the opposite orientation from that of the DNA
sequence data. To eliminate the possibility of fhlA gene in high copy
suppressing a mutation in a different gene in the 58 to 59 min region,
restriction endonuclease digests of chromosomal DNA from strain SE-2007
were probed with an internal Sail to Pst I fragment of the fhlA gene
(2,946 to 4,269 bases; Fig. 9). Initial results with Ca I "digests" of
chromosomal DNA from strain SE-2007 demonstrated that the lac fusion was
within the Ca I fragment (positions 800 to 4601; Fig. 9); the
hybridizing fragments were approximately 3.7 kb (strain MC4100) and
7.5 kb (strain SE-2007). Further analysis with Sall-Bgll, Sall-EcoRl,
and SoZI-CZoI digests of chromosomal DNA verified that the fusion was
within the fhlA gene between the Pstl and Bgll sites at the 3' end of

95
the open reading frame (positions 4,269 and 4,528 in Fig. 9; Fig. 13).
This again reemphasizes the necessity of the carboxy-terminus for
biological activity of the FHL-A protein.
Anaerobic induction of the fhlA gene. Regulation of expression of
the fhlA gene was monitored by measuring the levels of p-galactosidase
activity produced by strain SE-2007. When cultured under comparable
aerobic conditions to mutant strain SE-2011, maximal dioxygen repression
of fhlA gene expression was not obtained. Therefore, alternate
procedures were implemented to maximally aerate the culture at low cell
density (Table 7).
When cultured under microaerobic conditions, an overnight,
stationary (1 ml in 13 x 100 mm tube with a metal-cap) culture of strain
SE-2007 produced about 1,800 U of p-galactosidase activity (Table 7).
Upon transfer to "strict" aerobic conditions (10 ml LB in 125 ml flask
with 0.1 ml of overnight culture as inoculum; shaking at 250 rpm; 2 hr),
utilized routinely for culturing other laboratory strains, the p-
galactosidase activity of the culture decreased approximately 2-fold to
about 1,000 units. Subsequent transfer of this culture to fresh LB and
then aerobic growth for 1 hr further reduced the specific activity of
the enzyme over 3-fold (290 U). This aerobic culture was used to
inoculate LB and grown under anaerobic conditions. After 4 hr
incubation, the specific activity of pgal actos i dase of the culture was
comparable to initial "microaerobic" values. These results indicate

96
"-24, -12"
SPB
FNR
S CKB
r
"-35,-10"
PC
h-H h
-HH
H H
hyp A B C
D E
fhlA
hydE
F B
lac
SE-2007
V
SE-2001 *
SE-2002 V
Figure 13. Localization of the promoter lac fusions in strains SE-2007
WfhlA'-'lacZ*)], SE2001 [fhyp'-
,lacZ+)2]. The complete hypABCDE operon with the proposed a- and FNR-
dependent promoters has been redrawn with modifications from Lutz et al.
(144). Previously identified hydE, hydF and hydB genes are noted (35,
187, 188, 238). Additionally, the weak "-35 and -10" constitutive
promoter upstream of the fhlA gene, suggested in this study, is also
indicated.

97
Table 7. Effect of dioxygen on the expression of Q(fhlA'-'lacZ*) in
an fhlA mutant, strain SE-2007
Conditions
Innoculum
size
Incubation
time
p-Galactos i dase Activity3
Microaerobic
16 hr
1,800
Aerobic-1
1%
2 hr
990
Aerobic-2
1%
1 hr
290
Anaerobic
10%
4 hr
1,800
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
All cultures were grown at 37C in Luria Broth.
An overnight stationary (1 ml in 13 x 100 mm metal-cap tube) LB culture of
strain SE-2007 served as the inoculum for this experiment. This culture
is referred above as the microaerobic culture. During the first part, 0.1
ml of this culture was inoculated into 10 ml LB (125 ml flask) and shaken
at 250 rpm. After 2 hr of growth, 0.3 ml of this aerobic culture was
transferred to 30 ml of fresh LB (1 L flask) and grown in the shaker for
1 hr. This culture was used to inoculate LB at 10% (v/v) and grown under
anaerobic conditions. p-Galactosidase activities of all the cultures at
the time of transfer to fresh medium (except the anaerobic culture, which
was harvested after 4 hr growth) are presented in the Table.

98
that the fhlA gene is maximally induced at a higher oxygen tension in
the growth medium than the fhlB gene.
Constitutive fhlA gene expression under anaerobiosis. Anaerobic
expression of the fhlA gene was tested in LB medium supplemented with
various effectors of the FHL pathway. Addition of alternate electron
acceptors (nitrate and fumarate) did not influence the transcription of
the fhlA gene (Table 8). Lower levels of p-galactosidase activity
observed in cells grown in LB + nitrite medium may be a consequence of
growth inhibition by nitrite. It is interesting to note that the
exogenous formate and glucose did reduce the fhlA operon expression.
LBG medium buffered with 0.1 M sodium phosphate to pH 7.0 reversed the
effect of glucose on the levels of p-galactosidase produced by the
culture. Additional experiments were done to verify that pH had no
effect on fhlA gene expression using buffers ranging from pH 6.0 to 7.5
(0.1 M MES, pH 6.0; 0.1 M PIPES, pH 6.5; 0.1 M MOPS, pH 7.0; 0.1 M TES,
pH 7.5). Again the results were comparable; low pH did not enhance fhlA
gene transcription (data not shown).
Several genes known to be required for dihydrogen production,
including rpoN, narL, fnr, cya, molR, chlD and p/Z, were tested for
their requirement in transcription of (//?ZA'-' ZocZ4-). Appropriate
double-mutant strains were constructed to investigate the fhlA gene
regulation. Analysis of these strains suggested that fhlA transcription
is independent of known regulators of formate hydrogenlyase production
(Table 9). The only double-mutant strain exhibiting reduced levels of

99
Table 8. Effect of media composition on the expression
of Q(fhlA'-'lacZ*) in an fhlA mutant, strain SE-2007
Medium
p-Galactosidase
Activity3
Luria Broth
1,800
+ Formate (0.5%)
1,300
o

fH
i
CO
o
+
1,700
+ N02" (1.0%)
1,200
+ Glucose (0.3%)
1,200
+ Glucose + buffer13
2,000
+ Fumarate (0.5%)
1,700
All cultures were grown anaerobically at 37C in Luria Broth
supplements.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
b0.1M Phosphate buffer at pH 7.0.

Table 9. Expression of {fhlA'-' lacZ+) in different
genetic backgrounds
Strain
Relevant genotype
pGal actos i dase activity3
SE-2007
<^{fhlA,-lacZ+)
1,800
MJ101
${fhlA'-'lacZf) rpoN
2,100
MJ102
lacZ+) narL
1,200
MJ103
[fhlA'-' lacZ*) fnr
1,600
MJ107
Q(fhlA'-'lacZ*) cya
.Q
O
O
CO
MJ-108
QifhlA'-'lacZ+) molR
1,400
MJ-109
*{fhlA'-'lacZ+) pfl
2,300
SE-2007
HfhlA'-'lacZ+)
650/680
SE1762
*(fhlA'-'lacZ+) chlD
830/800
All cultures were grown for 4 hr anaerobically at 37C in Luria Broth
unless otherwise indicated.
aExpresssed as nanomoles of o-nitrophenol produced per minute per
milligram of protein.
bPoor cell growth.
independent experiment in which cells were grown in LB/and LB
supplemented with 1 mM Mo.

101
p-galactosidase activity is MJ107, (fhlAlacZ*), cya. This 2-fold
decrease in gene expression is probably due to the poor growth of the
culture; however, a cAMP effect cannot be ruled out at this point.
Localization of hyd gene fusions. It has recently been suggested
that the fhlA gene is regulated by the upstream a54- and FNR-dependent
promoters of the hyd [hyp) operon (144; Fig. 13). In order to
investigate this further, hyd gene fusions within this operon were
studied to compare transcriptional regulation. Mutant strains SE-2001,
SE-2002 and SE-2009 were similarly isolated as lac operon fusion
derivatives of strain MC4100, using ApiocMu53 (29). Biochemical
analysis revealed that strains SE-2001 and SE-2002 were deficient in all
three hydrogenase activities. Mutant strain SE-2009 was also
significantly affected in total hydrogenase activity, however basal
levels of hydrogenase were detectable. Therefore, these mutants were of
interest in studying FHL regulation since they were phenotypically
comparable to previously described hyd mutants.
Plasmid complementation analysis suggested that mutant strains SE-
2001 and SE-2002 were located in the 58-59 min hyd cluster. Plasmid
pSE-111 restored all three hydrogenase activities to parental levels in
strains SE-2001 and SE-2002. Plasmid pSE-130 complemented only to
approximately 35% of FHL activity in both mutants; whereas, HUP activity
was restored to 20% and 100% in strains SE-2001 and SE-2002,
respectively. Plasmids pSE-128 and pSE-125 did not complement the
mutation in either strain SE-2001 or SE-2002. Evidently, the complete

102
hyd {hyp) operon (59 min) is required for restoration of hydrogenase
activity in these fusion strains. Mutant strain SE-2009 was not
complemented for hydrogenase activity with any of the plasmids tested.
However, 250 pM NiC12 supplementation of the growth medium restored FHL
and HUP (H2 to BV) activities to wild type level. Phage Pl-transduction
experiments confirmed that the hyd mutations in strains SE2001 and SE-
2002 were located between the srl (58 min) and cys (59 min) genes.
However, the lac fusion in strain SE-2009 was not cotransducible within
this region. Mutant strain SE-2009 is phenotypically comparable to
strain HYD723, <\>{hydC'-' lacZ+), described by Wu et al. (239) which maps
at 77.6 min in the E. coli chromosome.
DNA hybridization experiments localized lac gene fusions in
strains SE-2001 and SE-2002 to a 0.9 kb Bgll-Sall fragment which
contains the partial hydE (hypB) coding region (360 amino acids) and
partial coding region of hypC {hydX) gene (20 amino acids) as
determined by Lutz et al. (144; Fig. 13). Neither mutant was suppressed
by nickel supplementation in the growth medium as in the case of point
mutations in hydE gene. This could be explained by the polar effect of
the fusions on the other hyd {hyp) genes whose products are also
essential for production of active hydrogenase.
Genetic regulation of the hyd {hyp) operon. Regulation of
expression of the hyd {hyp) operon was monitored by measuring the levels
of p-galactosidase activity produced by strains SE-2001 and SE-2002.
When cultured under strict aerobic conditions, both strains produced

103
about 20 U of pgal actos i dase activity. Upon transfer to anaerobic
conditions, hyd (hyp) operon expression was induced at a rate which
paralleled the growth of the culture in LB medium. The specific
activity of the enzyme reached maximum values of 170 U for SE2001 and
190 U for SE-2002 (LB; Table 10). This peak value was reached during
early stationary phase of growth (approximately 0.03 units A420nm or 10
pg cell protein) and remained constant throughout the remaining assay
period (5 hr total). Thus, the increase in specific activity of the
enzyme was coupled to growth (differential rate of induction approaching
1.0). In LB supplemented with glucose, formate or nitrate, the maximal
pgal actos i dase activity produced by strain SE2001 was not
significantly altered. The enzyme activity produced by strain SE-2002
also remained relatively constant in cells grown in LBG or LBN medium.
In medium supplemented with 15 mM formate, the enhancing effect was only
observed in strain SE-2002 in which the enzyme activity was slightly
higher. These results are in direct contrast to the formate
inducibility of the fhlB operon in which the levels of p-galactosidase
activity were increased by 3.5-fold in the presence of 15 mM formate,
and endogenously produced formate also had an enhancing effect.
To study the role of known effectors of dihydrogen metabolism,
appropriate double mutants were constructed and p-galactosidase activity
was monitored over a 5 hr time period. The only mutation which was
consistent in its effect on hyd (hyp) operon expression was fnr. The
fnr derivatives of strains SE-2001 and SE-2002 are strains SE-1651 and

104
Table 10. Anaerobic expression of (hyp'-' lacZ*'), strains SE-2001 and
SE-2002, in different genetic backgrounds and culture media
Strain
Relevant genotype
LBb
p-Galactosidase activity3
LBGb LBFb LBNb
SE-2001
(hyp'~
1lacZ*)1
170
170
210
150
SE1654
4'{hyp1-
1 lacZr+) 1 narL
240
240
300
200
SE1651
4>(/?yp'-
' lacZf)1 fnr
50
130
130
43
SE1657
4 {hyp'-
'lacZ*)1 rpoN
53
135
160
130
SE1658
4>(byp'-
' lacZ+)1 rpoN
250
125
130
105
SE1760
4>(/?yp'-
'lacZ+)1 chlD
135/189
NDd
ND
ND
SE-2002
4>(/?yp'-
'lacZ+)2
190
210
290
220
SE1655
<1'{hyp'-
'ZocZ+)2 narL
100
145
160
95
SE1652
4 {hyp1-
'lacZ+)2 fnr
22
140
120
50
SE1660
(hyp'~
'lacZ+)2 rpoN
125
130
135
135
SE1659
4'(hyp'-
llacZ+)2 rpoN
140
135
150
130
SE1761
4>(/?yp'-
1lacZ+)2 chlD
135/202
ND
ND
ND
aExpresssed as nanomoles of o-nitrophenol produced per minute per
milligram of protein
bLB, Luria Broth; LBG, supplemented with 0.3% glucose; LBF, supplemented
with 0.1% formate; LBN, supplemented with 0.1% nitrate.
CLB grown culture/ LB supplemented with 1 mM Mo.

105
SE1652, respectively. In LB with and without nitrate supplementation,
the maximum (J-galactosidase activity observed in these cultures was 50 U
which is a 3- to 4-fold reduction in activity from the parent hyd (hyp)
strains. Further analysis of these fnr double mutants, suggested that
the FNR-dependent induction of the hyd (hyp) operon was alleviated upon
formate or glucose supplementation (Table 10). When strains SE-1651 and
SE1652 were inoculated from microaerobic (1 ml standing LB cultures) to
anaerobic conditions and cultured for 4 hr, the maximum activity of the
cultures was comparable to activity in the presence of formate (data not
shown). Perhaps the formate or FHL-A protein produced during the period
of microaerobic conditions was sufficient for activation of the FNR-
independent promoter(s) of the hyd (hyp) operon. The rpoN gene product
was only required for anaerobic induction in one of the double mutants
tested, strain SE1657. The activity produced by this culture was
reduced 3-fold from parental values, but only in LB medium. The chlD
and narL mutations had no apparent effect on hyd (hyp) operon
expression. Additionally, nickel supplementation (250 pM) had no
influence which is in contrast to the repressive effect in strain SE-
2009, the hyd mutant which is not located in this 58-59 min region. It
would be interesting to test whether a hyd (58 min) mutation, presumed
to be necessary for nickel processing, would alleviate the nickel
repression observed in strain SE-2009. Potentially, a protein-bound
form of nickel would exert this negative effect on transcription.
These results are comparable to the DNA sequence data which

106
suggests multiple promoters for the hyp operon (FNR- and o54-dependent
promoters (144). The FNR-dependent promoter within the hypA gene is
apparently transcribed when formate concentrations are low as measured
by p-galactosidase activity of strains SE-1651 and SE-1652 grown in LB
medium. Formate supplementation alleviates the FNR requirement,
suggesting transcription from FNR-independent promoter(s) in later
stages of growth. The p-galactosidase produced by strain SE-1657 in LB
medium, similarly, suggests a role for a54 in hyp operon expression.
Hybridization studies to hyp operon mRNA suggest that the fhlA gene is
cotranscribed from the hyp promoters (A. Bock, personal communication).
This is in contrast to the genetic regulatory pattern observed in strain
SE-2007, HfhlA'-'lacZ*).
Two possibilities could mask the true physiological regulation of
the fhlA gene and hyd [hyp) operon from the data presented above
(strains SE-2007, SE2001 and SE-2002). First, it is possible that the
fhlA gene is autoregulated. The introduction of a gene fusion in the
fhlA gene would inactivate its product, as seen phenotypically in strain
SE-2007 (FHL activity). If FHL-A protein is required for regulation of
fhlA gene transcription, the p-galactosidase values obtained for strain
SE-2007 would not reflect this FHL-A requirement. Secondly, the fhlA
gene could be transcribed from the hyd [hyp) promoter(s). The polarity
of the hyd [hyp) mutations of strains SE-2001 and SE-2002 would inhibit
fhlA gene transcription if hyp promoters were indeed the normal
physiological promoters. Thus, if FHL-A is cotranscribed and is

107
required for formate inducibility of the hyd [hyp) operon, as is the
case for fhlB operon expression, the increase would be absent in strains
SE-2001 and SE-2002. Plasmid pSE-133, a Clal insert retaining the
partial hydF and complete hydB, fhlA genes, complements the fhlA mutants
(SE-1174 and SE2007). Even though the hyd [hyp) promoter(s) are absent
in this plasmid, it is conceivable that the cloned genes are transcribed
from a pBR-322 vector promoter.
Effect of multiple copies of the fhlA gene on fhlA and hyd [hyp)
transcription. In order to study these possibilities, the effect of
multiple copies of the fhlA gene on its own expression and transcription
of the hyd [hyp) operon were monitored using plasmid pSE-133 (Fig. 14).
The p-galactosidase activity of strains SE-2007 plasmid pSE-133 was
comparable, irregardless of the presence of high copy fhlA+ or medium
composition (Table 9). Interestingly, the fhlA gene is induced at very
early stages of the organism's anaerobic growth to approximately 500 U.
Then during late-log phase, a second induction occurs at a lower rate to
a maximum value of about 1,500 U. The FHL-A protein would be available
initially to activate the FHL pathway as formate is produced by the
organism. In later stages of growth, formate levels would be higher (pH
lower) and therefore, FHL-A protein would be necessary for maximum
activation of the FHL system. However, transcription of the fhlA gene
does not require formate as an inducer (Table 9).
From the data presented (Fig. 14), the hyd [hyp) operon expression
differs from the fhlA gene regulation. In the presence of multiple

108
PROTEIN (ng)
Figure 14. Differential rate of synthesis of 4>(fhlA-' lacZ*), strain SE-
2007, and (hypX'- lacZ*) 1, strain SE-2001, in LB medium supplemented
with 3 mM formate in the presence and absence of the fhlA+ gene in a
multicopy plasmid (pSE-133). p-Galactosidase activities and protein
concentrations are expressed as units per ml and pg per ml,
respectively.

109
copies of fhlA+ gene (pSE-133) and frmate (3 mM), the p-galactosidase
activity produced by strain SE-2001 is increased by 2-fold. This would
suggest that the fhlA gene is independent of the hyd {hyp) promoter(s)
and is transcribed from its own "-35 and -10" o70 promoter. The
increased production of p-galactosidase activity in the SE2001/pSE133
culture is probably due to the unnaturally high concentration of FHL-A
protein available in the cytoplasm. These results also suggest that the
putative transcription termination region observed between the hydB and
fhlA genes (Fig. 9) is physiologically significant.

110
Molybdate Metabolism and FHL Activity
CHL-D and MOL-R proteins are required for fhlB operon expression.
As was discussed earlier, both the chlD and molR gene products are
necessary for fhlB operon expression (Table 6). Further analysis was
done on the chlD and molR derivatives of strain SE-2011, strains SE1714
and MJ-40 respectively, to better understand this regulation (Table 11).
For these experiments, the strains were grown under anaerobic conditions
in LB-formate medium, and the cells were harvested after 4 hr for enzyme
assays. Under these conditions, a molR or chlD mutation reduced p-
galactosidase activity produced by strain SE-2011 by over 4- to 5-fold.
Supplementing the medium with 1 mM molybdate, restored enzyme levels to
those of the parent, strain SE-2011. In the presence of multiple copies
of the fhlA gene, the specific enzyme activity produced by strains MJ-40
and SE1714 was increased by over 5-fold the activity of strains without
plasmid pSE-133 (fhlA+). However, the enzyme levels of these cultures
(+pSE-133) were approximately 2-fold less than the SE-201l/pSE-133
culture. Molybdate (1 mM) supplementation was still required for peak
fhlB operon expression. These results suggest that the FHL-A mediated
formate inducibility of the fhlB operon and molybdate-mediated
regulation are separate and independent of each other.
Primary structure of the molR gene. Previous experiments
identified the molR gene product as a putative regulatory element of
both the fdhF and ant genes and essential for utilization of molybdate

Ill
Table 11. The effect of molybdenum on expression of (fhlB'-'lacZ+)
mutant, strain SE-2011 in chlD and molR genetic backgrounds
0-Galactosidase Activity3
ImM Mo

+

+
Strain
Relevant genotype pSE133b


+
+
SE-2011
Q(fhlB'-'lacZ*)
700
700
1400
1500
MJ-40
4>(//?Zfl'-' lacZ+) molR
130
640
720
1400
SE1714
(fhlB'-'lacZ*) chlD
160
700
900
1700
All cultures were anaerobically grown at 37C in LB medium supplemented
with 3 mM formate except SE1714 which was grown in 15 mM formate.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
Multicopy plasmid carrying the fhlA+ gene
Values represent maxiumum activity reached during 4 hours of anaerobiosis.

112
by E. coli (134, 136). The physiological data presented above (Table
11) show that the MOL-R protein is needed for optimum induction of the
fhlB operon. Therefore, the DNA sequence of the molR gene was
determined to identify possible conserved motifs in the gene product
(Fig. 15).
Plasmid pSE1009 and exonuclease III generated deletion
derivatives, plasmids 1 through 5 complemented a molR mutant (strain
SE1100) for FHL activity (plasmid derivatives 6 to 13 did not restore
activity). Therefore, derivatives 5 to 13 were initially used for
sequencing, since the complete molR gene was probably encoded by these
plasmids (fxoIII derivatives are identified in Fig. 15). By searching
GenBank and EMBL data bases with the preliminary sequence, it was
determined that the Sou3Al-l,609-base-pair restriction fragment in
pSJE301 which was cloned and sequenced by Johann and Hinton (110) is an
internal fragment of the plasmid pSE-1009 sequence (2,927-base-pair
Kpnl-EcoRV restriction fragment). Because the complete chlJ' and 0RF3'
DNA sequences have not yet been determined and the molR gene is upstream
of the chlD gene, the region sequenced was extended to include the Clal
restriction site of plasmid pSE-1004 (for a total of 3,876-base-pair DNA
sequenced).
Previous experiments have shown that the chlD expression is
repressed by molybdate whereas the molR gene is constituively
transcribed (134, 155). This raises several unanswered questions.
First if the molR gene product is regulatory, as the constitutive nature

113
CCCTGGCTAATGGAACCGGCAACGCAATGTGTGTTTTAGTAGCGAAATCGCGCGCGGTCG 60
CGGAATAATTTTTGTTGGAGCTTAAGGGTGAGAAGGATTTCGGCCTGCATAACAATGTCC 120
T GGCAAAAGTCTT ATT GT GACGGAAAACGAACGCCACGCAAAGCTGACCGCACAAAAGGG 180
GAGTGCTTTTCTGTGCTTAGCGGTTAGAATAGTCTCATGACTATATCTGGAGTTGACCAT 240
Kpnl
GTT AGAGTT ATT AAAAAGT CT GGT ATTCGCCGT AAT CAT GGTACCT GTCGT GAT GGCCAT 300
CATCCTGGGTCTGATTTACGGTCTTGGTGAAGTATTCAACATCTTTTCTGGTGTTGGTAA 360
|£xoIII-l
AAAAGACCAGCCCGGACAAAATCATTGATTCCCTGAATGCCCGCTTAGTCGGGCATTTTC 420
|£xoIII-2
TTTTTCTCAACTTCCTGCTTTTCCTGCCGATATTTTTTCTTATCTACCTCACAAAGGTTA 480
GCAATAACTGCTGGGAAAATTCCGAGTTAGTCGTTATATTGTCGCCTACATAACGTTACA 540
TTAAGGGGTTACCAATGGCTCGTAAATGGTTGAACTTGTTTGCCGGGGCGGCACTCTCTT 600
TCGCTGTTGCTGGCAATGCACTGGCAGATGAAGGGAAAATCACGGTGTTCGCCGCCGCAT 660
CACTGACTAACGCAATGCAGGACATTCTTACGCAGTTTAAAAAAGAGAAAGGCGTGGATG 720
|£xoII1-3
TGGTTTCTTCTTTCGCTTCGTCATCTACTCTCGCCCGTCAGATTGAAGCGGGTGCGCCTG 780
CGGAT CTGTTT ATTT CT GCCGAT CAGAAAT GGAT GGATT AT GCGGTTGAT AAAAAAGCGA 840
TCGATACACGTACGCGTCAGACACTGCTCGGCAATAGCCTGGTCGTTGTAGCACCGAAAG 900
CCAGCGTGCAGAAAGATTTCACCATCGACAGCAAAACCAACTGGACTTCACTGCTGAATG 960
|£xoIII-4
GCGGTCGCCTGGCGGTTGGCGATCCGGAACATGTTCCCGCTGGCATTTATGCAAAAGAAG 1020
CACTGCAAAAACTGGGCGCATGGGATACGCTCTCTCCGAAACTGGCCCCAGCGGAAGATG 1080
TTCGTGGGGCGCTGGCGCTGGTCGAACGTAACGAAGCGCCTCTGGGCATTGTCTACGGTT 1140
CTGACGCAGTTGCCAGCAAAGGGGTAAAAGTGGTTGCCACCTTCCCGGAAGATTCACATA 1200
AAAAAGTGGAATATCCGGTTGCTGTTGTGGAAGGGCATAACAATGCCACAGCGAAACCTT 1260
|£xoIII-5
TTATGATTATCTGAAGGCACCGCAGGCACCCAAATCTTTAAACGTTACGGATTTACAATC 1320
|£xoIII-6
AAGTAATGATACTGACCGATCCAGAATGGCAGGCAGTTTTATTAAGCCTGAAAGTTTCTT 1380
CCCTGGCTGTGCTGTTTAGCCTGCCGTTTGGGATCTTTTTTGCCTGGTTACTGGTGCGTT 1440
GCACGTTTCCGGGCAAAGCTCTGCTCGACAGCGTACTGCATCTACCGCTGGTGTTACCGC 1500
|£xoIII-7
CCGTGGTCGTCGGTTACTTATTATTAGTTTCGATGGGACGGCGCGGATTTATCGGTGAAC 1560
|£xoIII-8
GTCTGTATGACTGGTTTGGTATTACCTTCGCCTTTAGCTGGCGCGGCGCGGTTCTCGCTG 1620
CCGCCGTCATGTCGTTTCCGCTGATGGTGCGGGCAATTCGTCTGGCGCTGGAAGGGGTTG 1680
ATGTCAAACTGGAACAGGCCGCAAGAACACTGGGGGCCGGGCGCTGGCGCGTTTTCTTTA 1740
CTATCACGTTACCGCTGACCTTACCGGGAATTATTGTTGGTACGGTACTGGCTTTTGCTC 1800
Figure 15. Nucleic acid sequence of the mol (chi) operon. Restriction
endonuclease sites for some of the enzymes are highlighted. Exonuclease Ill
generated deletion derivatives, plasmids #1 through 13 are identified.
Translational start sites of 0RF1 and 0RF2 are double underlined (positions 555
to 557 and positions 1326 to 1328, respectively). "Shine-Dalgarno" sequence and
translational start site of the chlD gene are single underlined (positions 2005
to 2009 and positions 2018 to 2020).

114
|£xoIII-9
GTTCTCTCGGTGAGTTTGGTGCAACCATCACCTTTGTGTCGAACATTCCTGGTGAAACGC 1860
|fxoIII-10
GAACCATTCCTTCTGCCATGTATACCCTGATCCAGACCCCCGGCGGGGAAAGTGGAGCGG 1920
CGAGACTGTGCATTATTTCTATTGCGCTGGCGATGATCTCCCTGTTGATTTCAGAATGGC 1980
TGGCCAGAATCAGCCGTGAACGGGCGGGGCGCTAATCATGCTGGAACTGAATTTTTCCCA 2040
|£xoIII-ll
GACGTTGGGCAACCATTGCCTGACTATTAATGAAACGCTGCCCGCCAATGGCATCACTGC 2100
|£xoIII-12
TATCTTTGGCGTCTCCGGTGCCGGAAAAACTTCGCTGATTAACGCCATCAGTGGACTGAC 2160
GCGCCCGCAAAAAGGGCGGATTGTCCTCAATGGGCGGGTACTAAATGATGCCGAAAAAGG 2220
TATCTGCCTGACGCCGGAAAAGCGTCGCGTTGGCTATGTTTTTCAGGATGCGCGGCTGTT 2280
|£xoIII-13
CCCGCATTACAAAGTGCGTGGCAATCTGCGCTACGGCATGTCGAAAAGTATGGTCGATCA 2340
GTTCGATAAGCTGGTGGCGCTTTTAGGCATTGAACCGTTGCTTGACCGTTTACCAGGCAG 2400
CCTGTCCGGAGGCGAAAAACAGCGCGTGGCGATTGGTCGGGCTTTGCTGACAGCACCGGA 2460
ATTGCTGTTGCTGGATGAACCGCTGGCGTCACTGGATATTCCGCGTAAACGCGAACTGTT 2520
GCCTTATCTGCAACGGCTGACACGGGAAATCAACATTCCGATGTTGTATGTCAGCCATTC 2580
GCTGGATGAGATCCTCCATCTGGCAGACAGAGTGATGGTACTGGAAAACGGTCAGGTGAA 2640
AGCCTTTGCGCTGGAGGAAGTGTGGGGCAGTAGCGTGATGAATCCGTGGCTGCCGAAAGA 2700
GCAACAAAGTAGCATTCTGAAAGTGACGGTGCTTGAGCATCATCCGCATTACGCGATGAC 2760
CGCGCTGGCGCTGGGCGATCAGCATTTGTGGGTCAATAAGCTGGACGAACCGCTGCAAGC 2820
TGCGCTACGTATCCGCATTCAGGCTTCCGATGTTTCTCTGGTTTTACAACCGCCGCAGCA 2880
AACCAGCATTCGTAACGTATTGCGGGCAAAAGTTGTTAATAGTTATGACGACAACGGCCA 2940
GGTGGAAGTGGAACTGGAAGTCGGCGGTAAAACGCTGTGGGCGCGTATCAGCCCGTGGGC 3000
CAGGGATGAACTGGCGATCAAACCTGGCCTGTGGCTGTACGCGCAAATTAAAAGTGTGTC 3060
GATAACCGCCTGATTAAATCAGGTGGCTATAAATGAACTGGGCAATGCTGTCGGTGGTGT 3120
TATCACCAATCACAATGTTGGCGCGCGCTTTTACCGCGTCATCGGCGTTGCCCATCGCCA 3180
fcoRV
CGCCTGTACCAGCGGCTTCCAGCATACTGATATCATTAAAGTATCGCCGAATGCCACGAC 3240
GTTCTCCATCGACCAACCTTGCGCCTCAACCCATTTCGTCAAACGTTTACCTTTGCTGTT 3300
GCCGCCGCGTGCAATATCAACCTGATCGTGCCAGGACCATTCACACTCCAGTCCCAGTTC 3360
AT GTTCGACAT GCTT ACCAAAAT GCTGCAATT GCCGCAGGT CATCGT GCGT CAGGGCGAA 3420
CTTCCATACGGCGTTAACTTGTTGCGCCGTTTCAGCCAGAGAAGCGACTTGTGTGAAAGT 3480
CGGACGCTGTTCCGGCGGCAGGGTTTGCGCCCAGTTAGATGTGCGAATGACATGCCCGGT 3540
CGGGTGCTCATAGACCATTGCATCATCGACATACATCAGACCGTGAATGTGGTGTTCATT 3600
CAGCCATCTCAATGAGTTGCAGGGCTTTAATAACGGGCATTGGGTCCGCTTCCAGCACGG 3660
TTTTTGCATGATAATCATACAAATAGGTGCCATTACAGCAAATAGCAGGTGTATCCAGCG 3720
CCAGCGCCT GAT AAAAAGGAT GAAT AGCGACGT GAT GGCGACCTGT GACGAT GATT AATT 3780
Ca I
GATAGCCTGCTTCGCGAGCGCGGGCCAGGGCTTCTATCGATGAAGGAAGCAGGGTCTTTT 3840
TCGGGGTCAATAAGGTGCCGTCTAAGTCGAGAGCAA 3876
Figure 15continued.

115
of the operon would suggest, it is possible that the operon it regulates
when present on a high copy plasmid no longer requires MOL-R protein.
Another possibility is that the chlD mutants synthesize a "molybdenum
cofactor-binding protein" which binds MPT to form an active repressor
(6, 14, 189). The molR mutant may be deficient in synthesizing the
molybdenum-binding protein. Thus in the presence of molybdate, the
repressor is absent and the molR gene is constitutively transcribed.
Thirdly, the mol {chi) operon may be transcribed by multiple promoters.
The chlD gene may be cotranscribed with the genes encoding the
periplasmic-binding-protein dependent transport system for molybdate;
whereas, the molR gene may be a regulatory protein transcribed from its
own promoter. Further investigation will be required to answer these
questions.

CONCLUSION
Genetic regulation of the FHL system encompasses a number of
factors including anaerobiosis, nitrate, formate, low pH, molybdate,
nickel and selenium. Genetic and physiological analysis of the fhlB
operon can provide answers to the molecular mechanism by which these
various components influence the levels of FHL activity in the cell.
This study demonstrates that the fhlB operon is anaerobically inducible
and requires o54, FHL-A, formate and molybdate for expression.
Sigma-54 is an alternate sigma factor required for fhlB operon
expression and is encoded by the rpoN (ntrA, glnF) gene. By monitoring
the expression of the rpoN gene using lac gene fusions, it has been
demonstrated that this sigma factor is constitutively expressed in £.
coliy K. pneumoniae and R. meliloti (34, 54, 154, 182). Whereas, the
majority of alternate sigma factors which have been analyzed are
inducible (as is the case for sporulation in B. subtilis; ref. 129,
142). This implies an interesting mechanism of transcriptional
regulation at o54-dependent promoters. Currently it is presumed that
transcriptional control is mediated predominately by modulation of the
activator protein (receiver) and not by altering the levels of the o54-
subunit (for review, see 90, 131). Therefore, identification and
116

117
analysis of the FHL activator is essential for full understanding of the
genetic regulation of formate hydrogenlyase in E. coli.
This study shows that the fhlA gene encodes a putative
transcriptional regulator which is homologous to other DNA-binding
proteins of the two-component regulatory system. The "domain D"
(presumed to be the site of ATP-binding and o54 interaction) and helix-
turn-helix motif are conserved; however, the amino-terminus of the FHL-A
protein (first 353 amino acids) is unique among the various proteins
analyzed. The FHL-A protein does not contain the conserved secondary
structure or aspartate and lysine residues which have been shown to be
phosphorylated by the respective histidine-protein-kinase (sensor).
This suggests that FHL-A protein may be modulated by a mechanism
differing from the typical phosphorylation cascade identified in the
two-component systems.
Interestingly, the fhlA gene is induced by microaerobic conditions
and constitutively synthesized anaerobically, while the fhlB gene (as
well as fdhFy and hyc genes; ref. 171, 196) expression requires formate
and molybdate in the medium (anaerobically). Transcription of the fhlA
gene is apparently from the weak "-35 and -10" fhlA promoter
(constitutive) and not from the FNR- or o54-dependent promoters of the
hyp operon. Therefore, formate modulation of active FHL-A protein is
not at the transcriptional level (this is independent of molybdate
control; discussed below). Although recent data suggest that FHL-A
alone can bind to URS of fdhF and retard electrophoretic mobility of

118
appropriate DNA, in vivo transcription assays have been unsuccessful to
date (A. Bock, personal communication). This suggests that the FHL-A
t
protein alone is a DNA-binding protein (however, purification of FHL-A
as a formate complex cannot be ruled out at this time) but is not in an
active form for transcriptional activation of the FHL pathway. Possibly
another unidentified factor (formate and/or sensor protein) is required
in the reaction.
From the fhlB gene regulation data presented in this study it can
be deduced that formate is required for either (i) direct activation of
FHL-A, (ii) transcription of another unidentified regulatory protein
(sensor) which positively interacts with FHL-A, or (iii) direct
interaction with a protein (sensor) which associates with FHL-A for
transcription of FHL promoters. DNA sequence analysis of the nearby
hypCDE genes did not reveal a typical sensor protein (Fig. 9).
Additionally, transcription of the fdhF gene in the absence of active
FHL-B suggests that the fhlB gene is not this "unidentified sensor
protein." Instead the FHL-B could potentially function as a modulator
of the FHL pathway. Continued research will need to be done in this
area to narrow the possibilities (i.e. formate binding studies with FHL-
A protein, random mutagenesis of FHL-A protein to identify if FHL-A
derivatives are formate independent, identification of a potential
formate "sensor" protein).
The fhlB gene in molR or chlD genetic backgrounds still retained
formate inducibility in the presence of multiple copies of the fhlA+

119
gene (exogenous molybdate was required for expression to wild type
levels). This suggests that FHL is regulated by a dual regulatory
system, one involving molybdate and a second formate-dependent pathway.
DNA sequence analysis of the mol (chi) operon indicates that the molR
gene, encoding a potential regulatory protein of the molybdate-dependent
pathway (136) is located near genes encoding a periplasmic-binding-
protein dependent molybdate transport system. The constitutivity of the
molR gene suggests multiple promoters are involved in transcription of
this operon.
These results show that fermentative dihydrogen production is
regulated by a complex series of reactions involving the availability of
formate, molybdate and the absence of oxygen. Further studies will be
necessary in order to better understand the various mechanisms of
regulation. Localization of the fhlB and molR mutations, primer
extension analysis of the mol (chi) operon promoter region and
identification of the parameters required for in vitro activation of the
o -dependent promoters of the FHL pathway will be beneficial to
furthering our understanding of dihydrogen production in E. coli.

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BIOGRAPHICAL SKETCH
Julie Anne Maupin was born on July 30, 1963, in Richmond Heights,
Missouri. She received a bachelor's degree in biological sciences in
April 1985 and during that time received a honor society scholarship.
Working in a biochemistry laboratory at the University of Alabama in
Huntsville, she developed an enthusiasm for genetics and molecular
biology. Since 1988, she has been in the Ph.D. program in the
Department of Microbiology and Cell Science at the University of
Florida.
143

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
zf=*t
K. T. Shanmugam, Chairman
Professor of Microbiology and
Cell Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Professor of Biochemistry and
Molecular Biology
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Francis C. Davis, Jr.
Associate Professor of
Microbiology and Cell Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
'OiM
Johli Gander
'"ofessor of Microbiology and
Cell Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Lonnie 0. Ingram
Professor of Microbiology and
Cell Science
This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
December, 1991
at- <^/vy
Dean, college of Agriculture
Dean, Graduate School



70
Trans-Ac ting Factors of the fhlB Operon
Genetic regulation of the fhlB operon. Expression of the enzymes in
dihydrogen metabolism requires the products of several genes and these
include hyd, fnr, fhlA, rpoN, molR and chlD gene products (25, 35, 135,
136, 171, 185, 186, 187, 188, 196, 223, 224, 231, 238, 242). In order to
study the role of these gene products on the expression of 4>(//>Zfi'-
1 ZacZ4-), appropriate double mutant strains were constructed in which one
of the putative regulatory genes is defective. Analysis of these double
mutant strains revealed that the fhlA, rpoN, chlD and molR gene products
are needed for the anaerobic formate-dependent induction of Q(fhlB'-
ZocZ+) (Table 6). The differential rate of expression of p-galactosidase
activity in the double mutants, strains MJ-5, MJ-20 and MJ-40 (rpoN, fhlA
and molR, respectively) was unity indicating that the induction and cell
growth are coupled and the enhancing effect of formate was absent. In an
fnr mutant (strain MJ-6), although the differential rate of expression was
comparable to strain SE-2011, the maximum activity was reduced by about
35%. The reduction in the amount of p-galactosidase activity produced by
the fhlB, fnr double mutant could be a consequence of lower cell yield of
the culture since the production of p-galactosidase activity by (fhlB'~
'lacZ+) required growth of the organism. HydF and cya mutations had no
apparent effect on the expression of (fhlB'-'lacZ*) operon with and
without formate supplementation. Introducing narL::Tni0 (217) into strain


23
that chlA repression is probably mediated by a complex of MPT and a MPT-
binding protein which has been reported previously (6, 189).
The constitutively synthesized chlB gene (86 min) product has been
purified (Mr = 35 kDa) and is denoted as "association factor-FA" which
is essential for synthesis of functional MPT (143, 180). A mutation in
this gene inhibits insertion of the MPT into the apoprotein; therefore,
association factor-FA may have a direct role in mediating the terminal
step of apoprotein maturation (71, 146). Johnson et al. (112) recently
developed a more sensitive fluorescence technique to analyze cell
extracts for MPT and MGD derivatives. By comparing chlB mutant extracts
to wild type, it was concluded the chlB gene was required for the
formation of MGD, a step occurring late in the MPT maturation process.
The chlC (27 min) and chlF (32 min) operons code for nitrate reductase
and FDH-N respectively. Since neither are necessary for MPT
biosynthesis, they have been renamed narGHJI (chlC) and fdnGHI (chlF)
(11, 19, 219). Mutations in both the chlG (0 min) and chlD operons (17
min) are suppressed by Mo supplementation of the growth medium (11, 207,
219). Phenotypically, the chlG mutants lack nitrate reductase activity,
even though detectable levels of the protein were produced in the
original isolates, and retain wild type levels of Fhl activity (219).
It is currently postulated that the CHL-G proteinfunctions to
incorporate Mo into molybdoenzymes and Mo-binding proteins (94).
Mutations in the chlD operon can be phenotypically distinguished
from the chlG gene. Dihydrogen production is lacking in chlD mutants


24
due to a deficiency in both FDH-H and HYD-3 activities (33, 73, 76,
196). The partial sequence of the chlD operon discloses significant
homology to binding-protein-dependent transport systems (110).
Generally, this type of transport is a unidirectional process. It is
now known that this process requires ATP for energy instead of acetyl-
phosphate (20, 95, 97, 100), and the substrate does not undergo
modification during transport (4). In the systems which have been
analyzed, the number and location of proteins involved in the transport
process are conserved (for review, see 5, 52). These proteins include:
(i) a high-affinity, substrate-binding protein located in the
periplasmic space which is released upon cold osmotic shock (i.e. HISJ,
MAL-E), (ii) two integral membrane proteins present in lower
concentration than the binding protein (i.e. HIS-Q, HIS-M, MAL-F, MAL-
G), and (iii) another membrane-associated protein, presumed to be
peripheral, which contains a nucleotide-binding domain with a conserved
amino acid sequence (i.e. HIS-P, MAL-K). The central domain of the
complete CHL-D sequence revealed extensive homology to peripheral
transport proteins (110). The partial CHL-J sequence suggested that
this protein is an integral membrane component of Mo transport (110).
Both the chlD gene and the molR gene, recently described by Lee et
al. (136) are essential for fdhF and hyc (ant, hyd-17) operon expression
as measured by 0-galactosidase activity (134, 196). This implies that
transcription of these FHL operons either directly or indirectly
requires Mo or a Mo-derivative. Interestingly, both molR and chlD genes


115
of the operon would suggest, it is possible that the operon it regulates
when present on a high copy plasmid no longer requires MOL-R protein.
Another possibility is that the chlD mutants synthesize a "molybdenum
cofactor-binding protein" which binds MPT to form an active repressor
(6, 14, 189). The molR mutant may be deficient in synthesizing the
molybdenum-binding protein. Thus in the presence of molybdate, the
repressor is absent and the molR gene is constitutively transcribed.
Thirdly, the mol {chi) operon may be transcribed by multiple promoters.
The chlD gene may be cotranscribed with the genes encoding the
periplasmic-binding-protein dependent transport system for molybdate;
whereas, the molR gene may be a regulatory protein transcribed from its
own promoter. Further investigation will be required to answer these
questions.


6
levels). This indicates that FHL is regulated by a dual regulatory
system, one involving molybdate and a second formate-dependent pathway
(Fig. 1). The results presented show that fermentative dihydrogen
production is regulated by a complex series of reactions involving the
availability of formate, molybdate and the absence of oxygen.


I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
zf=*t
K. T. Shanmugam, Chairman
Professor of Microbiology and
Cell Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Professor of Biochemistry and
Molecular Biology
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Francis C. Davis, Jr.
Associate Professor of
Microbiology and Cell Science
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
'OiM
Johli Gander
'"ofessor of Microbiology and
Cell Science


78
Expression of QjfdhF'-'cat) in an fhlB mutant, strain SE2011.
The FDH-H activity was undetectable in strain SE2011. In order to
determine whether this effect is at the transcriptional or post-
transcriptional level, a plasmid carrying a 'CAT' gene fusion in the
fdhF gene was transformed into strain SE-2011 and the effect of the fhlB
mutation on fdhF gene mediated CAT activity was determined. Cells were
grown anaerobically (4 hr) in LB medium supplemented with glucose and
formate for maximal expression. Due to the multicopy nature of the
expression vector, trace metals (Se, Mo, and Fe) were included to insure
saturation. The fdhF transcription measured as CAT activity in strain
SE-2011 [QtfhlB'-'lacZ*)] was comparable to parent strain MC-4100 at 140
and 110, units respectively. Whereas, in strain SE-2007 [Q(fhlA'-
'lacZ+)] expression was reduced over 5-fold, to about 20 units
confirming the need for FHL-A protein. This suggests that FHL-B protein
is not required for transcription of fdhF gene but is required for the
production of active FDH-H (Table 3).


4
(fhlX)?
h y
fhIA
y i
fi- ii y
I URS URS T
-i 'transport proteins r-
HYD3J ^ |_FDH-H
FHL
FORMATE H2 + CO*
Figure 1. Model of genetic regulation of formate hydrogenlyase in E.
coli.


background still retained formate inducibi1ity; however, molybdate was
required at high concentrations for expression to wild type levels. DNA
sequence analysis of fhlA gene revealed that the FHL-A protein is
homologous to other known transcriptional activators. It is proposed
that formate, in association with the FHL-A protein, initiates
transcription of the fhlB, hyc, hyp and fdhF operons. Analysis of
mutant strain SE-2007 [${fhlAlacZ*)] indicated that the transcription
of the fhlA gene, located at the 3'-end of the hyp operon, is
constitutive. This is in contrast to the hyp operon transcription which
has dual controls: formate-dependent and FNR-dependent. The DNA
sequence of the mol operon (essential for molybdate transport, FHL
activity and fhlB expression) was also determined to investigate the
role of molybdate metabolism in the regulation of FHL in E. coli. These
studies revealed that the fermentative dihydrogen production is
regulated by a complex series of reactions involving the availability of
formate, Mo and absence of 02. It is postulated that the FHL-B protein
is one of the modulators of this control system.
xv


62
Figure 5. Effect of nitrate supplementation on fhlB gene expression.
Cultures were grown in LB, LB-formate, LB-nitrate, and LB-nitrate-
formate media (LB, LBF, LBN and LBNF, respectively). A. Nitrate
respiration (as measured by nitrite produced) in fhlB mutant strain SE-
2011 grown in LB and LBNF. B. Rate of induction of (3gal actos i dase
activity by 4>(fhlB'-'lacZÂ¥) strain SE-2011.


33
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
PC0287
thr-20, leu-32, proA35, argF58,
argI60, lacYl, gal-6, rpsL125,
tonA48, tsx-70, supE44
CGSC #5404
RK5278
narL215::lnlO
V. Stewart
SE-1000
cysC-43, srl-3000::lnl0, thr-1,
leu-6, thi-1, lacY-1, galK2, ara-14,
xyl-5, mtl-1, proA2, his-4, argE3,
rpsL31, tsx-33, supE44
Laborotory
collection
SE-67-1
hydFI02, cys::Tnl0
PI transduction
(SE67 x SE1300)
SE-1100
BW545, 4>{tnolR'-' lacZ*)
Laboratory
collection (136)
SE-1162
LS853, zif-4::JnlO
Laboratory
collection
SE-1174
fhlA::JnlO
Laboratory
collection (186)
SE-1188
JRG861a, zcj-5::lnlO
Laboratory
collection
SE1265
LCB898, zba-6::Tni0
Laboratory
collection
SE1300
BW545, cys::TnT0
Laboratory
collection
SE1651
4>{hyp'-' lacZ+) 1, fnr, zcj-5::JnlO
PI transduction
(SE2001 x SE-1188)
SE1652
4>(hyp'-' lacZ+)2, fnr, zcj-5: :Tni0
PI transduction
(SE-2002 x SE-1188)
SE1654
4>{hyp'-' lacZ*) 1, narL2l5::lr\10
PI transduction
(SE2001 x RK5278)


29
In this study, the mechanism by which formate and molybdenum
regulate the FHL pathway was investigated. This includes isolation of a
specific mutant, strain SE-2011 lacZ*)], physiological
characterization of this fhlB mutant, and trans-acting factors
regulating the fhlB gene. These trans-acting factors were found to be
the FHL-A protein of the formate-dependent pathway and an
uncharacterized component of the molybdate-dependent pathway.


132
134. Lee, J. H. 1987. Identification and characterization of a new gene
essential for production of formate hydrogenlyase activity in
Escherichia coli, Ph.D. dissertation, University of Florida.
135. Lee, J. H., P. Patel, P. Sankar, and K. T. Shanmugam. 1985.
Isolation and characterization of mutant strains of Escherichia
coli altered in H2 metabolism. J. Bacteriol. 162:344-352.
136. Lee, J. H., J. C. Wendt, and K. T. Shanmugan. 1990. Identification
of a new gene, molR, essential for utilization of molybdate by
Escherichia coli. J. Bacteriol. 172:2079-2087.
137. Li, S. F., and J. A. DeMoss. 1987. Promoter region of the nar
operon of Escherichia coli: nucleotide sequence and transcription
initiation signal. J. Bacteriol. 169: 4614-4620.
138. Lim, S. T. 1978. Determination of hydrogenase in free-living
cultures of Rhizobium japonicum and energy efficiency of soybean
nodules. Plant Physiol. 62: 609-611.
139. Lin, E. C. C., and D. R. Kuritzkes. 1987. Pathways for anaerobic
electron transport, p. 201-221. In F. C. Neidhardt, J. L.
Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E.
Umbarger (ed.), Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology. Am. Soc. Microbiol., Washington,
DC.
140. Ljungdahl, L. G. 1976. Tungsten, a biologically active metal. TIBS
1:63-65.
141. Ljungdahl, L. G. 1980. Formate dehydrogenases: role of
molybdenum, tungsten and selenium, p. 463-485. In M. P. Coughlan
(ed.), Molybdenum and Molybdenum-containing Enzymes. Pergamon
Press, Inc., Elmsford, NY.
142. Losick, R., P. Youngman, and P. J. Piggot. 1986. Genetics of
endospore formation in Bacillis subtilis. Ann. Rev. Genet. 29:625-
670.
143. Low, D. C., J. Pommier, G. Giordano, and D. H. Boxer. 1988.
Biosynthesis of molybdoenzymes in Escherichia coli: chlB is the
only chlorate resistance locus required for protein F activity.
FEMS Microbiol. Lett. 49:331-336.
144. Lutz, S. A. Jacobi, V. Schlensog, R. Bohm, G. Sawers, and A. Bock.
1991. Molecular characterization of an operon {hyp) necessary for
the activity of the three hydrogenase isoenzymes in Escherichia


57
Table 4. The effect of media composition on the expression of
Q(fhlB'-lacZ+) in an fhlB mutant, strain SE-2011
Medium composition
3-Gal actosi dase
activity3
Aerobic
Anaerobic
Luria Broth
110
280
+ Nitrate (1.0%)
90
120
+ Fumarate (0.5%)
110
250
+ Glucose (0.3%)
92
580
+ Formate (0.1%)
170
1,300
+ Formate and Glucose
190
1,400
+ Formate and bufferb
NDC
700
Cells were grown as described in the "Methods" section.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
b0.1 M Phosphate buffer at pH 7.0
CND, not determined


129
Phosphorylation and dephosphorylation of a bacterial activator by
a transmembrane receptor. Genes Dev. 3:1725-1734.
102. Ingledew, W. J., and R. K. Poole. 1984. The respiratory chains of
Escherichia coli. Microbiol. Rev. 48:222-271.
103. Inouye, S., A. Nakazawa, and T. Nakazawa. 1988. Nucleotide
sequence of the regulatory gene xylR of the TOL plasmid from
Pseudomonas put ida. Gene 66:301-306.
104. Iuchi, S., and E. C. C. Lin. 1987. The narL gene produce activates
the nitrate reductase operon and represses the fumarate reductase
and trimethyl amine N-oxide reductase operons in Escherichia coli.
Proc. Natl. Acad. Sci. USA 84:3901-3905.
105. Iuchi, S., and E. C. C. Lin. 1988. arcA (dye), a global regulatory
gene in Escherichia coli mediating repression of enzymes in
aerobic pathways. Proc. Natl. Acad. Sci. USA 85:1888-1892.
106. Iuchi, S., Z. Matsuda, T. Fujiwara, and E. C. C. Lin. 1990. The
arcB gene of Escherichia coli encodes a sensor-regulator protein
for anaerobic repression of the arc modulon. Mol. Microbiol.
4:715-727.
107. Jamieson, D. J., R. G. Sawers., P. A. Rugman, D. H. Boxer, and C.
F. Higgins. 1986. Effects of anaerobic regulatory mutations and
catabolite repression on regulation of hydrogen metabolism and
hydrogenase isoenzyme composition in Salmonella typhimurium. J.
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108. Jamieson, D. J., and C. F. Higgins. 1986. Two genetically distinct
pathways for transcriptional regulation of anaerobic gene
expression in Salmonella typhimurium. J. Bacteriol. 168:389-397.
109. Jerlstrom, P. G., J. Liu, and I. R. Beacham. 1987. Regulation of
Escherichia coli L-asparaginase II and L-aspartase by the fnr gene
product. FEMS Microbiol. Lett. 41:127-130.
110. Johann, S., and S. M. Hinton. 1987. Cloning and nucleotide
sequence of the chlD locus. J. Bacteriol. 169:1911-1916.
111. Johnson, J. L., B. E. Hainline, K. V. Rajagopalan, and B. H.
Arison. 1984. The pterin component of the molybdenum cofactor
structural characterization of two fluorescent derivatives. J.
Biol. Chem. 259:5414-5422.
112. Johnson, J. L., L. W. Indermaur, and K. V. Rajagopalan. 1991.


128
90. Hennecke, H. 1990. Regulation of bacterial gene expression by
metal-protein complexes. Mol. Microbiol. 4:1621-1628.
91. Hess, J. F., K. Oosawa, N. Kaplan, and M. I. Simon. 1988.
Phosphorylation of three proteins in the signaling pathway of
bacterial chemotaxis. Cell 53:79-87.
92. Hewitt, E. J., and B. A. Notton. 1980. Nitrate reductase systems
in eukaryotic and prokaryotic organisms, p. 273-326. In M. P.
Coughlan (ed.) Molybdenum and Molybdenum-containing Enzymes.
Pergamon Press, Inc. Elmsford, NY.
93. Hill, S., C. Kennedy, E. Kavanagh, R. B. Goldberg, and R. Hanau.
1981. Nitrogen fixation gene (nifL) involved in oxygen regulation
of nitrogenase synthesis in Klebsiella pneumoniae. Nature 290:424-
426.
94. Hinton, S. M. and D. Dean. 1990. Biogenesis of molybdenum
cofactors. Crit. Rev. Microbiol. 17:169-188.
95. Hobson, A., R. Weatherwax, and G. F. -L. Ames. 1984. ATP-binding
sites in the membrane components of histidine permease, a
periplasmic transport system. Proc. Natl. Acad. Sci. USA
81:7333-7337.
96. Horn, S. S. M., H. Hennecke, and K. T. Shanmugam. 1980. Regulation
of nitrogenase biosynthesis in Klebsiella pneumoniae: effect of
nitrate. J. Gen. Microbiol. 117:169-179.
97. Hong, J., A. Hunt, P. Masters, and M. Lieberman. 1979. Requirement
for acetyl-phosphate for the binding protein dependent transport
systems in Escherichia coli. Biochemistry 22:844-850.
98. Hsieh, L. -S., R. M. Burger, and K. Drlica. 1991. Bacterial DNA
supercoiling and [ADP]/[ATP] changes associated with a transition
to anaerobic growth. J. Mol. Biol. 219:443-450.
99. Huala, E. and F. M. Ausubel. 1989. The central domain of Rhizobium
meliloti NifA is sufficient to activate transcription from the R.
meliloti nifH promoter. J. Bacteriol. 171:3351-3365.
100. Hunt, A., and J. Hong. 1983. Properties and characterization of
binding protein dependent active transport of glutamine in
isolated membrane vesicles of Escherichia coli. Biochemistry
22:844-850.
101. Igo, M. M., A. J. Ninfa, J. B. Stock, and T. J. Silhavy. 1989.


64
Genetic Characteristics of Strain SE-2011, QtfhlB'-' lacZ*)
F1 complementation analysis. Initial complementation studies were
performed using F'143-1, an episomal element which carried DNA from 57
to 61 min in the E. coli chromosome. This was done since the previously
described hyd and fhlA genes, which demonstrated pleiotropic phenotypes
for dihydrogen metabolism, mapped to the 58 to 59 min region (185, 186,
187, 188). Mutant strain MJ-1, a cys::Tni0 derivative of SE-2011, was
used as a recipient for F'1431 transfer. Exconjugants were selected
for Cys+ phenotype and then screened for FHL activity. Approximately 20%
of the Cys+ clones were Fhl+. Among those complemented for dihydrogen
production, HUP activity was not restored (as measured by H2 to BV). It
is possible that the 20% complementation for FHL activity was because
the entire region necessary for restoration of strain SE-2011 to
parental phenotype was not completely transferred or that deletions may
have occurred in the plasmid during growth with uracil, a needed
nutrient. Therefore, strain MJ-50 [(fhlB'-'lacZ+) cys, srl] was used
to select a F* element carrying the entire region between cys+ and srl+
genes. A Fhl+ exconjugant (5% of the clones tested) was isolated and
used as a F*1431 donor strain to select for Cys+ in recipient strain
MJ-1, 4>(fhlB'-'lacZÂ¥) cys. Of the exconjugants analyzed, 100% were Fhl +
and Hup+ which suggested that the mutation mapped in this region.
PI transduction. Similar results were obtained with PI
transduction experiments using strain BW545 as the donor. Of the 72


25
are located in the same operon as determined by transduction and plasmid
complementation analysis (Shanmugam, unpublished results; this study).
However, MOL-R is expressed constitutively (134) while CHL-D synthesis
is Mo repressible (155) thus suggesting at least two promoters for this
operon.
Nickel
E. coli synthesizes three distinct nickel (Ni) containing
hydrogenase isoenzymes (12, 193). The HYD-1 protein and an
enzymatically active fragment of HYD-2 have been purified to homogeneity
(1, 13, 66, 168, 194). Biochemical characterization of these proteins
revealed that HYD-1 and HYD-2 contain nonheme iron, inorganic sulfur and
nickel. The HYD-3 protein, responsible for dihydrogen evolution, is
electrophoretically labile and has remained elusive to purification
(193). DNA sequence analysis of the large subunit of HYD-3 (hyc 0RF5),
however, displays significant homology to the "nickel-amino acid"
consensus sequence of other NiFe-hydrogenases (27, 195, 214). The large
subunits of all sequenced NiFe-hydrogenases contain the consensus R-X-C-
G-X-C-Xg-H in the amino-terminus and D-P-C-X2-C-X2-H at the carboxy-
terminus (177). Apparently the conserved sites at both termini are
somehow involved in liganding nickel at the active site (61, 87).
Several genes required for total hydrogenase activity (thus Fhl
activity) have been characterized and are presumed to be involved in
nickel transport, nickel processing, or hydrogenase regulation. These
include hydA (59 min), hydB or hypE (59 min), hydC (77.6 min), hydD


3
protein-kinase. Appropriate environmental stimulation results in sensor
autophosphorylation cascading to phosphorylation of the receiver
protein. Once in an active form, the receiver is a positive regulator
of specific modulon transcription (163, 183, 222).
Much remains to be understood about how the "appropriate
environmental stimulation" is transmitted to the sensor protein. It has
been proposed that the FHL system is regulated by five environmental
signals which include anaerobiosis, intracellular formate, pH,
molybdate, and nickel. Although the genes encoding the structural
components of the FHL complex [fdhF (FDH-H) and hyc (HYD-3) operons] are
located approximately 33 minutes apart in the E. coli chromosome, their
transcription appears to be coordinated (11, 24, 25, 171).
In order to decipher the mode by which these signals are
transmitted to the transcription of the formate modulon, a series of
promoter lac fusion strains were constructed and characterized for
anaerobic induction and dihydrogen related enzyme activities.
Mutational analysis revealed a unique gene, later denoted as the fhlB
gene, encoding a product essential for FHL activity including FDH-H and
total hydrogenase activity. However, FHL-B protein was not necessary
for expression of the fdhF gene and is therefore currently assumed
modulate transcription of the FHL pathway (Fig. 1). Transcription of
the fhlB gene was repressed by high-redox potential electron acceptors
(dioxygen and nitrate) and required formate for maximum anaerobic
induction. Further analysis revealed that rpoN (encoding the o54


47
Sodium dodecyl sulfate and proteinase K were added to a final
concentration of 0.5% and 0.1 mg/ml, respectively. This was incubated
for 1 hr at 37C. The NaCl concentration of the sample was then
adjusted to 0.7 M, mixed thoroughly and CTAB/NaCl solution (10%
hexadecyl trimethyl ammonium bromide in 0.7 M NaCl) was added to a final
concentration of 1% CTAB. This was heated to 65C for 10 min, extracted
with equal volume of chloroform: isoamyl alcohol (24:1) and extracted
with phenol/chloroform/isoamyl alcohol (25:24:1). The DNA was
precipitated with isopropanol and washed with 70% ethanol. The pellet
was vacuum-dried, resuspended in TE buffer (7.5 ml) and centrifuged in a
cesium chloride gradient (1 mg/ml cesium chloride; 0.625 mg/ml ethidium
bromide) using similar parameters to the large scale plasmid
preparation. Chromosomal DNA was removed from the gradient using a
syringe fitted with a 16G x needle and then extracted and
precipitated as with plasmid DNA. Concentration was determined by
agarose gel electrophoresis with known DNA standards and
fluorometrically with Hoechst 33258 dye (using the TK0 100-dedicated
Mini-Fluorometer; Hoeffer Scientific). Concentration as well as the
purity of the DNA was determined from the absorption spectrum between
the wavelengths 200 nm and 300 nm.
DNA sequence determination. DNA sequence was determined using the
Sanger dideoxy method with double stranded plasmid DNA (85, 184). The
plasmids used are listed in Table 2. Plasmids pSE-130, pSE-132 and pSE-
133 used in the fhlA gene sequencing experiments were described


87
sequence, the FHL-A protein does not have this domain and thus may not
undergo phosphorylation. The FHL-A region I probably determines the
specific regulatory function for this protein in response to FHL
inducers (i.e. formate and anaerobiosis).
The next region of about 24 amino acids, (region II, 356-374 amino
acids) aligns with the proposed "interdomain linker" of both NTR-C and
NIF-A proteins (58). The high content of glutamine (Q), serine (S),
glutamic acid (E), arginine (R) and proline (P) residues, 55% in NTR-C
and 74% in NIF-A, suggests a coil or turn conformation in the protein
structure. This could hypothetically link the region in the N-terminus
to the central ATP-binding region (discussed below). In FHL-A protein,
the extent of these residues is apparently random (26%) suggesting an
alternative secondary structure in this region.
The region III is about 240 amino acids (positions 380 to 619) and
is highly conserved among transcriptional activators of the two-
component regulatory systems which are proposed to interact at o54-
dependent promoters (80). Alignment with FHL-A protein is presented in
decreasing order of homology: NIF-A (53%), FLB-D (48%), NTR-C (47%),
XYL-R (46%), DCT-D (43%), and TYR-R (39%; Fig. 11). This region is
analogous to the "domain D" of the NIF-A and NTR-C proteins (58). The
boxed residues are potential ATP-binding sites (53, 67, 229). Current
evidence suggests that the activator protein binds to the o54-RNA
polymerase holoenzyme via this domain and catalyzes the ATP-dependent
isomerization of closed to open transcriptional complex (174). Non-


73
PROTEIN (jig)
Figure 6. Differential rate of induction of the lacZ*) fusion
in strain SE-2011 in the presence and absence of plasmid pSE-133. LB, LB
medium; LBF, LB medium supplemented with 30 mM formate. p-Galactosidase
and protein activities are expressed as units per ml and pg per ml,
respectively.


80
Sal
GTCGACGTCTGCGGCATTCAGCGCGATGTCGATTTAACGTTAGTCGGCAGCTGCGATGAA 60
VDVCGIQRDVDLTLVGSCDE
AACGGTCAGCCGCGCGTGGGCCAGTGGGTACTGGTACACGTTGGCTTTGCCATGAGCGTA 120
NGQPRVGQWVLVHVGFAMSV
ATTAATGAAGCCGAAGCACGCGACACTCTCGACGCCTTACAAAACATGTTTGACGTTGAG 180
INEAEARDTLDALQNMFDVE
CCGGATGTCGGCGCGCTGTTGTATGGCGAGGAAAAATAATGCGTTTTGTTGATGAATATC 240
PDVGALLYGEEK ***
MRFVDEYR
GCGCGCCGGAACAGGTGATGCAGTTAATTGAGCATCTGCGCGAACGTGCTTCACATCTCT 300
APEQVMQLIEHLRERASHLS
CTTACACCGCCGAACGCCCTCTGCGGATTATGGAAGTGTGTGGCGGTCATACCCACGCTA 360
YTAERPLRIMEVCGGHTHAI
TCTTTAAATTCGGCCTCGACCAGTTACTGCCGGAAAACGTTGAGTTTATCCACGGTCCGG 420
FKFGLDQLLPENVEFIHGPG
GGTGCCCGGTGTGCGTACTGCCGATGGGTAGAATCGACACCTGCGTGGAGATTGCCAGCC 480
CPVCVLPMGRIDTCVEIASH
ATCCGGAAGTCATCTTCTGTACCTTTGGCGACGCGATGCGCGTGCCGGGGAAACAGGGAT 540
PEVIFCTFGDAMRVPGKQGS
CGCTGTTGCAGGCAAAAGCACGCGGTGCCGATGTGCGCATCGTTTACTCGCCGATGGATG 600
LLQAKARGADVRIVYSPMDA
CGTTGAAACTGGCGCAGGAGAATCCAACCCGCAAAGTGGTGTTCTTCGGCTTAGGTTTTG 660
LKLAQENPTRKVVFFGLGFE
AAACCACTATGCCGACCACCGCTATCACTCTGCAACAGGCGAAAGCGCGTGATGTGCAGA 720
TTMPTTAITLQQAKARDVQN
ATTTTTACTTCTTCTGCCAGCACATTACGCTTATCCCGACGTTGCGCAGTTTGCTGGAAC 780
FYFFCQHITLIPTLRSLLEQ
Clal
AGCCGGATAACGGTATCGATGCGTTCCTCGCGCCGGGTCACGTCAGTATGGTTATCGGCA 840
PDNGIDAFLAPGHVSMVIGT
CCGACGCCTATAATTTTATCGCCAGCGATTTTCATCGTCCGCTGGTGGTTGCTGGATTCG 900
DAYNFIASDFHRPLVVAGFE
AACCCCTTGATCTACTACAAGGCGTGGTCATGCTGGTGCAGCAGAAAATAGCGGCCCACA 960
PLDLLQGVVMLVQQKIAAHS
Kpnl
GCAAGGTAGAGAATCAGTATCGTCGAGTGGTACCGGATGCCGGTAACCTGCTGGCGCAAC 1020
KVENQYRRVVPDAGNLLAQQ
Figure 9. Nucleic acid and predicted amino acid sequences of the
partial hypC gene and complete hydB, hydF and fhlA genes. The
termination codons are indicated by three asterisks. The "Shine-
Dalgarno" sequences and the weak "-35 and -10" region of the fhlA gene
are double underlined. Restriction sites for some of the enzymes are
highlighted. The inverted triangle between positions 4,404 and 4,405
represents the position of transposon Tn5 in plasmid pSE133-2, as
determined by DNA sequence analysis.


85
FHL-A MSYTPMSDLGQQGLFDITRTLLQQPDLASLCEALSQLVKRSALADNAAIVLWQAQTQRAS 60
FHL-A YYASREKDTPIKYEDETVLAHGPVRSILSRPDTLHCSYEEFCETWPQLDAGGLYPKFGHY 120
FHL-A CLMPLAAEGHIFGGCEFIRYDDRPWSEKEFNRLQTFTQIVSWTEQIQSRWNNVDYELL 180
NIF-A MIHKSDSDTTV 11
XYL-R MSLTYKPKMQHEDMQDLSSQIRFVAAEGKIWLG 33
FHL-A CRERDNFRILVAITNAVLSRLDMDELVSEVAKEIHYYFDIDDISIVLRSHRKNKLNIYST 240
NIF-A RRFDLSQQFTAMQRISWLSRATEASKTLQEVLSVLHNDAFMQHGMICLYDSQQEILSIE 71
XYL-R EQRMLVMQLSTLASFRREIISLIGVERAKGFFLRLGYQSGLMDAELARKLRPAMREEEVF 93
FHL-A HYLDKQHPAHEQSEVDEAGTLTERVFKSKEMLLINLHERDDLAPYERM-LFDTWGNQIQT 299
NTR-C MQRGIVWWDDDSSIRWVLERALAGAGLTCTTFENGAEVLEA-LASKTPDVLLS 53
NIF-A ALQQTEDQTLPGSTQIRYRPGEGLVGTVLAQGQSLVLPRVADDQRFLDRL-SLYDYDLPF 130
XYL-R LAGPQLYALKGMVKVRLLTMDIAIRDGRFNVEAEWIDSFEVDICRTELGL-MNEPVCWTV 152
FHL-A LCLLPLMSGDTMLGVLKLAQCEEKVFTTTN-LNLLRQIA ERVAIA-VDNALAYQEIH 354
NTR-C DIRMPGMDGLALLKQIKQRHPMLPVIIMTA-HSDLDAAVSAYQQGAFDYLPKPFDIDEAV 112
NIF-A IAVPLMGPHSRPIGVLAAHAMARQEERLPA-CTRFLETV ANLIAQ-TIRLMILPTSA 185
XYL-R LGYASGYGSAFMGRRIIFQETSCRGCGDDKCLIVGKTA EEWGDVSSFEAYFKSDPI- 208
FHL-A RLKERLVDENLALTEQLN-NVDSEFGEIIGRSEAMYSVLKQVEMVAQSDSTVLILGET 411
NTR-C ALVERAISHYQEQQQPRNVQLNGPTTDIIAKP-AMQDVFRIIGRLSRSSISVLINGES 169
NIF-A AQAPQQSPRIERPRACTP-SRGFGLENMVGKSPAMRQIMDIIRQVSRWDTTVLVRGES 242
XYL-R -VDERYELQTQVANLRNRLK-QYDGQYYG-IGHSPAYKRICETIDKAARGRVSVLLLGET 265
FHL-A GTGKELIARAIHNLSGRNNRRMVKMNCAAMPAGLLESDLFGHERGAFTGASAQRIGRFEL 471
NTR-C GTGKELVAHALHRHSPRAKAPFIALNMAAIPKDLIESELFGHEKGAFTGANTIRQGRFEQ 229
NIF-A GTGKELIANAIHHNSPRAAAAFVKFNCAALPDNLLESELFGHEKGAFTGAVRQRKGRFEL 302
XYL-R GVGKEVIARSVHLRSERAEQPFVAVNCAAIPPDLIESELFGVDKGAYTGAVNARAGRFER 325
FHL-A ADKSSLFLDEVGDMPLELQPKLLRVLQEQEFERLGSNKIIQTDVRLIAATNRDLKKMVAD 531
NTR-C ADGGTLFLDEIGDMPLDVQTRLLRVLADGQFYRVGGYAPVKVDVRIIAATHQNLEQRVQE 289
NIF-A ADGGTLFLDEIGESSASFQAKLLRILQEGEMERVGGDETLRVNVRIIAATNRHLEEEVRL 362
XYL-R ANGGTIFLDEVIELTPRAQATLLRVLQEGELERVGGDRTRKVDVRLITATNENLEEAVKM 385
FHL-A REFRSDLYYRLNVFPIHLPPLRERPEDIPLLAKAFTFKIARRLGRNIDSIPAETLRTLSN 591
NTR-C GKFREDLFHRLNVIRVHLPPLRERREDIPRLARHFLQVAARELGVEAKLLHPETEAALTR 349
NIF-A GHFREDLYYRLNVMPIALPPLRERQEDIAELAHFLVRKIAHSQGRTL-RISDGAIRLLME 421
XYL-R GRFRADLFFRLNVFPVHIPPLRERVEDIPLLVEHFLRRHHKEYGKKTLGLSDRAMEACLH 445
FHL-A MEWPGNVRELENVIERAVLLTRG-NVLQLSLPDIVLPE-PETPPAATWALE-GEDEY 646
NTR-C LAWPGNVRQLENTCRWLTVMAAGQEVLIQDLPGELFES-TVAESTSQMQPDSWATLLA 406
NIF-A YSWPGNVRELENCLERSAVLSES-GLIDRDVILFNHRDNPPKALASSGPAED-G 473
XYL-R YQWPGNIRELENALERGVILTES-NESINVESLFPG-LATATEGDRLSSE-GRLEEES 500
FHL-A QLIVRVLKETNGWAG PKGAAQRLGLKRTTLLSRMKRLG 685
NTR-C QWADRALRSGHQNLLSEAQPELERTLLTTALRHTQGHRUEAARLLGWGRNTLTKKLKELG 466
NIF-A -WLDNSLDERQRLIAALEKAGWV OMAAKL LGMTFRD VA Y R10IMD 518
XYL-R GDSWFRQIIDQGVSLEDLEAGLMRTAMDRCGQN1SQAAKLLGLTRPAMAYRLKKLD 556
FHL-A IDKSALI* 692
NTR-C ME* 468
NIF-A ITMPRL* 524
XYL-R PSLSVKAMGR* 566
Figure 10. Alignment of the predicted sequences of E. coli FHL-A, E.
coli NTR-C, K. pneumoniae NIF-A, and P. putida XYL-R proteins.
Identical and functionally similar amino acids are highlighted.
Underlined regions represent those residues of which two out of the
three aligined amino acids are homologous to FHL-A protein. The helix-
turn-helix motif is double underlined.


90
i
4 5 8 9 10
15 18 20
FHL-A
663
HYD-G
417
NTR-C
444
XYL-R
534
NIF-A
496
Pro-Lys-Gly-Ala-Ala-Gln-Arg-Leu-Gly-Leu-Lys-Arg-Thr-Thr-Leu-Leu-Ser-Arg-Met-LyB
Lys-Thr-Glu-Ala-Ala-Arg-Gln-Leu-Gly-Ile-Thr-Arg-Lys-Thr-Leu-Leu-Ala-LyB-Leu-Ser
Lys-Thr-Glu-Ala-Ala-Arg-Leu-I.eu-Gly-Trp-Gly-Arg-Asn-Thr-Leu-Thr-Arg-Lys-Leu-I.yB
Ile-Ser-Gln-Ala-Ala-Arg-Leu-Leu-Gly-Leu-Thr-Arg-Pro-Ala-Met-Ala-Tyr-Arg-Leu-Lys
Gln-Ala-Lys-Ala-Ala-Arg-Leu-Leu-Gly-Met-Thr-Pro-Arg-Gln-Val-Ala-Tyr-Arg-Ile-Gln
Figure 12. Alignment of potential DNA-binding motifs in the FHL-A, HYD-
G, NTR-C, XYL-R and NIF-A proteins. Identical and functionally similar
amino acids are highlighted. Critical amino acids for helix-turn-helix
formation are indicated.


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS
TABLE OF CONTENTS Hi
LIST OF FIGURES v
LIST OF TABLES vii
ABBREVIATIONS viii
LIST OF GENE SYMBOLS xi
ABSTRACT xiv
INTRODUCTION 1
LITERATURE REVIEW 7
Anaerobiosis 11
Nitrate 16
Formate 18
Molybdenum 21
Nickel 25
Selenium 27
MATERIALS AND METHODS 30
Materials 30
Bacterial Strains and Media 30
Isolation of Mutants 30
Enzyme Activities and their Respective Culture Conditions 36
Genetic and Molecular Biological Experiments 41
RESULTS AND DISCUSSION 53
Physiological Properties of an fhlB Mutant, Strain SE-2011 ... .53
Genetic Characteristics of Strain SE-2011, Q(fhlB'lacZ*) ... .64
Trans-acting Factors of the fhlB Operon 70
Analysis of the fhlA Gene 79


LIST OF ABBREVIATIONS
A Absorbance
Ac-CoA Acetyl coenzyme A
ATP Adenosine triphosphate
BV Benzyl viologen
CAT Chloramphenicol acetyl transferase
CRP cAMP repressor protein
CTAB Hexadecyltrimethyl ammonium bromide
DMS Dimethyl sulfide
DMSO Dimethyl sulfoxide
FDH-H Formate dehydrogenase (linked to H2
evolution)
FDH-N Formate dehydrogenase (linked to N03
reduction)
FHL Formate hydrogenlyase
FR Fumarate reductase
HUP Hydrogen uptake activity
HYD Hydrogenase
HYD-1 Hydrogenase isoenzyme-1
HYD-2 Hydrogenase isoenzyme-2
HYD-3 Hydrogenase isoenzyme-3
LB Luria Broth
vi i i


43
phage preparation. After 30 min at 25C, 1 ml of PI diluent was added
and the bacteria-phage mixture was vortexed. This was then centrifuged
(3,500 x g) for 5 min at 25C, resuspended in 0.1 ml of LB and incubated
for 1 hr at 25C before plating on selective medium for transductants
(25C). Tranduction of donor mutation was confirmed prior to and after
curing recipients off bacteriophage at 42C.
Conjugation. The F' complementation analysis was performed
according to Miller (156) using F'1431 (57 to 60 min), F126 (17 to 30
min), F' 116 (60 to 65 min), F' 128 (6 to 8 min; lacZ::lr\10), F 112 (90 to
98 min) and F1104 (0 to 7 min) provided by Dr. B. Bachmann. The Hfr-
mediated transfer of chromosomal DNA was accomplished by aerobically
(250 rpm) growing the donor and recipient strains to approximately 5 x
o
10 CFU/ml at 37C. Mating was initiated by mixing 1.0 ml of donor with
1.0 ml of recipient in 3.0 ml of fresh LBG (125 ml flask; 50 rpm).
Immediately upon removal, samples were vortexed, diluted in minimal
medium and centrifuged (3,500 x g) for 5 min at 25C. Exconjugants were
then selected on appropriate media.
Transformation by CaClg-heat shock method. Routine transformation
was performed by two methods, depending on the amount of competent cells
required. Both were a modification of the calcium chloride-heat shock
method described by Mandel and Higa (149) which was later established
for transforming episomal elements (43). The first method was basic and
expedient. An overnight LB culture of the desired host strain was
inoculated into fresh medium in 13 x 100 mm tubes (0.02 ml into 2.0 ml)


114
|£xoIII-9
GTTCTCTCGGTGAGTTTGGTGCAACCATCACCTTTGTGTCGAACATTCCTGGTGAAACGC 1860
|fxoIII-10
GAACCATTCCTTCTGCCATGTATACCCTGATCCAGACCCCCGGCGGGGAAAGTGGAGCGG 1920
CGAGACTGTGCATTATTTCTATTGCGCTGGCGATGATCTCCCTGTTGATTTCAGAATGGC 1980
TGGCCAGAATCAGCCGTGAACGGGCGGGGCGCTAATCATGCTGGAACTGAATTTTTCCCA 2040
|£xoIII-ll
GACGTTGGGCAACCATTGCCTGACTATTAATGAAACGCTGCCCGCCAATGGCATCACTGC 2100
|£xoIII-12
TATCTTTGGCGTCTCCGGTGCCGGAAAAACTTCGCTGATTAACGCCATCAGTGGACTGAC 2160
GCGCCCGCAAAAAGGGCGGATTGTCCTCAATGGGCGGGTACTAAATGATGCCGAAAAAGG 2220
TATCTGCCTGACGCCGGAAAAGCGTCGCGTTGGCTATGTTTTTCAGGATGCGCGGCTGTT 2280
|£xoIII-13
CCCGCATTACAAAGTGCGTGGCAATCTGCGCTACGGCATGTCGAAAAGTATGGTCGATCA 2340
GTTCGATAAGCTGGTGGCGCTTTTAGGCATTGAACCGTTGCTTGACCGTTTACCAGGCAG 2400
CCTGTCCGGAGGCGAAAAACAGCGCGTGGCGATTGGTCGGGCTTTGCTGACAGCACCGGA 2460
ATTGCTGTTGCTGGATGAACCGCTGGCGTCACTGGATATTCCGCGTAAACGCGAACTGTT 2520
GCCTTATCTGCAACGGCTGACACGGGAAATCAACATTCCGATGTTGTATGTCAGCCATTC 2580
GCTGGATGAGATCCTCCATCTGGCAGACAGAGTGATGGTACTGGAAAACGGTCAGGTGAA 2640
AGCCTTTGCGCTGGAGGAAGTGTGGGGCAGTAGCGTGATGAATCCGTGGCTGCCGAAAGA 2700
GCAACAAAGTAGCATTCTGAAAGTGACGGTGCTTGAGCATCATCCGCATTACGCGATGAC 2760
CGCGCTGGCGCTGGGCGATCAGCATTTGTGGGTCAATAAGCTGGACGAACCGCTGCAAGC 2820
TGCGCTACGTATCCGCATTCAGGCTTCCGATGTTTCTCTGGTTTTACAACCGCCGCAGCA 2880
AACCAGCATTCGTAACGTATTGCGGGCAAAAGTTGTTAATAGTTATGACGACAACGGCCA 2940
GGTGGAAGTGGAACTGGAAGTCGGCGGTAAAACGCTGTGGGCGCGTATCAGCCCGTGGGC 3000
CAGGGATGAACTGGCGATCAAACCTGGCCTGTGGCTGTACGCGCAAATTAAAAGTGTGTC 3060
GATAACCGCCTGATTAAATCAGGTGGCTATAAATGAACTGGGCAATGCTGTCGGTGGTGT 3120
TATCACCAATCACAATGTTGGCGCGCGCTTTTACCGCGTCATCGGCGTTGCCCATCGCCA 3180
fcoRV
CGCCTGTACCAGCGGCTTCCAGCATACTGATATCATTAAAGTATCGCCGAATGCCACGAC 3240
GTTCTCCATCGACCAACCTTGCGCCTCAACCCATTTCGTCAAACGTTTACCTTTGCTGTT 3300
GCCGCCGCGTGCAATATCAACCTGATCGTGCCAGGACCATTCACACTCCAGTCCCAGTTC 3360
AT GTTCGACAT GCTT ACCAAAAT GCTGCAATT GCCGCAGGT CATCGT GCGT CAGGGCGAA 3420
CTTCCATACGGCGTTAACTTGTTGCGCCGTTTCAGCCAGAGAAGCGACTTGTGTGAAAGT 3480
CGGACGCTGTTCCGGCGGCAGGGTTTGCGCCCAGTTAGATGTGCGAATGACATGCCCGGT 3540
CGGGTGCTCATAGACCATTGCATCATCGACATACATCAGACCGTGAATGTGGTGTTCATT 3600
CAGCCATCTCAATGAGTTGCAGGGCTTTAATAACGGGCATTGGGTCCGCTTCCAGCACGG 3660
TTTTTGCATGATAATCATACAAATAGGTGCCATTACAGCAAATAGCAGGTGTATCCAGCG 3720
CCAGCGCCT GAT AAAAAGGAT GAAT AGCGACGT GAT GGCGACCTGT GACGAT GATT AATT 3780
Ca I
GATAGCCTGCTTCGCGAGCGCGGGCCAGGGCTTCTATCGATGAAGGAAGCAGGGTCTTTT 3840
TCGGGGTCAATAAGGTGCCGTCTAAGTCGAGAGCAA 3876
Figure 15continued.


51
the manufacturers were followed. The DNA sequence was manipulated and
homology with other known sequences in the Genbank and EMBL library was
determined using the computer programs provided by Genetics Computer
Group, University of Wisconsin (56, 234) and Genepro (Hoeffer
Scientific).
DNA sequencing gels (both 21 x 50 cm and 38 x 50 cm) were poured
and run according to the procedures supplied by BioRad Laboratories with
modifications. A 6% polyacrylamide gel (acrylamide:bis 19:1) in 1 X TBE
(0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) with 8 M urea
was used to separate the reaction products. For increasing the length
of DNA sequence determined, a variety of successful techniques were
implemented. These included the use of wedge spacers provided by BioRad
laboratories, "double loading", and the addition of sodium acetate
(final concentration of approximately 0.1 M) to the bottom chamber in
order to form an electrolyte gradient (201).
Southern transfer and hybridization. Restriction endonuclease
digested chromosomal DNA was separated by electrophoresis in a vertical
1.0% agarose (Sigma type 1: low electroendosmosis grade) gel. The DNA
was stained with ethidium bromide and photographed for calculating the
fragment size of the hybridizing fragments. The DNA was depurinated
(0.25 M HC1 for 10 min) and denatured (0.5 N NaOH; 1 M NaCl for 30 min),
and then the gel was neutralized (0.5 M Tris-HCl pH 7.4; 3 M NaCl for 30
min). Southern transfer to Zeta-Probe membranes (Bio-Rad Laboratories)
was implemented using standard blotting techniques with 10 X SSC (1.5 M


96
"-24, -12"
SPB
FNR
S CKB
r
"-35,-10"
PC
h-H h
-HH
H H
hyp A B C
D E
fhlA
hydE
F B
lac
SE-2007
V
SE-2001 *
SE-2002 V
Figure 13. Localization of the promoter lac fusions in strains SE-2007
WfhlA'-'lacZ*)], SE2001 [fhyp'-
,lacZ+)2]. The complete hypABCDE operon with the proposed a- and FNR-
dependent promoters has been redrawn with modifications from Lutz et al.
(144). Previously identified hydE, hydF and hydB genes are noted (35,
187, 188, 238). Additionally, the weak "-35 and -10" constitutive
promoter upstream of the fhlA gene, suggested in this study, is also
indicated.


17
to other known DNA-binding proteins (receivers; ref. 81, 164, 220). The
NAR-Q protein was proposed by Egan and Stewart (60) as a second protein
kinase which could also activate NAR-L via phosphorylation at aspartate
residue 59; however, this role has not been confirmed. Mutations in the
narX gene confer variable phenotypes. In-frame deletions are
phenotypically indistinguishable from the wild type (59), while
narX::Jn5 and point mutants relieve (to varying levels) nitrate
inhibition at the frd operon (60, 118). Kalman and Gunsalus (118) have
sequenced three such point mutants which no longer require nitrate for
repression and have localized these single-amino acid changes to an 11-
residue domain. Molybdate is required for frd operon repression in one
*
out of the three narX strains tested, suggesting that NAR-X may have an
additional role in molybdenum sensing. The narL gene function is less
ambiguous, since all mutants studied to date render frd operon
expression nitrate insensitive (104, 117).
When cells are grown in buffered, rich medium with glucose, pfl
operon expression is repressed approximately 2.5-fold in the presence of
nitrate. The repression is directly meditated by NAR-L and not relieved
by the addition of formate (190). The physiological significance of
this partial NAR-L repression is unclear since formate is the substrate
for nitrate respiration. The NAR-L protein may serve as a modulator of
formate levels at the site of pfl transcription to prevent formic acid
accumulation during nitrate respiration. Transcription of the hyc (hyd-
17) and fdhF genes are repressed in the presence of nitrate (171);


124
46. Cole, J. A., and J. W. T. Wimpenny. 1966. The interrelationships
of low redox potential cytochrome c552 and hydrogenase in
facultative anaerobes. Biochim. Biophys. Acta. 128:419-425.
47. Cole, S. T., and J. R. Guest. 1979. Amplification and aerobic
synthesis of fumarate reductase in ampici11 in-resistant mutants of
Escherichia coli K-12. FEMS Microbiol. Lett. 5:65-67.
48. Coppard, J. R., and M. J. Merrick. 1991. Cassette mutagenesis
implicates a helix-turn-helix motif in promoter recognition by the
novel RNA polymerase sigma factor o. Mol. Microbiol.
5:1309-1317.
49. Cornish, E. C., V. P. Argyropoulos, J. Pittard, and B. E.
Davidson. 1986. Structure of the Escherichia coli K12 regulatory
gene tyrR. Nucleotide sequence and sites of initiation of
transcription and translation. J. Biol. Chem. 261:403-410.
50. Cotter, P. A., V. Chepuri, R. B. Gennis, and R. P. Gunsalus. 1990.
Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in
Escherichia coli is regulated by oxygen, pH, and the fnr gene
product. J. Bacteriol. 172:6333-6338.
51. Cotter, P. A., and R. P. Gunsalus. 1989. Oxygen, nitrate, and
molybdenum regulation of dmsABC gene expression in Escherichia
coli. J. Bacteriol. 171:3817-3823.
52. Cronan, J. E. Jr., R. B. Gennis, and S. R. Maloy. 1987.
Cytoplasmic membrane, p. 31-55. In F. C. Neidhardt, J. L.
Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E.
Umbarger (ed.), Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology. Am. Soc. Microbiol., Washington,
DC.
53. Debarbouille, M., I. Martin-Verstraete, A. Klier, and G. Rapoport.
1991. The transcriptional regulator LevR of Bacillus subtilis has
domains homologous to both o and phosphotransferase
system-dependent regulators. Proc. Natl. Acad. Sci. USA
88:2212-2216.
54. de Bruijn, F. J., and F. Ausubel. 1983. The cloning and
characterization of the glnF (ntrA) gene of Klebsiella pneumoniae:
role of glnF (ntrA) in the regulation of nitrogen fixation (nif)
and other nitrogen assimilation genes. Mol. Gen. Genet. 192:342-
353.
55. del Campillo-Campbell, A. and A. Campbell. 1982. Molybdenum


138
198. Shaw, D. J., and J. R. Guest. 1982. Amplification and product
identification of the fnr gene of Escherichia coli. J. Gen.
Microbiol. 128:2221-2228.
199. Shaw, D. J., D. W. Rice, and J. R. Guest. 1983. Homology between
CAP and FNR, a regulator of anaerobic respiration in Escherichia
coli. J. Mol. Biol. 166:241-247.
200. Shaw, W. V. 1975. Chloramphenicol acetyl transferase from
chloramphenicol-resistant bacteria. Meth. Enzymol. 43:737-755.
201. Sheen, J. Y., and B. Seed. 1988. Electrolyte gradient gels for DNA
sequencing. BioTechniques 6:942-944.
202. Shimokawa, 0., and M. Ishimoto. 1979. Purification and properties
of inducible tertiary amine N-oxide reductase from Escherichia
coli. J. Biochem. 86:1709-1717.
203. Showe, M. K., and J. A. DeMoss. 1968. Localization and regulation
of synthesis of nitrate reducatse in Escherichia coli. J.
Bacterid. 95:1305-1313.
204. Siegele, D. A., J. C. Hu, W. A. Walter, and C. A. Gross. 1989.
Altered promoter recognition by mutant forms of the o70 subunit of
Escherichia coli RNA polymerase. J. Mol. Biol. 206:591-603.
205. Smith, M. W. and F. C. Neidhardt. 1983. Proteins induced by
anaerobiosis in Escherichia coli. J. Bacterid. 154:336-343.
206. Snoep, J. L., M. Joost, T. de Mattos, and 0. M. Neijssel. 1991.
Effect of the energy source on the NADH/NAD ratio and on pyruvate
catabolism in anaerobic chemostat cultures of Enterococcus
faecalis NCTC 775. FEMS Microbiol. Lett. 81:63-66.
207. Sperl, G. T., and J. A. DeMoss. 1975. chlD gene function in
molybdate activation of nitrate reductase. J. Bacteriol.
122:1230-1238.
208. Spencer, M. E., and J. R. Guest. 1973. Isolation and properties of
fumarate reductase mutants of Escherichia coli. J. Bacteriol.
114:563-570.
209. Spiro, S., and J. R. Guest. 1987. Regulation and over-expression
of the fnr gene of Escherichia coli. J. Gen. Microbiol. 133:3279-
3288.
210. Spiro, S., and J. R. Guest. 1988. Inactivation of the FNR protein


82
MGCTAATAAAATTCTAAATCTCCTATAGTTAGTCAATGACCTTTTGCACCGCTTTGCGG 2400
0 ******
TGCTTTCCTGGAAGAACAAAATGTCATATACACCGATGAGTGATCTCGGACAACAAGGGT 2460
MSYTPMSDLGQQGL
TGTTCGACATCACTCGGACACTATTGCAGCAGCCCGATCTGGCCTCGCTGTGTGAGGCTC 2520
FDITRTLLQQPDLASLCEAL
TTTCGCAACTGGTAAAGCGTTCTGCGCTCGCCGACAACGCGGCTATTGTGTTGTGGCAAG 2580
SQLVKRSALADNAAIVLWQA
CGCAGACTCAACGTGCGTCTTATTACGCGTCGCGTGAAAAAGACACCCCCATTAAATATG 2640
QTQRASYYASREKDTPI KYE
AAGACGAAACTGTTCTGGCACACGGTCCGGTACGCAGCATTTTGTCGCGCCCTGATACGC 2700
DETVLAHGPVRSI LSRPDTL
TGCATTGCAGTTACGAAGAATTTTGTGAAACCTGGCCGCAGCTGGACGCAGGTGGGCTAT 2760
HCSYEEFCETWPQLDAGGLY
ACCCAAAATTTGGTCACTATTGCCTGATGCCACTGGCGGCGGAAGGGCATATTTTTGGTG 2820
PKFGHYCLMPLAAEGHIFGG
GCTGTGAATTTATTCGTTATGACGATCGCCCCTGGAGCGAAAAAGAGTTCAATCGTCTGC 2880
CEFIRYDDRPWSEKEFNRLQ
Hpal
AAACATTTACGCAGATCGTTTCTGTCGTCACCGAACAAATCCAGAGCCGCGTCGTTAACA 2940
TFTQIVSVVTEQIQSRVVNN
Sail
ATGTCGACTATGAGTTGTTATGCCGGGAACGCGATAACTTCCGCATCCTGGTCGCCATCA 3000
VDYELLCRERDNFRILVAIT
CCAACGCGGTGCTTTCCCGCCTGGATATGGACGAACTGGTCAGCGAAGTCGCCAAAGAAA 3060
NAVLSRLDMDELVSEVAKEI
TCCATTACTATTTCGACATTGACGATATCAGTATCGTCTTACGCAGCCACCGTAAAAACA 3120
HYYFDIDDISIVLRSHRKNK
AACTCAACATCTACTCCACTCACTATCTTGATAAACAGCATCCCGCCCACGAACAGAGCG 3180
LNIYSTHYLDKQHPAHEQSE
AAGTCGATGAAGCCGGAACCCTCACCGAACGCGTGTTCAAAAGTAAAGAGATGCTGCTGA 3240
VDEAGTLTERVFKSKEMLLI
TCAATCTCCACGAGCGGGACGATTTAGCCCCCTATGAACGCATGTTGTTCGACACCTGGG 3300
NLHERDDLAPYERMLFDTWG
GCAACCAGATTCAAACCTTGTGCCTGTTACCGCTGATGTCTGGCGACACCATGCTGGGCG 3360
NQIQTLCLLPLMSGDTMLGV
TGCTGAAACTGGCGCAATGCGAAGAGAAAGTGTTTACCACTACCAATCTGAATTTACTGC 3420
LKLAQCEEKVFTTTNLNLLR
GCCAGATTGCCGAACGTGTGGCAATCGCTGTCGATAACGCCCTCGCCTATCAGGAAATCC 3480
QIAERVAIAVDNALAYQEIH
ATCGTCTGAAAGAACGGCTGGTTGATGAAAACCTCGCCCTGACCGAGCAGCTCAACAATG 3540
RLKERLVDENLALTEQLNNV
TTGATAGTGAATTTGGCGAGATTATTGGCCGCAGCGAAGCCATGTACAGCGTGCTTAAAC 3600
DSEFGEI IGRSEAMYSVLKQ
Figure 9--continued.


58
Upon detailed analysis of fhlB gene expression, monitored at
specific time intervals, it was found that there was an exponential
increase in p-galactosidase activity which paralleled the growth. The
specific activity of the culture reached maximum value during early
stationary phase and remained constant over an additional 8 hr of
incubation (data not shown). In LB medium, maximum activity observed
was about 250 U (Fig. 3). In this medium, the increase in specific
activity of the enzyme was coupled to growth and the differential rate
of induction was about 1.0. In LB medium supplemented with glucose, the
differential rate of p-galactosidase production increased exponentially
during growth, probably due to continued production of formate by the
growing culture (since the amount of formate produced by the culture is
proportional to the cell density). The maximum activity reached was
about 600 units during the early stationary phase of growth when the
cell density was about 100 pg protein/ml. With the addition of formate
to the medium, the growth rate and final cell yield decreased, although
the differential rate of synthesis of p-galactosidase was enhanced to as
high as 130 units/pg cell protein. In this medium, the maximum activity
produced by the culture increased to about 1,200 units. In LB medium
supplemented with both glucose and formate, the differential rate of
induction of p-galactosidase activity was similar to the values obtained
with LB-formate cultures but the total amount of the enzyme produced by
strain SE-2011 was slightly higher (about 1,400 units). The final cell
yield of the culture in the latter two media was comparable. These


130
Molybdenum cofactor biosynthesis in Escherichia coli: requirement
of the chlB gene product for the formation of molybdopterin
guanine dinucleotide. J. Biol. Chem. 266:12140-12145.
113. Johnson, J. L., and K. V. Rajagopalan. 1982. Structural and
metabolic relationship between the molybdenum cofactor and
urothine. Proc. Natl. Acad. Sci. USA 79:6856-6860.
114. Johnson, M. E., and K. V. Rajagopalan. 1987. Involvement of chlA,
£, M and N loci in Escherichia coli molybdoprotein biosynthesis.
J. Bacteriol. 169:117-125.
115. Jones, H. M., and R. P. Gunsalus. 1987. Regulation of Escherichia
coli fumarate reductase (frdABCD) operon expression by respiratory
electron acceptors and the fnr gene product. J. Bacteriol.
169:3340-3349.
116. Jones, R. W. 1980. The role of the membrane-bound hydrogenase in
the energy-conserving oxidation of molecular hydrogen by
Escherichia coli. Biochem. J. 188:345-350.
117. Kalman, L. V., and R. P. Gunsalus. 1989. Identification of a
second gene involved in global regulation of fumarate reductase
and other nitrate-controlled genes for anaerobic respiration in
Escherichia coli. J. Bacteriol. 171:3810-3816.
118. Kalman, L. V., and R. P. Gunsalus. 1990. Nitrate- and molybdenum-
independent signal transduction mutations in narX that alter
regulation of anaerobic respiratory genes in Escherichia coli. J.
Bacteriol. 172:7049-7056.
119. Karube, I., M. Tomiyama, and A. Kikuchi. 1984. Molecular cloning
and physical mapping of the hyd gene of Escherichia coli K-12.
FEMS Microbiol. Lett. 25:165-168.
120. Kenner, J., and S. Kustu. 1988. Protein kinase and phosphoprotein
phosphatase activities of nitrogen regulatory proteins NTRB and
NTRC of enteric bacteria: roles of the conserved amino-terminal
domain of NTRC. Proc. Natl. Acad. Sci. USA 85:4976-4980.
121. Khoury, G., and P. Gruss. 1983. Enhancer elements. Cell
33:313-314.
Knappe, J. 1987. Anaerobic dissimilation of pyruvate, p. 151-155.
In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M.
Schaechter, and H. E. Umbarger (ed.), Escherichia coli and
Salmonella typhimurium: Cellular and Molecular Biology. Am. Soc.
122.


Ill
Table 11. The effect of molybdenum on expression of (fhlB'-'lacZ+)
mutant, strain SE-2011 in chlD and molR genetic backgrounds
0-Galactosidase Activity3
ImM Mo

+

+
Strain
Relevant genotype pSE133b


+
+
SE-2011
Q(fhlB'-'lacZ*)
700
700
1400
1500
MJ-40
4>(//?Zfl'-' lacZ+) molR
130
640
720
1400
SE1714
(fhlB'-'lacZ*) chlD
160
700
900
1700
All cultures were anaerobically grown at 37C in LB medium supplemented
with 3 mM formate except SE1714 which was grown in 15 mM formate.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
Multicopy plasmid carrying the fhlA+ gene
Values represent maxiumum activity reached during 4 hours of anaerobiosis.


INTRODUCTION
The production of dihydrogen by the members of the
Enterobacteriaceae family has been known since 1901 (166). Although
much has been learned since that time, many avenues of research remain
to be explored. It is now understood that the formate hydrogenlyase
complex is composed of FDH-H, HYD-3, and associated electron transport
proteins (10, 27, 72, 173, 193, 215, 243). This complex catalyzes the
oxidation of formate to H2 and C02 (2, 166) and is found only in
anaerobically growing cells. The reaction catalyzed by the FHL complex
operates at a standard free energy (AG0') of zero and does not appear to
be involved in cellular energy production (2). It is presumed that the
FHL system plays an important role in later stages of the
microorganism's fermentative growth when organic acids such as formate
accumulate and lower the pH of the culture medium. The cell apparently
synthesizes the FHL complex in order to modulate the external pH, thus
facilitating growth (77).
The FHL pathway is an attractive research field due to the
potential of harnessing dihydrogen production as an alternative energy
source. Current events, including the Persian Gulf War and concern over
the greenhouse effect, have aroused the public's attention. Although
lobbyists continue to solicit Congress for the oil industry, the
1


103
about 20 U of pgal actos i dase activity. Upon transfer to anaerobic
conditions, hyd (hyp) operon expression was induced at a rate which
paralleled the growth of the culture in LB medium. The specific
activity of the enzyme reached maximum values of 170 U for SE2001 and
190 U for SE-2002 (LB; Table 10). This peak value was reached during
early stationary phase of growth (approximately 0.03 units A420nm or 10
pg cell protein) and remained constant throughout the remaining assay
period (5 hr total). Thus, the increase in specific activity of the
enzyme was coupled to growth (differential rate of induction approaching
1.0). In LB supplemented with glucose, formate or nitrate, the maximal
pgal actos i dase activity produced by strain SE2001 was not
significantly altered. The enzyme activity produced by strain SE-2002
also remained relatively constant in cells grown in LBG or LBN medium.
In medium supplemented with 15 mM formate, the enhancing effect was only
observed in strain SE-2002 in which the enzyme activity was slightly
higher. These results are in direct contrast to the formate
inducibility of the fhlB operon in which the levels of p-galactosidase
activity were increased by 3.5-fold in the presence of 15 mM formate,
and endogenously produced formate also had an enhancing effect.
To study the role of known effectors of dihydrogen metabolism,
appropriate double mutants were constructed and p-galactosidase activity
was monitored over a 5 hr time period. The only mutation which was
consistent in its effect on hyd (hyp) operon expression was fnr. The
fnr derivatives of strains SE-2001 and SE-2002 are strains SE-1651 and


137
187. Sankar, P., and K.T. Shanmugam. 1988. Biochemical and genetic
analysis of hydrogen metabolism in Escherichia coli: the hydB
gene. J. Bacteriol. 170:5433-5439.
188. Sankar, P., and K.T. Shanmugam. 1988. Hydrogen metabolism in
Escherichia coli: biochemical and genetic evidence for a hydF
gene. J. Bacteriol. 170:5446-5451.
189. Saracino, L., M. Violet, D. H. Boxer, and G. Giordano. 1986.
Activation in vitro of respiratory nitrate reductase of
Escherichia coli K12 grown in the presence of tungstate:
involvement of molybdenum cofactor. Eur. J. Biochem. 158:483-490.
190. Sawers, G., and A. Bock. 1988. Anaerobic regulation of pyruvate
formate-lyase from Escherichia coli K-12. J.Bacteriol.
170:5330-5336.
191. Sawers, G., and A. Bock. 1989. Novel transcriptional control of
the pyruvate formate-lyase gene: upstream regulatory sequences
and multiple promoters regulate anaerobic expression. J.
Bacteriol. 171:2485-2498.
192. Sawers, G., J. Heider, E. Zehelein, and A. Bock. 1991. Expression
and operon structure of the sel genes of Escherichia coli and
identification of a third selenium-containing formate
dehydrogenase isoenzyme. J. Bacteriol. 173:4983-4993.
193. Sawers, R. G., S. P. Ballantine, and D. H. Boxer. 1985.
Differential expression of hydrogenase isoenzymes in Escherichia
coli K-12: evidence for a third isoenzyme. J. Bacteriol.
164:1324-1331.
194. Sawers, R. G., and D. H. Boxer. 1986. Purification and properties
of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown
Escherichia coli K12. Eur. J. Biochem. 56:265-275.
195. Sayavedra-Soto, L. A., G. K. Powell, H. J. Evans, and R. 0.
Morris. 1988. Nucleotide sequence of the genetic loci encoding
subunits of Bradyrhizobium japonicum uptake hydrogenase. Proc.
Natl. Acad. Sci. USA 85:8395-8399.
196. Schlensog, V. A. Birkmann and A. Bock. 1989. Mutations in trans
which affect the anaerobic expression of a formate dehydrogenase
(fdhF) structural gene. Arch. Microbiol. 152:83-89.
197. Shaw, D. J., and J. R. Guest. 1981. Molecular cloning of the fnr
gene of Escherichia coli K-12. Mol. Gen. Genet. 181:95-100.


94
Regulation of the fhlA Gene
Localization of an fhlA gene fusion. In order to study the
regulation of fhlA gene, mutant strain SE-2007 was isolated as a lac
operon fusion derivative of strain MC4100, using AplacMu53 (29). Upon
detailed analysis, this strain was found to be defective in the
production of dihydrogen. Plasmid complementation studies suggested
that the fusion was located in the fhlA gene. The FHL activity of
strain SE-2007 was restored by plasmids pSE-133 and pSE-133-1
[hydB::Tn5) but not by pSE133-2 [fhlA::Tn5). Construction of a Hfr
PO(fhlA) suggested the fusion mapped between the srl (58 min) and cys
(59 min) genes; however, chromosomal transfer implied that direction of
transcription was in the opposite orientation from that of the DNA
sequence data. To eliminate the possibility of fhlA gene in high copy
suppressing a mutation in a different gene in the 58 to 59 min region,
restriction endonuclease digests of chromosomal DNA from strain SE-2007
were probed with an internal Sail to Pst I fragment of the fhlA gene
(2,946 to 4,269 bases; Fig. 9). Initial results with Ca I "digests" of
chromosomal DNA from strain SE-2007 demonstrated that the lac fusion was
within the Ca I fragment (positions 800 to 4601; Fig. 9); the
hybridizing fragments were approximately 3.7 kb (strain MC4100) and
7.5 kb (strain SE-2007). Further analysis with Sall-Bgll, Sall-EcoRl,
and SoZI-CZoI digests of chromosomal DNA verified that the fusion was
within the fhlA gene between the Pstl and Bgll sites at the 3' end of


93
length, but the 17 base pairs region between this secondary structure
and the start codon of the fhlA gene is 65% AT rich. Whether this is
physiologically important has not been determined. The hydB gene
product was extremely hydrophobic with a hydrophilic region in the
amino-terminus. No significant homology of any hyd {hyp) genes
sequenced could be found with known sequences in the GenBank or EMBL
data bases and thus the potential mechanism by which these proteins
participate in H2 metabolism cannot be deduced.


49
Table 2.
continued
Plasmid
Relevant Genotype or Phenotype
Reference
pSE1009
Apr, 2.9 kb Kpnl-EcoRV fragment from
pSE-1007, complementing molR and chlD
mutants, in pUC19
Laboratory
collection (136)
pSE-1009
Exo #1-13
Apr, ExoIII deletions from Kpnl, in pUC19
This study
pSE-1004
Apr, 4.7 kb Pvull-Clal fragment from
pSE-1001, complementing molR and chlD
mutants, in pBR322
Laboratory
collection (136)
pSE1213
Apr, 6.5 kb Sau3M fragment with
partial hyb operon in pBR-322
Laboratory
collection
33pBR
Tcr, 6.2 kb Pstl-EcoRI fragment with
complete hyb operon
A. E. Przybyla
pSV208
Apr, Cmr, EcoRl-BamHl promoter fragment
from pBN208 [Apr, 240 bp upstream of fdhF] into pKK232-8
A. Bock (196)


8
electron transport pathways are not synthesized in the presence of
dioxygen. Furthermore when nitrate is present in an anaerobic
environment, the nitrate respiratory pathway is induced and alternate
respiratory systems are repressed (for review, see 82, 102, 139, 175).
Presumably, a complex interconnected regulation involving transcription,
translation, protein processing, and allosteric effectors is
conceivable.
Respiration involves the generation of a proton motive force
(PMF), consisting of a pH gradient (ApH) and electrochemical gradient
(Aip), by electron transport (for review, see 84, 160). In general,
respiratory components are organized into "modules" or pathways. These
include a substrate dehydrogenase, which transfers electrons to a
quinone pool, and ultimately a membrane-bound reductase; thus coupling
substrate oxidation to the reduction of an electron acceptor (139). A
fermenting cell, on the other hand, produces endogenous electron
acceptors and derives most of its energy from substrate-level
phosphorylation (42). Although it is generally believed that the FHL
system is a fermentative process discrete from anaerobic respiration,
the precise function of the FHL pathway remains unclear.
E. coli is a mixed-acid fermentor producing acetate, lactate,
formate, ethanol, succinate, H2 and C02 (Fig. 2). The ratio of these
major end products is regulated (conceivably by redox-mediated
modulation) to balance the reducing equivalents generated during
glycolysis (3, 206). All of these fermentation products are derived


14
159), DMSO:TMAO reductase (216, 232), PFL (190, 191), FDH-N (19, 25);
anaerobic glycerol-3-phosphate dehydrogenase (130), fumarase B (18,
237), aspartase, and asparaginase II (109). The FNR protein represses
its own synthesis (autoregulation; ref. 209) as well as cytochrome o
(low-affinity oxidase; ref. 50, 68) and NADH dehydrogenase II (213).
Recent evidence is conflicting on FNR regulation at the cyd promoter.
By monitoring cyd expression at varying levels of dissolved dioxygen
concentrations, Fu et al. (68) suggest that FNR, a weaker
transcriptional activator at this operon, outcompetes ARC-A for operator
binding as the culture shifts from microaerobic to anaerobic conditions.
The FNR protein has been shown through sequence analysis to be
homologous to cAMP receptor protein (CRP) in the helix-turn-helix and
nucleotide binding domains (199). However, the FNR amino-terminus
differs. It is cysteine rich and three of the four cysteine residues
are proposed to be involved in redox-sensitive iron-binding (210). The
addition of ferrozine, an iron specific chelator, to the growth medium
reduced the level of transcription of the frd operon (encoding fumarate
reductase). Since this operon is positively regulated by FNR, iron in
association with FNR may be involved in this regulation. In support of
this possibility, Green et al. (79) purified FNR in the presence of
externally added iron and demonstrated specific in vitro binding of the
pure protein to a synthetic (postulated) FNR-binding site by both DNasel
and methylation-protection (DMS) footprinting. Although direct evidence
is still lacking, parallels can be drawn with the thoroughly


34
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
SE1655
(hyp'-' lacZ+)2, nor/,215: :Tni0
PI transduction
(SE-2002 x RK5278)
SE1657
(/?yp'-'lacZ+)\i rpoNulnlO
PI transduction
(SE-2001 x YMC18)
SE1658
Q(hyp'- lacZ*) 1, rpoN::lr\10
PI transduction
(SE-2001 x YMC18)
SE1659
[hyp'-' lacZ+)2i rpoN: :lnlO
PI transduction
(SE-2002 x YMC18)
SE1660
4>(/jyp'-'lacZ+)2, rpoN::JnlO
PI transduction
(SE-2002 x YMC18)
SE-1704
moZ/?::Tn5, zgg::lr\10
Laboratory
collection
SE1714
QtfhlB'-'lacZ*), chlD::lnlO
PI transduction
(SE-2011 x VJS720)
SE1760
${hyp'-'lacZ*) 1, chlD::lr\10
PI transduction
(SE-2001 x VJS720)
SE1761
(hyp'-'lacZ+)2, chlD::lnlO
PI transduction
(SE-2002 x VJS720)
SE1762
PI transduction
(SE-2007 x VJS720)
SE-2001
MC4100, (hyp'-'lacZ+)l
This study
SE-2002
MC4100, This study
SE-2007
MC4100, QifhlA'-'lacZ*)
This study
SE-2009
MC4100, (hydC'-'lacZ+)
This study
SE2011
MC4100, Q{fhlB'lacZ*)
This study
VJS720
chlDalnlO
V. Stewart


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
GENETIC REGULATION OF FORMATE HYDROGENLYASE IN ESCHERICHIA COLI
By
Julie Anne Maupin
December, 1991
Chairman: Dr. K. T. Shanmugam
Major Department: Microbiology and Cell Science
A new gene (fhlB) whose product regulates the production of
formate hydrogenlyase (FHL) has been identified in Escherichia coli.
Biochemical analysis of a mutant, strain SE-2011 [$>(//IB'-' lacZ*)],
revealed that this mutant lacks formate dehydrogenase activity
associated with FHL (FDH-H) and hydrogenase activity. As a consequence,
dihydrogen production and uptake were undetectable in strain SE-2011.
Expression of the fhlB gene (measured as p-galactosidase activity) was
increased 2- to 3-fold by anaerobic conditions, and enhanced by formate,
but only under anaerobic conditions. Maximum expression of (fhlB'~
'lacZ+) required rpoN, fhlA, chlD, and molR gene products. The
concentration of formate required for maximum expression of the fhlB
gene was reduced in the presence of fhlA gene in a multicopy plasmid
from about 15 mM to 3 mM. The fhlB gene in molR or chlD genetic
xiv


133
coli. Mol. Microbiol. 5:123-135.
145. Lutz, S., R. Bohm, A. Beier, and A. Bock. 1990. Characterization
of divergent NtrA-dependent promoters in the anaerobically
expressed gene cluster coding for hydrogenase 3 components of
Escherichia coli. Mol. Microbiol. 4:13-20.
146. MacGregor, C. H., and C. A. Schanitman. 1973. Reconstitution of
nitrate reductase activity and formation of membrane particles
from cytoplasmic extracts of chlorate-resistant mutants of
Escherichia coli. J. Bacteriol. 114:1164-1176.
147. MacNeil, T., G. P. Roberts, D. MacNeil, and B. Tyler. 1982. The
products of glnL and glnG are bifunctional regulatory proteins.
Mol. Gen. Genet. 188:325-333.
148. Macy, J., H. Kulla, and G. Gottschalk. 1976. H2-dependent
anaerobic growth of Escherichia coli on malate: succinate
formation. J. Bacteriol. 125:423-428.
149. Mandel, M., and A. Higa. 1970. Calcium dependent bacteriophage DNA
infection. J. Mol. Biol. 53:159-162.
150. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
151. Menon, N. K., J. Robbins, J. C. Wendt, K. T. Shanmugam, H. D.
Peck, Jr., and A. E. Przybyla. 1991. Mutational analysis and
characterization of the E. coli hya operon encoding [NiFe]
hydrogenase-1. J. Bacteriol. 173:4851-4861.
152. Merrick, M. J., J. Gibbins, and A. Toukdarian. 1987. The
nucleotide sequence of the sigma factor gene ntrA (rpoN) of
Azotobacter vinelandii: analysis of conserved sequences in NtrA
protein. Mol. Gen. Genet. 210:323-330.
153. Merrick, M., S. Hill, H. Henecke, M. Hahn, R. Dixon, and C.
Kennedy. 1982. Repressor properties of the nifL gene product in
Klebsiella pneumoniae. Mol. Gen. Genet. 185:75-81.
154. Merrick, M. J., and W. D. P. Stewart. 1985. Studies on the
regulation and function of the Klebsiella pneumoniae ntrA gene.
Gene 35:297-303.
155. Miller, J. B., D. J. Scott, and N. K. Amy. 1987. Molybdenum-
sensitive transcriptional regulation of the chlD locus of


60
experiments clearly show that the transcription of the fhlB operon is
dependent on formate, either produced internally or added externally to
the medium.
The amount of p-galactosidase activity produced by the Q(fhlB-
,lacZ+) strain increased linearly with increasing formate concentration
up to about 5 mM (Fig. 4). The activity continued to increase at a
lower rate until the maximum was reached at about 15 mM formate in the
medium. For these experiments, strain SE-2011 was grown under anaerobic
conditions, in LB-formate medium, and the cells were harvested after 4
hr for enzyme assays. Similar results were also obtained with strain
MJ-9 [(fhlB'-' lacZ*), pfl] which lacks the ability to produce formate
internally due to a loss of pyruvate formatelyase activity. At 15 mM
formate, strain MJ-9 produced only about 60% of the p-galactosidase
activity observed in the pfl+ parent strain. At higher formate
concentrations (about 30 mM), the specific activity of p-galactosidase
detected in strain MJ-9 was comparable to the pfl+ strain. These
results suggest that the internally produced formate plays a significant
role in the transcription of the fhlB operon.
Nitrate repression of fhlB gene expression. Further analysis of
the repressive effect of nitrate on fhlB operon expression, was carried
out by monitoring the p-galactosidase activity and nitrite produced by
strain SE-2011 over a 6 hr growth period in an anaerobic environment.
The fhlB mutant was grown in LB, LB-formate, LB-nitrate and LB-formate-
nitrate medium (LB, LBF, LBN and LBFN respectively; Fig. 5). Nitrite


95
the open reading frame (positions 4,269 and 4,528 in Fig. 9; Fig. 13).
This again reemphasizes the necessity of the carboxy-terminus for
biological activity of the FHL-A protein.
Anaerobic induction of the fhlA gene. Regulation of expression of
the fhlA gene was monitored by measuring the levels of p-galactosidase
activity produced by strain SE-2007. When cultured under comparable
aerobic conditions to mutant strain SE-2011, maximal dioxygen repression
of fhlA gene expression was not obtained. Therefore, alternate
procedures were implemented to maximally aerate the culture at low cell
density (Table 7).
When cultured under microaerobic conditions, an overnight,
stationary (1 ml in 13 x 100 mm tube with a metal-cap) culture of strain
SE-2007 produced about 1,800 U of p-galactosidase activity (Table 7).
Upon transfer to "strict" aerobic conditions (10 ml LB in 125 ml flask
with 0.1 ml of overnight culture as inoculum; shaking at 250 rpm; 2 hr),
utilized routinely for culturing other laboratory strains, the p-
galactosidase activity of the culture decreased approximately 2-fold to
about 1,000 units. Subsequent transfer of this culture to fresh LB and
then aerobic growth for 1 hr further reduced the specific activity of
the enzyme over 3-fold (290 U). This aerobic culture was used to
inoculate LB and grown under anaerobic conditions. After 4 hr
incubation, the specific activity of pgal actos i dase of the culture was
comparable to initial "microaerobic" values. These results indicate


LITERATURE REVIEW
Facultative anaerobes, such as Escherichia coli, have evolved
respiratory networks organized such that the most energetically
favorable electron transport pathway is utilized for the specific redox
state of the environment. E. coli is capable of using a variety of
electron acceptors which include in order of decreasing potential:
l/202/H20 (E0'= +0.82 V), N03"/N02" (E0'= +0.42 V), N02"/NH4+
(E0'= +0.36 V), DMSO/DMS (EQ' = +0.16 V), TMAO/TMA (E0'= +0.13 V),
fumarate/succinate (E0'= +0.03 V), pyruvate/lactate (E0'= -0.19 V),
acetaldehyde/ethanol (E0*= -0.2 V), H+/H2 (E0' = -0.42 V; ref. 16, 228).
Pathway preference is determined by the difference between the standard
oxidation-reduction potential of the initial electron-donor (NADH) and
terminal acceptor system (AE0') available in the growth medium. The
standard free energy change (AG0) of a reaction is given by
AG0I= -nfAE0'
where n is the number of electrons transferred and F is Faraday's
constant. Therefore the greater the AE0, the more free energy can be
harnessed by the organism for biosynthetic purposes.
This establishes a hierarchy with aerobic respiration being the
most energetically favorable for the cell's survival. Thus, other
7


136
o54-dependent flagellar gene promoters. Proc. Natl. Acad. Sci. USA
87:2369-2373.
177. Reeve, J. N., G. S. Beckler, D. S. Cram, P. T. Hamilton, J. W.
Brown, J. A. Krzycki, A. F. Kolodziej, L. Alex, W. H. Orme-
Johnson, and C. T. Walsh. 1989. A hydrogenase-1inked gene in
Methanobacterium thermoautotrophicum strain AH encodes a
polyferredoxin. Proc. Natl. Acad. Sci. USA 86:3031-3035.
178. Reiss, J. A. Kleinhofs and W. Klingmuller. 1987. Cloning of seven
differenly complementing DNA fragments with chi functions from
Escherichia coli K12. Mol. Gen. Genet. 206:352-355.
179. Reitzer, L. J., and B. Magasanik. 1986. Transcription of glnA in
E. coli is stimulated by activator bound to sites far from the
promoter. Cell 45:785-792.
180. Riviere, C. G. Giordano, J. Pommier and E. Azoulay. 1975. Membrane
reconstitution in chl-r mutants of Escherichia coli K12 VIII.
Purification and properties of the FA factor, the product of the
chlB gene. Biochim. Biophys. Acta. 389:219-235.
181. Ronson, C. W., P. M. Astwood, B. T. Nixon, and F. M. Ausubel.
1987. Deduced products of C-4 dicarboxylate transport regulatory
genes of Rhizobioum leguminosarum are homologous to nitrogen
regulatory gene products. Nucleic Acids Res. 15:7921-7934.
182. Ronson, C. W., B. T. Nixon, L. M. Albright, and F. M. Ausubel.
1987. Rhizobium meliloti ntrA (rpoN) gene is required for diverse
metabolic functions. J. Bacteriol. 169:2424-2431.
183. Ronson, C. W., B. T. Nixon, and F. M. Ausubel. 1987. Conserved
domains in bacterial regulatory proteins that respond to
environmental stimuli. Cell 49:579-581.
184. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing
with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA
74:5463-5467.
185. Sankar, P., J. H. Lee, and K. T. Shanmugam. 1985. Cloning of
hydrogenase genes and fine structure analysis of an operon
essential for H2 metabolism in Escherichia coli. J. Bacteriol.
162:353-360.
186. Sankar, P., J. H. Lee, and K. T. Shanmugam. 1988. Gene product
relationships of fhlA anb fdv genes of Escherichia coli. J.
Bacteriol. 170:5440-5445.


54
Table 3. Biochemical characteristics of strain MC4100 and an fhlB
mutant, strain SE-2011
Specific activity
Enzyme MC4100 SE-2011
(parent) <\>(fhlB'-' lacZ*)
Hydrogenase3
J Q 3
( H2-exchange)
1,200
44
Hydrogen uptake
(H2 to BV)b
700
29
(H2 to fumarate)0
290
UD9
Formate hydrogenlyased
170
UD
Formate dehydrogenase-He
490
38
Fumarate reductasef
600
100
o 3
Expressed as nanomoles of H20 produced per minute per milligram of cell
protein.
Expressed as nanomoles of BV reduced per minute per milligram of cell
protein.
Expressed as nanomoles of H2 consumed per minute per milligram of cell
protein.
Expressed as nanomoles of H2 produced per minute per milligram of cell
rotein.
Expressed as nanomoles of BV reduced per minute per milligram of cell
protein.
Expressed as nanomoles of BVred oxidized per minute per milligram of cell
rotein.
UD, Undetectable


37
these cultures were harvested after 4 hr of incubation at 37C
(standing) and used for enzyme assays.
The amount of p-galactosidase present in cells was determined as
described by Miller (156), after permeabilization with SDS and
chloroform. The specific activity of the enzyme is expressed as
nanomoles ortho-nitrophenol produced per min per mg cell protein. The
differential rate of synthesis of p-galactosidase activity was
calculated as units of activity per pg cell protein produced by the
culture and represents the increase in the amount of p-galactosidase
activity produced by the culture in relation to the increase in total
cell protein.
Dihydrogen related metabolic activities and culture conditions.
Whole cells were utilized for all biochemical determinations (135).
Cells were inoculated (5% V/V) from overnight LB-grown cultures into
fresh LB6 medium in 250 ml screw cap bottles or 20 ml screw cap tubes
(16 x 150 mm) filled to the top. Cultures were grown anaerobically for
4 hr at 37C. Cells were collected by centrifugation (3,000 x g for 10
min) at 4C, washed with half volume of phosphate buffer (0.1 M sodium
phosphate, pH 7.0; 1 mM glutathione; 0.1 mg/ml chloramphenicol). The
cell pellet was resuspended in 1.0 ml buffer and diluted to 1.75 mg cell
protein per ml, as determined in a Spectronic 710 spectrophotometer
(Baush and Lomb) in which 1 A420nm unit equalled 350 pg cell protein
per ml. Samples were maintained under N2 at 4C and assayed
immediately.


108
PROTEIN (ng)
Figure 14. Differential rate of synthesis of 4>(fhlA-' lacZ*), strain SE-
2007, and (hypX'- lacZ*) 1, strain SE-2001, in LB medium supplemented
with 3 mM formate in the presence and absence of the fhlA+ gene in a
multicopy plasmid (pSE-133). p-Galactosidase activities and protein
concentrations are expressed as units per ml and pg per ml,
respectively.


39
chromatograph as described above. Assay mixture consisted of 0.1 M
phosphate buffer (pH 7.0) and 50 mM fumarate brought to a final volume
of 1.0 ml with cells. "Wheaton" vials (10 ml) with serum stoppers and
aluminum seals were used to establish anaerobic conditions (10% H2/90%
N2 gas phase). The HUPfumarate activity was calculated as nanomoles of
H2 consumed per min per mg cell protein.
Formate-dependent reduction of BV (FDH-H, FDH activity associated
with FHL), was assayed in an assay mixture containing 0.33 M sodium
phosphate buffer (pH 7.0), 6.5 mM BV, 40 mM formate and cells in a final
volume of 4.0 ml (135). Test tubes (13 x 100 mm) with "subaseal"
stoppers were used to monitor the reaction in a N2 atmosphere. Assay
mixture was reduced with sodium dithionite until a faint purple color
developed. Reaction was initiated by adding whole cells and the
AAgsonm was correlated to BV reduction. The FDH-H activity was
calculated as nanomoles BV reduced per min per mg of cell protein.
Formate hydrogenlyase activity was measured in 70 mM sodium
phosphate buffer (pH 6.5) containing 0.1 M formate (final volume of 1.0
ml with cells; 96). Wheaton" vials (10 ml) with serum stoppers and
aluminum seals were used to monitor the production of dihydrogen from
formate in a dinitrogen atmosphere. Dihydrogen evolution was quantified
using a gas chromatograph. The FHL activity was calculated as nanomoles
of H2 produced per min per mg of cell protein.
Fumarate reductase activity was assayed in 92.5 mM sodium
phosphate buffer (pH 7.0), 30 mM fumarate, and 0.35 mM BV under a N2


67
absence of formate was increased 2- to 3-fold. The significance of
these results remains to be determined.
Plasmid complementation. Previously described plasmid pSE-133,
which carries the partial hydF and complete hydB, fhlA genes (186),
restored FHL activity of strain SE-2011 to low levels (data not shown).
As later experiments would show, this effect is due to a physiological
effect of the FHL-A protein, a transcriptional activator for the FHL
system.
It is now known that the two divergently transcribed operons in
the 58 to 59 min region (the hyc and hyp operons) span approximately
15.5 kb of DNA. Plasmid pRBH, which carries the complete hyc operon on
low copy vector pACYC184 restored the Fhl+ activity of strain SE-2011 to
parental levels. Although the H2 to BV activity was increased, this
activity was attributable to HYD-3 (Table 5). Additionally, the p-
galactosidase activity produced by strain SE-2011/pRBH was 2-fold lower
than strain SE-2011 (LB-formate; 10 mM). Lutz et al. (144) have
recently reported that the first gene of the hyc operon is a putative
repressor and this is probably responsible for this decrease in fhlB
expression.
Interestingly, multicopy plasmid pSE-1213 which carries the
partial hyb operon (encoding HYD-2) and complements hyb mutants restored
strain SE-2011 to parental levels of HUP and low levels of FHL activity
(10% of wild type levels). Multicopy plasmid 33pBR which carries the
complete hyb operon complemented strain SE-2011 for both FHL and HUP


Figure
LIST OF FIGURES
page
1 Model of the genetic regulation of formate hydrogenlyase in
E. coli 4
2 Mixed-acid fermentation of E. coli 9
3 Differential rate of synthesis of p-galactosidase activity by
[fhlB'-'lacZ+) strain SE-2011 grown in LB medium with
different supplements 59
4 Effect of formate on the induction of p-galactosidase
activity by QifhlB-lacZ*) strain SE-2011 and a pfl
derivative, strain MJ-9 61
5 Effect of nitrate supplementation on fhlB gene expression. .62
6 Differential rate of induction of the Q(fhlB'-'lacZ+)
fusion in strain SE-2011 in the presence and absence of
plasmid pSE-133 73
7 Effect of formate concentration on the levels of 0-galacto-
sidase activity produced by a Q(fhlB'-'lacZ*) pfl double
mutant, strain MJ-9, in the presence of plasmids pSE-133 and
pSE-133-2 (fhlA::Jn5) 74
8 Differential rate of synthesis of <\>(fdhF'-'lacZ+) t strain
M9s, and $(fhlB'-'lacZ+), strain SE-2011, in LB medium
supplemented with 3 mM formate in the presence and absence
of the fhlA+ gene in a multicopy plasmid (pSE-133-1) 77
9 Nucleic acid and predicted protein sequences of the
partial hypC gene and complete hydB, hydF and fhlA genes . .80
10Alignment of the predicted seguences of E. coli FHL-A,
E. coli NTR-C, K. pneumoniae NIF-A, and P. put ida XYL-R
proteins 85
v


27
fusion expression and transcript levels under the same conditions (144;
this study).
Recent studies have established that nickel is not essential for
transcription of the electrophoretically stable HYD-1 and HYD-2
isoenzymes, but necessary for hydrogenase activity. Lutz et al. (144)
reported that mutations in hypBCDE did not alter hydrogenase
transcription as monitored by immunoblotting analysis with anti-HYD-1
and HYD-2 antibodies; however, the electrophoretic mobility was altered
in a 10% SDS-polyacrylamide gel. Potentially the lack of nickel
processing and insertion could lead to a modified hydrogenase
conformation and proteolytic susceptibility. Menon et al. (151)
obtained similar results for HYD-1 in a hydE (hypB) mutant. Although
HYD-1 transcription was comparable to wild type in these mutants in the
absence of Ni, activity was absent. Additionally, the hydE gene product
appeared essential for membrane localization of HYD-1, and this
requirement was nickel repressible. Labile HYD-3 is probably regulated
by a similar post-translational mechanism requiring nickel incorporation
for Fhl activity.
Selenium
A number of enzymes from both prokaryotic and eukaryotic
organisms, including FDH-H, require selenium in the form of
selenocysteine for activity (172; for review, see 26). DNA sequence
analysis of the fdhF gene [coding for the 701 amino acid FDH-H subunit
(Mf=79 kDa)] established that a UGA "nonsense" codon at amino acid


TMAO Trimethyl amine-N-oxide
Tris Tris-(hydroxymethyl)-ami nomethane
X-gal 5-Bromo-4-chloro-3-indolyl-p-D-
galactopyranoside
x


109
copies of fhlA+ gene (pSE-133) and frmate (3 mM), the p-galactosidase
activity produced by strain SE-2001 is increased by 2-fold. This would
suggest that the fhlA gene is independent of the hyd {hyp) promoter(s)
and is transcribed from its own "-35 and -10" o70 promoter. The
increased production of p-galactosidase activity in the SE2001/pSE133
culture is probably due to the unnaturally high concentration of FHL-A
protein available in the cytoplasm. These results also suggest that the
putative transcription termination region observed between the hydB and
fhlA genes (Fig. 9) is physiologically significant.


101
p-galactosidase activity is MJ107, (fhlAlacZ*), cya. This 2-fold
decrease in gene expression is probably due to the poor growth of the
culture; however, a cAMP effect cannot be ruled out at this point.
Localization of hyd gene fusions. It has recently been suggested
that the fhlA gene is regulated by the upstream a54- and FNR-dependent
promoters of the hyd [hyp) operon (144; Fig. 13). In order to
investigate this further, hyd gene fusions within this operon were
studied to compare transcriptional regulation. Mutant strains SE-2001,
SE-2002 and SE-2009 were similarly isolated as lac operon fusion
derivatives of strain MC4100, using ApiocMu53 (29). Biochemical
analysis revealed that strains SE-2001 and SE-2002 were deficient in all
three hydrogenase activities. Mutant strain SE-2009 was also
significantly affected in total hydrogenase activity, however basal
levels of hydrogenase were detectable. Therefore, these mutants were of
interest in studying FHL regulation since they were phenotypically
comparable to previously described hyd mutants.
Plasmid complementation analysis suggested that mutant strains SE-
2001 and SE-2002 were located in the 58-59 min hyd cluster. Plasmid
pSE-111 restored all three hydrogenase activities to parental levels in
strains SE-2001 and SE-2002. Plasmid pSE-130 complemented only to
approximately 35% of FHL activity in both mutants; whereas, HUP activity
was restored to 20% and 100% in strains SE-2001 and SE-2002,
respectively. Plasmids pSE-128 and pSE-125 did not complement the
mutation in either strain SE-2001 or SE-2002. Evidently, the complete


139
of Escherichia coli by targeted mutagenesis in the N-terminal
region. Mol. Microbiol. 2:701-707.
211. Spiro, S., and J. R. Guest. 1990. FNR and its role in
oxygen-regulated gene expression in Escherichia coli. FEMS
Microbiol. Rev. 75:399-428.
212. Spiro, S., and J. R. Guest. 1991. Adaptive responses to oxygen
limitation in Escherichia coli. TIBS 16:310-314.
213. Spiro, S., R. E. Roberts, amd J. R. Guest. 1989. FNR-dependent
repression of the ndh gene of Escherichia coli and metal ion
requirement for FNR-regulated gene expression. Mol. Microbiol.
3:601-608.
214. Steigerwald, V. J., G. S. Beckler, and J. N. Reeve. 1990.
Conservation of hydrogenase and polyferredoxin structures in the
hyperthermophi1ic archaebacterium Methanothermus fervidus. J.
Bacteriol. 172:4715-4718.
215. Stephenson, M., and L. H. Stickland. 1931. Hydrogenase: a
bacterial enzyme activating molecular hydrogen. I. The properties
of the enzyme. Biochem. J. (London) 2:205-214.
216. Stewart, V. 1982. Requirement of Fnr and NarL functions for
nitrate reductase expression in Escherichia coli K12. J.
Bacteriol. 151:1320-1325.
217. Stewart, V. 1988. Nitrate respiration in relation to facultative
metabolism in Enterobacteria. Microbiol. Rev. 52:190-232.
218. Stewart, V., and B. L. Berg. 1988. Influence of nar (nitrate
reductase) genes on nitrate inhibition of formate-hydrogen lyase
and fumarate reductase gene expression in Escherichia coli K-12.
J. Bacteriol. 170:4437-4444.
219. Stewart, V., and C. H. MacGregor. 1982. Nitrate reductase in
Escherichia coli K-12: involvement of chlC, chlE and chlF loci.
J. Bacteriol. 151:788-799.
220. Stewart, V., J. Parales, and S. M. Merkel. 1989. Structure of
genes narL and narX of the nar (nitrate reductase) locus in
Escherichia coli K-12. J. Bacteriol. 171: 2229-2234.
221. Stock, A. M., J. M. Mottonen, J. B. Stock, and C. E. Schutt. 1989.
Three-dimensional structure of CheY, the response regulator of
bacterial chemotaxis. Nature 337:745-749.


110
Molybdate Metabolism and FHL Activity
CHL-D and MOL-R proteins are required for fhlB operon expression.
As was discussed earlier, both the chlD and molR gene products are
necessary for fhlB operon expression (Table 6). Further analysis was
done on the chlD and molR derivatives of strain SE-2011, strains SE1714
and MJ-40 respectively, to better understand this regulation (Table 11).
For these experiments, the strains were grown under anaerobic conditions
in LB-formate medium, and the cells were harvested after 4 hr for enzyme
assays. Under these conditions, a molR or chlD mutation reduced p-
galactosidase activity produced by strain SE-2011 by over 4- to 5-fold.
Supplementing the medium with 1 mM molybdate, restored enzyme levels to
those of the parent, strain SE-2011. In the presence of multiple copies
of the fhlA gene, the specific enzyme activity produced by strains MJ-40
and SE1714 was increased by over 5-fold the activity of strains without
plasmid pSE-133 (fhlA+). However, the enzyme levels of these cultures
(+pSE-133) were approximately 2-fold less than the SE-201l/pSE-133
culture. Molybdate (1 mM) supplementation was still required for peak
fhlB operon expression. These results suggest that the FHL-A mediated
formate inducibility of the fhlB operon and molybdate-mediated
regulation are separate and independent of each other.
Primary structure of the molR gene. Previous experiments
identified the molR gene product as a putative regulatory element of
both the fdhF and ant genes and essential for utilization of molybdate


42
agar) at 50C was added to the mixture and after mixing was overlayed on
LB+thymine (25 pg/ml) plates. After 9 hr of incubation at 42C, the
overlay from the plates with confluent lysis was harvested after
addition of 2.5 ml Pl-diluent (10 g/L trypticase peptone; 10 mM
MgCl2.6H20). Chloroform (0.5 ml) was added to the lysate and mixed with
a glass pipet. This was centrifuged (12,100 x g) for 15 min at 4C.
The supernatant was again extracted with choloroform, and the final
supernatant was stored at 4C with 0.1 ml chloroform. Two consecutive
series of PI infection were done to ensure enrichment of host strain
mutation.
Procedures implemented for replication of bacteriophage A were
comparable to PI with some critical exceptions. Strain LE392 (supF,
supE) was used as the host strain and was grown in LBM medium. Cells
were pelleted and resuspended in an equal volume of 10 mM MgS04.7H20.
Water-Thy agar (0.6% agar; 0.1 mg/ml thymine) was used as the soft agar
overlay, and plates were incubated at 37C. Lambda diluent (10 mM Tris-
HC1, pH 7.5; 10 mM MgS04.7H20; 50 mM NaCl; 0.1% gelatin) was used to
titer and harvest phage.
Transduction using bacteriophage PI Cm cZrlOO. Transduction
experiments were carried out according to Miller (156) with
modifications. Two milliliters of a "mid-log phase" culture of
recipient cells were centrifuged (3,500 x g) for 5 min at 25C and
resuspended in 1.0 ml of PI adsorption medium. Then 0.2 ml of the
resuspension was infected at a M.0.1. of 1 to 10 with the appropriate


142
244. Zinoni, F., Heider, J., and A. Bock. 1990. Features of the formate
dehydrogenase mRNA necessary for decoding of the U6A codon as
selenocysteine. Proc. Natl. Acad. Sci. USA 87:4660-4664.


9
2[H]
GLUCOSE
ADP
ATP
PEP
LACTATE"
2[H]
I
12
ADP
ATP
PYRUVATE -
2[H]
V
10
ACETYL-CoA
si
CO,
FORMATE16
2[H]
I \
.Lp
h* CoA
ACETALYDEHYDE ACETYL-P
V- adp
2[H]H 11 9 [
ETHANOL
ATP
ACETATE
- OAA
2[H]
MALATE
sl
H* -A 7
A *
FUMARATE
*
2[H]
SUCCINATE
Figure 2. Mixed-acid fermentation of E. coli. 1, Phosphoenolypyruvate
carboxylase; 2, malate dehydrogenase; 3, fumarase; 4, fumarate
reductase; 5, pyruvate formatelyase; 6, formate hydrogenlyase complex
(formate dehydrogenase-H, hydrogenase isoenzyme-3, and associated
electron transport proteins); 7, hydrogenase isoenzyme-2; 8,
phosphotransacetylase; 9, acetate kinase; 10, acetaldehyde
dehydrogenase; 11, alcohol dehydrogenase; 12, lactate dehydrogenase.
Redrawn with modifications from Alam and Clark (3).


65
Cys+ clones tested, 12.5% were restored for FHL activity. One lysogen
from this experiment tested positive for HUP activity but the clone
cured of the lysogenic phage was deficient in both FHL and HUP
activities.
Hfr mediated conjugation analysis of the fhlB gene. Because of
these inconclusive results the approximate map location of the altered
gene in the E. coli chromosome was determined by Hfr-mediated
conjugation analysis. For these experiments, an Hfr-derivative of
strain SE-2011 in which the origin of DNA transfer is the fhlB gene was
constructed, using lac homology, as described before (40, 136). In a 30
min conjugation period, strain MJ-18, transferred argl gene (96.6 min;
11) and not frdA gene (94.4 min; 11) indicating that the fhlB gene was
located between these two genes. Both thr and leu were also transferred
at high frequency during this 30 min duration. Since the orientation of
the lac operon with respect to the origin of transfer is known (40),
results of these experiments were also used to determine the direction
of transcription of the fhlB gene. According to the results, it
appeared that the fhlB gene was transcribed in a clockwise direction,
towards argl, thr and leu.
The accuracy of utilizing this procedure in certain lac fusion
strains is currently under investigation (Shanmugam, personal
communication). Therefore after reevaluating the physiological data and
mapping results of SE-2011, it appeared probable that the localization
of fhlB gene to the 96.6 min position was due to homology of the


106
suggests multiple promoters for the hyp operon (FNR- and o54-dependent
promoters (144). The FNR-dependent promoter within the hypA gene is
apparently transcribed when formate concentrations are low as measured
by p-galactosidase activity of strains SE-1651 and SE-1652 grown in LB
medium. Formate supplementation alleviates the FNR requirement,
suggesting transcription from FNR-independent promoter(s) in later
stages of growth. The p-galactosidase produced by strain SE-1657 in LB
medium, similarly, suggests a role for a54 in hyp operon expression.
Hybridization studies to hyp operon mRNA suggest that the fhlA gene is
cotranscribed from the hyp promoters (A. Bock, personal communication).
This is in contrast to the genetic regulatory pattern observed in strain
SE-2007, HfhlA'-'lacZ*).
Two possibilities could mask the true physiological regulation of
the fhlA gene and hyd [hyp) operon from the data presented above
(strains SE-2007, SE2001 and SE-2002). First, it is possible that the
fhlA gene is autoregulated. The introduction of a gene fusion in the
fhlA gene would inactivate its product, as seen phenotypically in strain
SE-2007 (FHL activity). If FHL-A protein is required for regulation of
fhlA gene transcription, the p-galactosidase values obtained for strain
SE-2007 would not reflect this FHL-A requirement. Secondly, the fhlA
gene could be transcribed from the hyd [hyp) promoter(s). The polarity
of the hyd [hyp) mutations of strains SE-2001 and SE-2002 would inhibit
fhlA gene transcription if hyp promoters were indeed the normal
physiological promoters. Thus, if FHL-A is cotranscribed and is


11
Formate hydrogenlyase is composed of FDH-H, HYD-3, and redox
carriers linking the two enzymes (27, 77, 173, 193). The fdhF gene
(92.4 min) encodes the 80 kDa selenopolypeptide constituent of FDH-H
(11, 243). Additionally, the FDH-H protein requires molybdenum
(molybdopterin-guanylate) for activity (33, 73, 112). The hyc operon
(59 min) consists of 8 ORFs and is presumed to encode the remaining FHL
components. Five of these ORFs code for electron carriers (previously
termed ant and hyd-17; ref. 11, 27, 171, 242). The 0RF5 (hyd-17) shows
significant sequence homology with the large subunit from other Ni/Fe
hydrogenases and is the presumptive structural gene for HYD-3.
Numerous mutants have been isolated with defects in H2 metabolism
(Fhl-), the majority of which have been deficient in all three
hydrogenase isoenzymes (35, 37, 76, 107, 128, 135, 167, 171, 231).
Through the characterization of mutant strains, DNA sequence analysis
and mapping of the genes affected, FHL regulation can be more clearly
understood. It is proposed that the FHL system is regulated by signals
which include anaerobiosis, nitrate, intracellular formate, low pH,
molybdate, nickel, and selenium. How these signals are transmitted to
the transcription, translation and post-translational modification of
the components of the FHL complex remains poorly understood.
Anaerobiosis
The presence or absence of terminal electron acceptors, such as
dioxygen or nitrate, play a major role in regulating genes involved in
respiration and fermentation. It was estimated, by using 2-dimensional


63
production was measured in the cultures supplemented with nitrate to
estimate the rate of nitrate respiration. Results of these experiments
indicate that significant levels of nitrate respiration are not evident
until 2 hr after initiation of anaerobiosis and growth (Fig. 5A). After
this initial lag, nitrite accumulated in the medium throughout the
remaining time of the experiment. In the absence of exogenous formate,
nitrate repression of fhlB gene transcription (as measured by p-
galactosidase activity) paralleled the initiation of nitrate respiration
(LBN; Fig. 5B). Formate supplementation (73 mM) partially suppressed
this repressive effect, and a narL mutation did not affect the fhlB
operon regulation. This is comparable to the regulatory patterns
observed for other genes of the FHL pathway (hyd-17 and fdhF genes;
171). These results suggest that both formate and anaerobiosis are
necessary for maximal induction of fhlB operon transcription. Although
nitrate and neutral pH repress this expression, this effect can be
overcome by formate.


131
Microbiol., Washington, DC.
123. Knappe, J. F., A. Neugebauer, H. P. Blaschkowski, and M. Ganzler.
1984. Post-translation activation introduces a free radical into
pyruvate formate-lyase. Proc. Natl. Acad. Sci. USA 81:1332-1335.
124. Knappe, J., J. Schacht, W. Mockel, T. Hopner, H. Vetter, Jr., and
R. Edenharder. 1969. Pyruvate formate-lyase reaction in
Escherichia coli: the enzymatic system converting an inactive form
of the lyase into the catalytically active form. Eur. J. Biochem.
11:316-327.
125. Kohara, Y., K. Akiyama, and K. Isomo. 1987. The physical map of
the whole E. coli chromosome: application of a new strategy for
rapid analysis and sorting of a large genomic library. Cell
50:495-508.
126. Kozak, M. 1983. Comparison of initiation of protein synthesis in
procaryotes, eucaryotes, and organelles. Microbiol. Rev. 47:1-45.
127. Krasna, A. I. 1980. Regulation of hydrogenase activity in
enterobacteria. J. Bacteriol. 144:1094-1097.
128. Krasna, A. I. 1984. Mutants of Escherichia coli with altered
hydrogenase activity. J. Gen. Microbiol. 130:779-787.
129. Kroos, L., B. Kunkel, and R. Losick. 1989. Switch protein alters
specificty of RNA polymerase containing a compartment-specific
sigma factor. Science 243:526-528.
130. Kuritzkes, D. R., X. -Y. Zhang, and E. C. C. Lin. 1984. Use of
(glp-lac) in studies of respiratory regulation of the Escherichia
coli anaerobic s/7-glycerol-3-phosphate dehydrogenase genes (glpAB)
J. Bacteriol. 157:591-598.
131. Kutsu, S., E. Santero, J. Keener, D. Popham, and D. Weiss. 1989.
Expression of sigma 54 (ntrA)-dependent genes is probably united
by a common mechanism. Microbiol. Rev. 53:367-376.
132. Kutsu, S., K. Sei, and J. Keener. 1986. Nitrogen regulation in
enteric bacteria, p. 139-154. In I. Booth, and C. Higgins (eds)
Regulation of Gene Expression. Symposium of the Soc. Gen.
Microbiol. Cambridge University Press, Cambridge, England.
133. Lambden, P. R., and J.R. Guest. 1976. Mutants of Escherichia coli
K12 unable to use fumarate as an anaerobic electron acceptor. J.
Gen. Microbiol. 97:145-160.


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120


61
FORMATE (mM)
Figure 4. Effect of formate on the induction of p-galactosidase activity
by <\>(fhlB-lacZ+) strain SE-2011 and a pfl derivative, strain MJ-9.
Specific activity represents the maximum value observed at each formate
concentration.


5
subunit of RNA polymerase), fhlA, chlD and molR gene products were
necessary for optimum fhlB gene transcription (Fig. 1).
Subsequent investigation of the fhlB gene transcription
demonstrated that high cytoplasmic concentrations of FHL-A protein
(using multicopy plasmid pSE-133) decreased but did not eliminate the
formate requirement. Because of these observations, the DNA sequence of
the fhlA gene was determined to identify the characteristics of the gene
and its product. The FHL-A protein was found to have sequence homology
with putative o54-dependent transcriptional activators (receivers) of
the two-component regulatory system. However, the amino terminus of the
FHL-A protein was unique and did not contain the conserved secondary
structure or aspartate and lysine residues which have been shown to be
phosphorylated by the respective histidine-protein-kinase (sensor).
This would suggest that phosphorylation is not required for active FHL-A
protein, and a sensor protein may not be involved. Interestingly,
transcription of the fhlA gene was shown to be from the weak
"-35 and -10" fhlA promoter (constitutive) and not from the upstream
54
FNR- or o -dependent promoters of the hyp operon. It is therefore
possible that the FHL-A protein is synthesized in an inactive form and
activated in the presence of formate (Fig. 1).
During investigation of the molybdate requirement for FHL, it was
determined that the fhlB gene in molR or chlD genetic backgrounds still
retained formate inducibility in the presence of multiple copies of the
fhlA+ gene (exogenous molybdate was required for expression to wild type


125
cofactor requirement for biotin sulfoxide reduction in Escherichia
coli. J. Bacteriol. 149:469-478.
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12:387-395.
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repression in Escherichia coli. J. Bacteriol. 91:2263-2269.
58. Drummond, M., P. Whitty, and J. Wootton. 1986. Sequence and domain
relationships of ntrC and nifA from Klebsiella pneumoniae:
homologies to other regulatory proteins. EMBO J. 5:441-447.
59. Egan, S. M., and V. Stewart. 1990. Nitrate regulation of anaerobic
respiratory gene expression in narX deletion mutants of
Escherichia coli K-12. J. Bacteriol. 172:5020-5029.
60. Egan, S. M., and V. Stewart. 1991. Mutational analysis of nitrate
regulatory gene narL in Escherichia coli K-12. J. Bacteriol.
173:4424-4432.
61. Eidness, M. K., R. A. Scott, B. C. Prickril, D. V. DerVartanian,
J. Legal 1, I. Moura, J. J. G. Moura, and H. D. Peck, Jr. 1989.
Evidence for selenocysteine coordination to the active site nickel
in the [NiFeSe] hydrogenases from Desulfovibrio baculatus. Proc.
Natl. Acad. Sci. USA 86:147-151.
62. Fimmel, A. L., and B. A. Haddock. 1979. Use of chlC-lac fusions to
determine regulation of gene chlC in Escherichia coli. J.
Bacteriol. 138:726-738.
63. Fisher, H. -M., and H. Hennecke. 1987. Direct response of
Bradyrhizobium japonicum /? /-mediated nif gene regulation to
cellular oxygen status. Mol. Gen. Genet. 209:621-626.
64. Forchhammer, K., and A. Bock. 1991. Selenocysteine synthase from
Escherichia coli: analysis of the reaction sequence. J. Biol.
Chem. 266:6324-6328.
65. Forchhammer, K., W. Leinfelder, K. Boesmiller, B. Veprek, and A.
Bock. 1991. Selenocysteine synthase from Escherichia coli:
nucleotide sequence of the gene (selA) and purification of the
protein. J. Biol. Chem. 266:6318-6323.
66. Francis, K., P. Patel, J. C. Wendt, and K. T. Shanmugam. 1990.
Purification and characterization of two forms of hydrogenase


119
gene (exogenous molybdate was required for expression to wild type
levels). This suggests that FHL is regulated by a dual regulatory
system, one involving molybdate and a second formate-dependent pathway.
DNA sequence analysis of the mol (chi) operon indicates that the molR
gene, encoding a potential regulatory protein of the molybdate-dependent
pathway (136) is located near genes encoding a periplasmic-binding-
protein dependent molybdate transport system. The constitutivity of the
molR gene suggests multiple promoters are involved in transcription of
this operon.
These results show that fermentative dihydrogen production is
regulated by a complex series of reactions involving the availability of
formate, molybdate and the absence of oxygen. Further studies will be
necessary in order to better understand the various mechanisms of
regulation. Localization of the fhlB and molR mutations, primer
extension analysis of the mol (chi) operon promoter region and
identification of the parameters required for in vitro activation of the
o -dependent promoters of the FHL pathway will be beneficial to
furthering our understanding of dihydrogen production in E. coli.


ACKNOWLEDGEMENTS
I would like to sincerely thank my advising professor Dr. K. T.
Shanmugam. His continual encouragement, guidance and enthusiasm for
science have definitely influenced me for a lifetime. I would like to
thank the K. T. S. laboratory group including both old and new. I would
also like to extend my thanks to Drs. Richard Boyce, Francis Davis Jr.,
John Gander, and Lonnie Ingram for their helpful questions and counsel
while serving on my committee. Additionally, I would like to thank Drs.
A. Bock and B. Bachmann for providing several strains and plasmids
necessary for this study. Finally, special thanks are extended to my
family and fianc for their willingness to listen to my ups and downs
throughout the course of this work.


MATERIALS AND METHODS
Materials
Biochemicals were purchased from Sigma Chemical Co. Analytical-
grade inorganic and organic chemicals were from Fisher Scientific.
Bacterial Strains and Media
The bacterial strains are derivatives of E. coli K12 and are
listed in Table 1. Basal minimal, dihydrogen/fumarate and
glycerol/fumarate media and LB were prepared as described previously
(135). Cultures were grown at 37C in LB which was supplemented with
glucose (0.3%) or sodium formate (0.1% to 0.5%), as needed. Ampicillin
(100 pg/ml), kanamycin (50 pg/ml), streptomycin (100 pg/ml),
tetracycline (15 pg/ml), chloramphenicol (5 pg/ml) or X-gal (20-40
pg/ml) were added as needed.
Isolation of Mutants
Strain MC4100, grown in LB+maltose (0.3%; LBM) medium was
mutagenized with XpZocMu53 and ApMu507, as described by Bremer et al.
(29). Kanamycin resistant mutants were transferred by replica plating
techniques to LB + X-Gal medium (156) and incubated under aerobic or
anaerobic conditions. Mutants which are Lac+ only under anaerobic
growth conditions were identified and inoculated into 1 ml of LBG medium
in 12 x 75 mm tubes. These tubes were sealed with serum stoppers and
30


18
therefore, FHL activity is also reduced. However, this control does not
appear to be mediated by the NAR-X, NAR-L cascade (218). High
concentrations of formate relieve both nitrate and fumarate repression
of the FHL structural genes (171). Interestingly, mutations in nor/.,
narK (hypothetical nitrate transport gene) and narGHJI partially
relieved nitrate inhibition at the level of hyc and fdhF operon
expression when tested in the absence of formate (218). Likewise,
strain WL24, deficient in the FDH-N selenopeptide encoded by the fdhGHI
operon, is derepressed for the synthesis of FDH-H when grown
anaerobically in the presence of nitrate (192). These results suggest
that the nitrate effect at the fdhF and hyc level may be a consequence
of formate (an obligate inducer) being channeled to nitrate respiration.
Formate
Irrespective of whether high redox potential electron acceptors
(dioxygen or nitrate) are present in the environment, glucose is
actively transported into the cytoplasm by the phosphotransferase system
(PTS) and catabolized to pyruvate (for review, see 75). Therefore, the
pivotal metabolic step signalling anaerobiosis could potentially be
pyruvate degradation by PFL to formate which occurs in the absence of
dioxygen (42). Pyruvate dehydrogenase (PDH) and pyruvate formatelyase
(PFL) are tightly controlled by dioxygen both transcriptionally and
allosterically (123, 124, 205). Anaerobiosis represses and inhibits PDH
while inducing PFL synthesis and catalytic activity (83, 170). Pyruvate
formatelyase constitutes up to 3% of the cytoplasmic protein in


32
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
MJ-8
lacZ+), cya-2, zif-4::JnlO
PI transduction
(SE2011 x SE1162)
MJ-9
*{fhlB'-'lacZ+), pfl-1, zba-6: :TnJ0
PI transduction
(SE2011 x SE1265)
MJ-18
MJ-19, Hfr PO(fhlB)
Conjugation
(MJ-19 x TT627)
MJ-19
CSH26, *{fhlB'-'lacZ+)
PI transduction
(CSH26 x SE2011)
MJ-20
Q(fhlB'-'lacZ*), fhlA::Tni0
PI transduction
(SE2011 x SE1174)
MJ-21
SE1000, metBl, melA7,
thr+, arg+, leu+, F~
Conjugation
(SE-1000 x M2508)
MJ-40
^(fhlB'-'lacZ+)t molR
PI transduction
(SE2011 x SE1704)
MJ-50
SE-1000, PI transduction
(SE-1000 x SE2011)
MJ101
(fhlA'-' lacZ+), rpoN::lr)10
PI transduction
(SE-2007 x YMC18)
MJ-102
4>(fhlA,-'lacZ+), nor/,215: :TnJ0
PI transduction
(SE-2007 x RK5278)
MJ-103
Q>(fhlA'-' lacZ+), /nr, zcj-5::TnI0
PI transduction
(SE-2007 x SE-1188)
MJ107
(fhlA'-'lacZ+), cya-2, zi/-4::TnJ0
PI transduction
(SE-2007 x SE1162)
MJ-108
Q(fhlA'-' lacZ+), moZ/?
PI transduction
(SE-2007 x SE1704)
MJ-109
(fhlA'-' lacZ+), pfl-1, zba-6: :TnJZ?
PI transduction
(SE-2007 x SE1265)


135
166. Pakes, W. W. C., and W. H. Jollyman. 1901. The bacterial
decomposition of formic acid into carbon dioxide and hydrogen. J.
Chem. Soc. 79: 386-391.
167. Pascal, M. -C., F. Casse, M. Chippaux, and M. Lepelletier. 1975.
Genetic analysis of mutants of Escherichia coli K12 and
Salmonella typhimurium LT2 deficient in hydrogenlyase activity.
Mol. Gen. Genet. 141:173-179.
168. Patel, P. S. 1985. Biochemical genetics of hydrogen metabolism in
Escherichia coli: purification and characterization of
hydrogenase. Ph.D. dissertation, University of Florida.
169. Pateman, J. A., D. J. Cove, B. M. Rever, and D. B. Roberts. 1964.
A common co-factor for nitrate reductase and xanthine
dehydrogenase which also regulates the synthesis of nitrate
reductase. Nature. 201:58-60.
170. Pecher, A., H. P. Blaschkowski, K. Knappe, and A. Bock. 1982.
Expression of pyruvate formate-lyase of Escherichia coli from the
cloned structural gene. Arch. Microbiol. 132:365-371.
171. Pecher, A., F. Zinoni, C. Jatisatienr, R. Wirth, H. Hennecke, and
A. Bock. 1983. On the redox control of synthesis of anaerobically
induced enzymes in enterobacteriaceae. Arch. Microbiol.
136:131-136.
172. Pecher, A., F. Zinoni, and A. Bock. 1985. The selenopeptide of
formate dehydrogenase (formate hydrogen-lyase linked) from
Escherichia coli: genetic analysis. Arch. Microbiol. 141:359-363.
173. Peck, H. D. Jr., and H. Gest. 1957. Formic dehydrogenase and the
hydrogenlyase enzyme complex in coli-aerogenes bacteria. J.
Bacteriol. 73:706-721.
174. Popham, D. L., D. Szeto, J. Kenner, and S. Kustu. 1989. Function
of a bacterial activator protein that binds to transcriptional
enhancers. Science 243:629-635.
175. Poole, R. K., and W. J. Ingledew. 1987. Pathways of electrons to
oxygen, p. 170-200. In F. C. Neidhardt, J. L. Ingraham, K. B. Low,
B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia
coli and Salmonella typhimurium: Cellular and Molecular Biology.
Am. Soc. Microbiol., Washington, DC.
176. Ramakrishnan, G. and A. Newton. 1990. FIbD of Caulobacter
crescentus is a homologue of the NtrC (NR() protein and activates


112
by E. coli (134, 136). The physiological data presented above (Table
11) show that the MOL-R protein is needed for optimum induction of the
fhlB operon. Therefore, the DNA sequence of the molR gene was
determined to identify possible conserved motifs in the gene product
(Fig. 15).
Plasmid pSE1009 and exonuclease III generated deletion
derivatives, plasmids 1 through 5 complemented a molR mutant (strain
SE1100) for FHL activity (plasmid derivatives 6 to 13 did not restore
activity). Therefore, derivatives 5 to 13 were initially used for
sequencing, since the complete molR gene was probably encoded by these
plasmids (fxoIII derivatives are identified in Fig. 15). By searching
GenBank and EMBL data bases with the preliminary sequence, it was
determined that the Sou3Al-l,609-base-pair restriction fragment in
pSJE301 which was cloned and sequenced by Johann and Hinton (110) is an
internal fragment of the plasmid pSE-1009 sequence (2,927-base-pair
Kpnl-EcoRV restriction fragment). Because the complete chlJ' and 0RF3'
DNA sequences have not yet been determined and the molR gene is upstream
of the chlD gene, the region sequenced was extended to include the Clal
restriction site of plasmid pSE-1004 (for a total of 3,876-base-pair DNA
sequenced).
Previous experiments have shown that the chlD expression is
repressed by molybdate whereas the molR gene is constituively
transcribed (134, 155). This raises several unanswered questions.
First if the molR gene product is regulatory, as the constitutive nature


narL
Putative DNA binding protein of nitrate regulation
narX
Putative membrane-bound histidine-protein-kinase of
nitrate regulation
narQ
Second proposed histidine-protein-kinase of nitrate
regulation
narGHJI
Nitrate reductase
nifA
Positive activator for nitrogen fixation; Klebsiella
pneumoniae
ntrC
glnG, nitrogen metabolism
pfl
Pyruvate formatelyase
pgi
oxrC, phosphoglucose isomerase
tyrR
Aromatic amino acid biosynthesis and transport
xylR
Degradative pathway of aromatic hydrocarbons; Pseudomonas
put ida


141
supercoiling by DNA gyrase. A static head analysis. Cell Biophys.
12:157-181.
234. Wilbur, W. J., and D. J. Lipman. 1983. Rapid similarity searches
of nucleic acid and protein data banks. Proc. Natl. Acad. Sci.
80:726-730.
235. Wimpenny, J. W. T., and J. A. Cole. 1967. The regulation of
metabolism in facultative bacteria. III. The effect of nitrate.
Biochim. Biophys. Acta 148:233-243.
236. Wimpenny, J. W. T., and D. K. Necklen. 1971. The redox environment
and microbial physiology. I. The transition from anaerobiosis to
aerobiosis in continuous cultures of facultative anaerobes.
Biochim. Biophys. Acta 253:352-359.
237. Woods, S. A., and J. R. Guest. 1987. Differential role of the
Escherichia coli fumarases and /nr-depencent expression of
fumarase B and aspartase. FEMS Microbiol. Lett. 48:219-224.
238. Wu, L. F., and M. -A. Mandrand-Berthelot. 1986. Genetic and
physiological characterization of new Escherichia coli mutants
impaired in hydrogenase activity. Biochimie. 68:167-179.
239. Wu, L. -F., M. -A. Mandrand-Berthelot, R. Waugh, C. J. Edmonds, S.
E. Holt, and D. H. Boxer. 1989. Nickel deficiency gives rise to
the defective hydrogenase phenotype of hydC and far mutants in
Escherichia coli. Mol. Microbiol. 3:1709-1718.
240. Wylie, D., A. Stock, C. -Y. Wong, and J. Stock. 1988. Sensory
transduction in bacterial chemotaxis involves phosphotransfer
between Che proteins. Biochem. Biophys. Res. Commun. 151:891-896.
241. Yamamoto, N., and M. L. Droffner. 1985. Mechanisms determining
aerobic and anaerobic growth in the facultative anaerobe
Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 82:2077-2081.
242. Yerkes, J. H., L. P. Casson, A. K. Honkanen, and G. C. Walker.
1984. Anaerobiosis induces expression of ant, a new Escherichia
coli locus with a role in anaerobic electron transport. J.
Bacteriol. 158:180-186.
243. Zinoni, F. A. Birkmann, T. C. Stadtman, and A. Bock. 1986.
Nucleotide sequence and expression of the selenocysteine-
containing polypeptide of formate dehydrogenase (formate-
hydrogen-lyase-linked) from Escherichia coli. Proc. Natl. Acad.
Sci. USA. 83:4650-4654.


2
American population is more aware of this crisis and open to
environmentally and economically sound options. Research in these
fields must continue in order for alternative fuels to become more
competitive. Unfortunately, historical events, i.e. the "Hindenberg",
have curbed the use of dihydrogen as an energy source. When properly
controlled, dihydrogen is a clean burning fuel. The end product being
H20, instead of CO, C02, S02 and nuclear waste (the current waste
products of energy production in our country).
Additionally, the question of how an organism responds to various
environmental stimuli such as dioxygen and regulates the metabolism by
activating or repressing specific genes remains to be fully elucidated.
By studying the FHL system of Escherichia coli, one can not only
integrate regulatory similarities from this system to other pathways,
but also study modes of control which are unique to FHL in order to gain
a clear understanding of gene expression. Associating this with the
physiological role of anaerobic H2 and C02 production is also of
interest. The metabolic fate of the end products of FHL are poorly
understood.
Following the advent of DNA sequencing techniques and computer
programs designed to organize the sequences generated, studies on
protein homology have enabled the researcher to identify apparent
function from the primary structure. It is now understood that there
are two categories of proteins involved in signal transduction: sensor
and receiver. Generally, the sensor is a membrane-bound, histidine-


10
from pyruvate. It has been proposed that PDH is inhibited by the high
[NADH]/[NAD+] ratio reached upon shift to anaerobic conditions (83).
Therefore anaerobically active PFL, encoded by the pfl operon, plays a
central role in glucose metabolism (122, 170, 191). The PFL complex
catalyzes the nonoxidative cleavage of pyruvate leading to transfer of
"acetyl" group to coenzyme A (Ac-CoA) and formate (75). Consequently,
the C02 and reduced NADH produced aerobically by PDH is replaced
anaerobically by formate. This is critical since the cell must maintain
an acceptable ratio of [NADH]/[NAD+] (42). The FHL catalyzes the
oxidation of formate to H2 and C02. This minimizes the accumulation of
formic acid which would otherwise lower the pH (77). The metabolic
fates of the gaseous products, H2 and C02, are poorly understood. It
is hypothesized that FHL is the major anaerobic enzyme producing C02 for
the synthesis of oxaloacetate (OAA) from phosphoenol pyruvate (PEP; ref.
42). OAA can be further metabolized to succinate or 2-oxoglutarate, all
three of these metabolites being essential for biosynthetic reactions
such as amino acid production. E. coli can utilize the H2 produced from
formate oxidation by FHL to reduce fumarate to succinate (HUP; ref.
148). This energy yielding pathway is HYD-2 dependent (12, 151, 193).
This would link FHL to the membrane-bound fumarate reductase and thus a
respiratory process which provides the cell with 1 mole of ATP per mole
of fumarate reduced, presumably by the generation of PMF (116).
Therefore, FHL appears to be linked to both fermentative and respiratory
pathways.


117
analysis of the FHL activator is essential for full understanding of the
genetic regulation of formate hydrogenlyase in E. coli.
This study shows that the fhlA gene encodes a putative
transcriptional regulator which is homologous to other DNA-binding
proteins of the two-component regulatory system. The "domain D"
(presumed to be the site of ATP-binding and o54 interaction) and helix-
turn-helix motif are conserved; however, the amino-terminus of the FHL-A
protein (first 353 amino acids) is unique among the various proteins
analyzed. The FHL-A protein does not contain the conserved secondary
structure or aspartate and lysine residues which have been shown to be
phosphorylated by the respective histidine-protein-kinase (sensor).
This suggests that FHL-A protein may be modulated by a mechanism
differing from the typical phosphorylation cascade identified in the
two-component systems.
Interestingly, the fhlA gene is induced by microaerobic conditions
and constitutively synthesized anaerobically, while the fhlB gene (as
well as fdhFy and hyc genes; ref. 171, 196) expression requires formate
and molybdate in the medium (anaerobically). Transcription of the fhlA
gene is apparently from the weak "-35 and -10" fhlA promoter
(constitutive) and not from the FNR- or o54-dependent promoters of the
hyp operon. Therefore, formate modulation of active FHL-A protein is
not at the transcriptional level (this is independent of molybdate
control; discussed below). Although recent data suggest that FHL-A
alone can bind to URS of fdhF and retard electrophoretic mobility of


75
an F1 element carrying fhlA+ gene (F143-1) into strain SE-2011, did not
significantly alter the rate or the level of p-galactosidase activity.
These results suggest that increasing the copy number of the fhlA+ gene
either decreased the concentration of formate required for transcription
of lac2+) or eliminated the need for formate.
Formate is required for FHL-A activation of the fhlB operon. In
order to distinguish between the two possibilities, plasmid pSE-133 was
transferred to strain MJ-9 [Q(fhlB'-'lacZ+), pfl] and the amount of p-
galactosidase activity produced by the culture was determined after
culturing the cells in either LB or LB-formate medium. The strain MJ-
9/pSE-133 produced about 100 units of p-galactosidase activity when
grown in LB medium and about 1,300 units when grown in LB-formate medium
(Fig. 7). The minimum amount of formate needed for this transcription
was about 3 mM. At formate concentrations higher than 3 mM, the amount
of p-galactosidase activity produced by strain MJ9/pSE133 increased
slowly reaching a maximum value of about 1,500 units at about 30 mM.
The amount of p-galactosidase activity produced by strain MJ-9/pSE-133-2
(fhlA::Tn5) was actually lower than strain MJ-9 itself and under the
conditions used in these experiments, this activity never exceeded 300
units. These results show that in the presence of multiple copies of
fhlA gene, the concentration of formate required for optimum expression
of fhlB gene is considerably reduced but is still a needed inducer.
The FHL-B protein is cytoplasmic. With 3 mM formate in the
medium, the differential rate of synthesis of p-galactosidase activity


38
Total hydrogenase activity, measured as tritium exchange (7, 138),
was determined using 50 pg cell protein adjusted to 0.2 ml volume in
sodium phosphate buffer (0.1M; pH 7.0). Assays were carried out in 12 x
75 mm thick walled test tubes, sealed with 11 x 17 mm serum stoppers.
The gas phase was replaced with helium. The reactions were initiated
with 0.7 ml of dihydrogen and 0.55 pCi tritium gas (11.2 mCi/mmol; New
England Nuclear Corp.) and terminated after 1 hr incubation at room
temperature. After removing the stopper, the reaction mixture was
agitated with a vortex mixer and allowed to stand for 10 min. Tritium
in a 50 pi sample in 2.5 ml Scintiverse-E scintillation fluid was
determined, and total hydrogenase activity was calculated as nanomoles
of tritiated water produced per min per mg cell protein.
Hydrogen uptake (HUP) was measured both as the ability to reduce
benzyl viologen (BV) (96, 167) and fumarate with dihydrogen as electron
donor (148). The reduction of BV was monitored in 13 x 100 mm test
tubes with "subaseal" stoppers. The assay mixture (0.1 M sodium
phosphate buffer, pH 7.0; 4 mM BV) was degassed, the gas phase replaced
with dihydrogen and reduced with sodium diothionite until a faint purple
color appeared. The reaction was initiated by the addition of whole
cells to a final volume of 2.5 ml. The rate of BV reduction was
correlated to AA550nm using a DW2C spectrophotometer (SLM Instruments;
Urbana, Illinois). The HUPBV activity was expressed as nanomoles BV
reduced per min per mg cell protein. Fumarate reduction was measured as
the rate of dihydrogen consumption from the gas phase using a gas


46
Plasmids carrying the transposon Tn5 in the gene of interest were
selected as Kmr Ampr transformants and then screened for lack of
complementation in the appropriate mutant. Location of the transposon
Tn5 was determined by analyzing "restriction endonuclease" digests of
the DNA. The relevant genotype of plasmid pSE-133-1 is hydB::Jn5, fhlA+
and pSE133-2 carries the transposon in the fhlA gene (hydB+,
fhlA::Tn5).
Plasmid and chromosomal DNA preparations. Small and large scale
plasmid isolation were carried out following standard alkaline lysis
protocol with some modifications (150). The cesium chloride gradient
consisted of (final concentration) 1 g/ml cesium chloride and 0.625
mg/ml ethidium bromide. This was centrifuged at 50,000 rpm (160,000 x
g), for 18 hr, at 18C. Plasmid band was recovered with syringe fitted
with a 22G x 1" needle, and ethidium bromide was removed with water
saturated 1-butanol. Extracted plasmid solution was diluted with 2
volumes of deionized water and then ethanol precipitated at -20C for 18
hr. After centrifugation (12,100 x g; 30 min; 4C), the pellet was
washed with 70% cold ethanol and vacuum dried. This was resuspended in
a small volume of sterile H20.
Chromosomal DNA was prepared according to Ausubel et al. (9) with
modifications. A culture of the E. coli strain of interest (1 liter)
was grown to mid-log phase (3 x 108 CFU/ml). The cells were harvested
by centrifugation at 2,300 x g, for 10 min at 4C. The pellet was
resuspended in 95 ml of TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0).


140
222. Stock, J. B., A. J. Ninfa, and A. M. Stock. 1989. Protein
phosphorylation and regulation of adaptive responses in bacteria.
Microbiol. Rev. 53:450-490.
223. Stoker, K., L. F. Oltmann, and A. Stouthamer. 1989. Randomly
induced Escherichia coli K-12 Tn5 insertion mutants defective in
hydrogenase activity. J. Bacteriol. 171:831-836.
224. Stoker, K., W. N. M. Reijnders, L. F. Oltmann, and A. H.
Stouthamer. 1989. Initial cloning and sequencing of hydHG, an
operon homologous to the ntrBC and regulating the labile
hydrogenase activity in Escherichia coli K-12. J. Bacteriol.
171:4448-4456.
225. Stokes, H. W., P. W. Betts, and B. G. Hall. 1985. Sequence of the
ebgA gene of Escherichia coli: comparison with the lacZ gene.
Mol. Biol. Evol. 2:469-477.
226. Stokes, H. W., and B. G. Hall. 1985. Sequence of the ebgR gene of
Escherichia coli. Evidence that the EBG and LAC operons are
decended from a common ancestor. Mol. Biol. Evol. 2:478-483.
227. Thony, B., and H. Hennecke. 1989. The -24/-12 promoter comes of
age. FEMS Mocrobiol. Rev. 63:341-358.
228. Tinoco, I. Jr., K. Sauer, and J. C. Wang. 1985. Physical
Chemistry: Principles and Applications to Biological Sciences.
Prentice-Hall, Inc., Englewood Cliffs, NJ.
229. Walker, J. E., M. Saraste, M. J. Runswick, and N. J. Gay. 1982.
Distantly related sequences in the a- and 8-subunits of ATP
synthase, myosin, kinases and other ATP-requiring enzymes and a
common nucleotide binding fold. EMBO J 1:945-951.
230. Warren, R. A. J. 1972. Lactose-utilizing mutants of lac deletion
strains of Escherichia coli. Can. J. Microbiol. 18:1439-1444.
231. Waugh, R., and D. H. Boxer. 1986. Pleiotropic hydrogenase mutants
of Escherichia coli K12: growth in the presence of nickel can
restore hydrogenase activity. Biochimie. 68:157-166.
232. Weiner, J. H., D. P. Maclsaac, R. E. Bishop, and P. T. Bilous.
1988. Purification and properties of Escherichia coli dimethyl
sulphoxide reductase, an iron-sulfur molybdoenzyme with broad
substance specificity. J. Bacteriol. 170:1505-1510.
233. Westerhoff, H., M. O'Dea, A. Maxwell, and M. Gellert. 1988. DNA


92
Therefore, the upstream DNA was sequenced in order to identify the
characteristics of the hydF and hydB genes and their products. The
sequence of a partial gene was also determined since it was present in
the plasmid used for these experiments. Sequence analysis revealed that
the hyd genes were transcribed in the same orientation as the fhlA gene
(Fig. 9). The first open reading frame (bases 1 to 216) encodes the
partial hypC product (3' end). The 72 amino acid residues identified
have an anhydrous molecular weight of 7,848 Da. The second open reading
frame, positions 219 to 1337, comprises the hydF gene. The gene codes
for a protein of 373 amino acids with an anhydrous molecular weight of
41,363 Da which is comparable to the apparent molecular weight of
44,000 Da obtained by "maxicell" experiments (188). Positions 1379 to
2344 constitute the hydB gene which codes for a 322 amino acids product
of an anhydrous molecular weight of 33,712 Da which again is comparable
to the apparent molecular weight obtained by Sankar and Shanmugam (187).
A typical "-10 and -35" or "-12 and -24" promoter sequence was not
evident (between positions 1 to 2311); however, "Shine-Dalgarno"
consensus sequences (positions 208 to 212 and 1367 to 1371) were located
in front of both complete genes (hydF and hydB) sequenced. A 38 base
pair intergenic region was located between the hydF and hydB genes. An
inverted repeat (underlined in Fig. 9; positions 2387 to 2393 and
positions 2397 to 2403) that can produce a 7-base-pairs stem and a 3-
base (2394 to 2396) loop structure followed the hydB open reading frame.
This region is not followed by a thymine residue stretch of significant


104
Table 10. Anaerobic expression of (hyp'-' lacZ*'), strains SE-2001 and
SE-2002, in different genetic backgrounds and culture media
Strain
Relevant genotype
LBb
p-Galactosidase activity3
LBGb LBFb LBNb
SE-2001
(hyp'~
1lacZ*)1
170
170
210
150
SE1654
4'{hyp1-
1 lacZr+) 1 narL
240
240
300
200
SE1651
4>(/?yp'-
' lacZf)1 fnr
50
130
130
43
SE1657
4 {hyp'-
'lacZ*)1 rpoN
53
135
160
130
SE1658
4>(byp'-
' lacZ+)1 rpoN
250
125
130
105
SE1760
4>(/?yp'-
'lacZ+)1 chlD
135/189
NDd
ND
ND
SE-2002
4>(/?yp'-
'lacZ+)2
190
210
290
220
SE1655
<1'{hyp'-
'ZocZ+)2 narL
100
145
160
95
SE1652
4 {hyp1-
'lacZ+)2 fnr
22
140
120
50
SE1660
(hyp'~
'lacZ+)2 rpoN
125
130
135
135
SE1659
4'(hyp'-
llacZ+)2 rpoN
140
135
150
130
SE1761
4>(/?yp'-
1lacZ+)2 chlD
135/202
ND
ND
ND
aExpresssed as nanomoles of o-nitrophenol produced per minute per
milligram of protein
bLB, Luria Broth; LBG, supplemented with 0.3% glucose; LBF, supplemented
with 0.1% formate; LBN, supplemented with 0.1% nitrate.
CLB grown culture/ LB supplemented with 1 mM Mo.


77
PROTEIN (fig)
Figure 8. Differential rate of synthesis of and lacZ*), strain SE-2011, in LB medium supplemented with 3 mM
formate in the presence and absence of the fhlA+ gene in a multicopy
plasmid (pSE-133-1). p-Galactosidase and protein activities are
expressed as units per ml and pg per ml, respectively.


22
biotin-sulfoxide reductase (55, 92, 111, 140, 141, 202, 219; for
review, see 217). Molybdopterin is a complex composed of a nonprotein
organic moiety (6-alkyl-pterin) with a Mo atom (111, 113). Initially,
Pateman et al. (169) linked anaerobic chlorate-resistance to nitrate
respiration in Aspergillus nidulans by demonstrating that the nitrate-
respiration defective mutant is unable to reduce chlorate to toxic
chlorite. The chlorate-resistance (chi) genes exhibited pleiotropic
phenotypes and were later identified as essential for molybdenum
transport, regulation and formation of functional molybdopterin
containing enzymes (for review, see 94). Complementation analysis and
deduced sequence homology to other periplasmic binding and transport
systems estimated at least 11 chi genes mapping at 5 loci which are
necessary for MPT biosynthesis in E. coli (110, 178).
The chlA operon, consisting of three complementation groups (18
min), and the chlE operon, composed of two (18 min), are presumed to be
necessary for synthesis of the organic portion of the MPT (11, 113, 178,
219). Mutations in either one of these operons give rise to defects in
pterin biosynthesis (114). Baker and Boxer (14) constructed
merodiploids containing chlA+ / (chlA'- lacZ*) to study the
transcription of the chlA locus. Various chi mutations (chlA, chlB,
chlD, chlG and chlE) were introduced into the merodiploid strains.
These results suggested that the chlA operon is anaerobically inducible
and repressed in the presence of MPT. These investigators concluded


50
previously (187, 188). Plasmid pSE-190 carries a 1.0 kb Sall-Pstl
internal fragment of fhlA gene from previously described plasmid pSE-128
(187) in vector plasmid pUC19. Sequencing of the hydX'FB {hypC'DE)
genes, upstream of the fhlA gene, was accomplished using plasmid pSE-
137, a Sall-Kpnl fragment containing the partial hydX (hypC) and hydF
(hypD) genes in vector plasmid pUC19, pSE-132 which was described
previously, and plasmid pSE125-1 which was constructed as a 2.38 kb
Clal deletion of previously described plasmid pSE-125 (185). The mol
operon was sequenced using previously described plasmid pSE-1009 (134,
136) and exonuclease Ill-generated deletion derivatives from the Kprtl
(the 5'-end of the open reading frames) towards the fcoRV site (88).
Procedures from Promega "Erase-a-base system" technical manual were
followed in these experiments. Plasmid pSE1004 was also used to
establish the fcoRV to Clal DNA sequence. Both strands of chromosomal
DNA present in these plasmids were sequenced using appropriate primers.
New oligonucleotide primers were synthesized, as needed, based on the
partial DNA sequence of the genes, by the DNA synthesis core laboratory,
Interdisciplinary Center for Biotechnology Research, and by Dr. F.C.
Davis, Department of Microbiology and Cell Science, University of
Florida. Commercially available sequencing primers were obtained from
US Biochemical Corporation, Pharmacia-LKB or New England BioLabs. DNA
sequence was determined using T7 DNA polymerase (Sequenase), obtained
from either US Biochemical Corporation or Pharmacia-LKB, and ^S-dATP
was supplied by DuPont-New England Nuclear. The protocols supplied by


15
investigated, metal-dependent regulation of FUR (iron uptake) and MER-R
(mercury resistance) systems (for review, see 90). It is possible that
molybdenum and nickel (necessary for transcriptional repression of chlD
and hydC genes, respectively) be also involve a metal-protein complex.
Formate hydrogenlyase synthesis is independent of the ARC system,
and FNR-dependence appears to be indirect (44, 105, 171). The
transcription of the pfl gene which is essential for the production of
formate is partially FNR dependent (190, 191). The gene coding for
nickel transport (hydC) is also FNR dependent (239). The hyp operon,
whose products presumably process the nickel into forms suitable for
hydrogenase, appears to be FNR regulated when the intracellular formate
concentration is limiting (this study).
DNA supercoiling has been suggested to be essential for
transcription from several promoters including the FNR-independent
promoters of the FHL pathway. Early studies have shown that mutants
lacking the DNA gyrase activity are also impaired in anaerobic growth
(241). Additionally, FHL activity is absent and plasmid supercoiling is
altered in a glucose-grown pgi (oxrC) mutant. Phosphoglucose isomerase,
a glycolytic enzyme encoded by the pgi (oxrC) gene, is necessary for the
synthesis of FDH-H, peptidase T, tripeptide permease, HYD-1 and HYD-3 in
Salmonella typhimurium (107, 108). Recently, Hsieh et al. (98) have
compared changes in [ATP]/[ADP] ratios and negative supercoiling of both
chromosomal and plasmid DNA upon shifting f. coli from aerobic to
anaerobic conditions. They monitored the effect of dioxygen tension on


91
however, binding site specificity may be determined by this region,
since R. meliloti nifH promoter activation was reduced 2-fold in E. coli
and 10-fold in R. meliloti (99). Likewise, mutations in the helix-turn-
helix region eliminate NTR-C repressive function in which binding to a
specific operator sequence is required (147).
Primary structure of the hydX'FB [hypC DE) genes upstream of the
fhlA gene. Amino acid sequence data of the FHL-A protein suggests that
a typical "sensor" protein (histidine-protein-kinase) is probably not
required for FHL-A activity. However, the possibility of a nearby
region encoding a sensor protein can not be ruled out. For example, the
K. pneumoniae nifLA operon encodes both the activator (NIF-A) and its
repressor (NIF-L). Sankar and Shanmugam (187, 188) described two genes,
hydF and hydB, which were upstream of the fhlA gene. Based on genetic
analysis, the 44 kDa and 32 kDa polypeptides coded by the hydF and hydB
genes, respectively, appeared to interact with the fhlA gene product.
Partial hydF gene products were capable of restoring total hydrogenase
activity but only in the presence of multiple copies of fhlA gene (188).
There appeared to be a direct correlation between the size of the
overexpressed, partial HYD-F protein and the level of hydrogenase
activity produced by the hydF mutants, strains SE-65 and SE-67.
Additionally, the hydB gene appeared to substitute for the hydF gene in
specific genetic backgrounds (SE-203 hydF, x~). This suggested that
hydF, hydB, fhlA, and an unidentified gene product could potentially
interact in the production of FHL activity.


72
SE2011 did not alter the amount of p-galactosidase activity produced by
the culture both in the presence and absence of nitrate (data not shown).
Effect of multiple copies of the fhlA gene on fhlB transcription.
The effect of increasing the copy number of the fhlA gene on the
expression of (fhlB'-'lacZ+) was investigated using plasmid pSE-133
which carries the complete fhlA+ and hydB+ genes (186, 187). In the
presence of plasmid pSE-133, the differential rate of production of p-
galactosidase activity by (fhlB'-' lacZ+) was about 700 units / pg cell
protein in LB-formate medium (Fig. 6). This value is greater than 5
times the rate of about 130 units / pg cell protein for strain SE-2011
cultured in the same medium. The maximum activity produced by strain
SE201l/pSE-133 is also greater than 2 times the values obtained with
strain SE-2011 and this increase was detected immediately after
establishing anaerobic conditions. The maximum activity observed in
strain SE-2011/pSE133, grown in LB medium without formate
supplementation, was also increased to about 1,100 units of p-
galactosidase activity. This level of activity is comparable to the
values obtained with strain SE-2011 grown in LB medium with 30 mM
formate (about 1,000 units) although the differential rate of induction
observed with strain SE-2011/pSE133, in LB medium, was lower.
Inserting transposon Tn5 into the fhlA gene in the plasmid (pSE-133-2;
Fig. 7) abolished this enhancing effect of plasmid pSE-133 while a
hydB::lr\5 mutation in the plasmid (pSE-133-1) had no effect indicating
that the plasmid-mediated increase is due to the fhlA gene. Transferring


35
Table 1.
continued
Strain
Relevant Genotype or Phenotype
Source or Reference
YMC18
endA, thi, hsdR, h(lacU)169,
rpoN:JnlO
B. Magasanik
Salmonella typhimurium
TT627 strAl, pyrC7/F'tsll4 zz/::TniO
J. Roth (40)


11 Alignment of the predicted "domain D" amino acid sequences
of E. coli FHL-A, K. pneumoniae NIF-A, S. typhimurium FLB-D,
E. coli NTR-C, P. putida XYL-R, R. leguminosarum DCT-D, and
E. coli TYR-R 88
12 Alignment of potential DNA-binding motifs in the FHL-A,
HYD-G, NTR-C, XYL-R and NIF-A proteins 90
13 Localization of the promoter lac fusions in strains SE-2007
[Q(fhlA'-'lacZ*)], SE-2001 [*{hyp'-'lacZ+)1], and SE-2002
[Q{hyp'lacZ+)2] 96
14 Differential rate of synthesis of Q(fhlAlacZ*), strain
SE-2007, and (hypX'-' lacZ+)li strain SE-2001, in LB
medium supplemented with 3 mM formate in the presence and
absence of the fhlA+ gene in a multicopy plasmid (pSE-133). 108
15 Nucleic acid sequence of the mol {chi) operon 113
VI


83
AAGTTGAAATGGTGGCGCAAAGTGACAGTACCGTGCTGATCCTCGGTGAAACTGGCACGG 3660
VEMVAQSDSTVLI LGETGTG
GTAAAGAGCTGATTGCCCGTGCGATCCATAATCTCAGTGGGCGTAATAATCGCCGCATGG 3720
KELIARAIHNLSGRNNRRMV
TCAAAATGAACTGCGCGGCGATGCCTGCCGGATTGCTGGAAAGCGATCTGTTTGGTCATG 3780
KMNCAAMPAGLLESDLFGHE
AGCGTGGGGCTTTTACCGGTGCCAGCGCCCAGCGTATCGGTCGTTTTGAACTGGCGGATA 3840
RGAFTGASAQRIGRFELADK
AAAGCTCCCTGTTCCTCGACGAAGTGGGCGATATGCCACTGGAGTTACAGCCGAAGTTGC 3900
SSLFLDEVGDMPLELQPKLL
TGCGTGT ATTGCAGGAACAGGAGTTTGAACGTCTCGGCAGCAACAAAATCATTCAGACGG 3960
RVLQEQEFERLGSNKI IQTD
ACGTGCGTCTAATCGCCGCGACTAACCGCGATCTGAAAAAAATGGTCGCCGACCGTGAGT 4020
VRLIAATNRDLKKMVADREF
TCCGTAGCGATCTCTATTACCGCCTGAACGTATTCCCGATTCACCTGCCGCCACTACGCG 4080
RSDLYYRLNVFPIHLPPLRE
AGCGTCCGGAAGATATTCCGCTGCTGGCGAAAGCCTTTACCTTCAAAATTGCCCGTCGTC 4140
RPEDIPLLAKAFTFKIARRL
TGGGGCGCAATATCGACAGCATTCCTGCCGAGACGCTGCGCACCTTGAGCAACATGGAGT 4200
GRNIDSIPAETLRTLSNMEW
GGCCGGGTAACGTACGCGAACTGGAAAACGTCATTGAGCGCGCGGTATTGCTAACACGCG 4260
PGNVRELENVI ERAVLLTRG
Pst I
GTAACGTGCTGCAGCTGTCATTGCCAGATATTGTTTTACCGGAACCTGAAACGCCGCCTG 4320
NVLQLSLPDIVLPEPETPPA
CCGCAACGGTTGTCGCCCTGGAGGGCGAAGATGAATATCAGTTGATTGTGCGCGTGCTGA 4380
ATVVALEGEDEYQLIVRVLK
Tn5

AAGAAACCAACGGCGTGGTTGCCGGGCCTAAAGGCGCTGCGCAACGTCTGGGGCTGAAAC 4440
ETNGVVAGPKGAAQRLGLKR
GCACGACCCTGCTGTCACGGATGAAGCGGCTGGGAATTGATAAATCGGCATTGATTTAAC 4500
TTLLSRMKRLGIDKSALI ***
TGCAAATTGCCGGACAGATCTGCCTGTCCGGCATACTATTCATGAGGTTTTTTCGGACGA 4560
Clal
TATTTTTCCGGCAGTTCTGGCACCGGACGCTTGTCATCGAT 4601
Figure 9continued.


52
NaCl; 0.15 M trisodium citrate) buffer (150). Membranes were dried
under vacuum at 80C for 3 hr and stored dry between two pieces of
"Whatman" 3MM filter paper in plastic bags until used for hybridization.
DNA probes were labeled by random primed incorporation of digoxigenin-
labeled deoxyuridine-triphosphate (Dig-dUTP), hybridized under stringent
conditions to the transferred DNA, and immunologically detected using an
alkaline phosphatase linked antibody-conjugate. Procedures provided
with the Boehringer-Mannheim Biochemicals "Genius"-kit were performed to
carry out these experiments.
For localization of the lac fusion in strain SE2007, an internal
fhlA gene fragment (1.3 kb Sall-Pstl) from plasmid pSE-190 was labeled
and used to probe chromosomal DNA from strains SE-2007 and MC4100
(parent) digested with either a single or two restriction endonucleases
(Sail and Bgll, Sail and fcoRI, Sail and Clal, or Clal). Similarly lac
fusion strain SE-2001 (strains MC-4100 and SE-2009 were used as
controls) chromosomal DNA was digested with endonucleases Kpnl and SoZI,
Clal and Bgll, Bgll, or SoZI and EcoRI and probed with a 0.7 kb SoZI-
Kpnl fragment from plasmid pSE-137 (carrying the partial hypCD genes).
Further localization of the lac fusion was done on both mutant strains,
SE-2001 and SE-2002, by digesting chromosomal DNA with Pst I and then
using 4.0 kb and 4.6 kb Pstl fragments from plasmid pSE-111 as probes.


98
that the fhlA gene is maximally induced at a higher oxygen tension in
the growth medium than the fhlB gene.
Constitutive fhlA gene expression under anaerobiosis. Anaerobic
expression of the fhlA gene was tested in LB medium supplemented with
various effectors of the FHL pathway. Addition of alternate electron
acceptors (nitrate and fumarate) did not influence the transcription of
the fhlA gene (Table 8). Lower levels of p-galactosidase activity
observed in cells grown in LB + nitrite medium may be a consequence of
growth inhibition by nitrite. It is interesting to note that the
exogenous formate and glucose did reduce the fhlA operon expression.
LBG medium buffered with 0.1 M sodium phosphate to pH 7.0 reversed the
effect of glucose on the levels of p-galactosidase produced by the
culture. Additional experiments were done to verify that pH had no
effect on fhlA gene expression using buffers ranging from pH 6.0 to 7.5
(0.1 M MES, pH 6.0; 0.1 M PIPES, pH 6.5; 0.1 M MOPS, pH 7.0; 0.1 M TES,
pH 7.5). Again the results were comparable; low pH did not enhance fhlA
gene transcription (data not shown).
Several genes known to be required for dihydrogen production,
including rpoN, narL, fnr, cya, molR, chlD and p/Z, were tested for
their requirement in transcription of (//?ZA'-' ZocZ4-). Appropriate
double-mutant strains were constructed to investigate the fhlA gene
regulation. Analysis of these strains suggested that fhlA transcription
is independent of known regulators of formate hydrogenlyase production
(Table 9). The only double-mutant strain exhibiting reduced levels of


36
the gas phase was replaced with dinitrogen. After 16 hr of incubation
at 37C, dihydrogen in the gas phase of the culture tubes was determined
using a gas chromatograph (Varian; Model 920) fitted with a 50 nm
molecular sieve column. From a total of 68 mutants, 13 were found to be
defective in dihydrogen production (Fhl_) and were analyzed further.
Enzyme Activities and their Respective Culture Conditions
P-qalactosidase activities and culture conditions. For anaerobic
induction of p-galactosidase activity in mutant strains SE-2001, SE-2002
and SE-2011, 120 ml of medium in a 160 ml "Wheaton" bottle was
inoculated (1% V/V) with a 1.5 hr old aerobic culture, grown at 37C, in
a shaker, at 250 rpm. However, with strain SE-2007 or derivatives
thereof, alternate procedures were used to maximally aerate the culture
at low cell density before starting the experiment. A 2 hr old aerobic
culture (37C; 250 rpm) was transferred to fresh LB medium (1% V/V) and
grown again in the shaker for 1 hr. This culture was used to inoculate
the experimental medium at 10% (V/V) and then grown under anaerobic
conditions in a "Wheaton" bottle. The bottles were closed with rubber
stoppers and secured with aluminum seals. The gas phase was replaced
with argon. Samples were removed at different time periods with a
syringe and needle and growth of the culture and p-galactosidase
activity of the cells were determined. In another set of experiments,
the aerobic cultures were used to inoculate (1% V/V) the appropriate
medium in 13 x 100 mm screw cap tubes filled to the top. Cells from


105
SE1652, respectively. In LB with and without nitrate supplementation,
the maximum (J-galactosidase activity observed in these cultures was 50 U
which is a 3- to 4-fold reduction in activity from the parent hyd (hyp)
strains. Further analysis of these fnr double mutants, suggested that
the FNR-dependent induction of the hyd (hyp) operon was alleviated upon
formate or glucose supplementation (Table 10). When strains SE-1651 and
SE1652 were inoculated from microaerobic (1 ml standing LB cultures) to
anaerobic conditions and cultured for 4 hr, the maximum activity of the
cultures was comparable to activity in the presence of formate (data not
shown). Perhaps the formate or FHL-A protein produced during the period
of microaerobic conditions was sufficient for activation of the FNR-
independent promoter(s) of the hyd (hyp) operon. The rpoN gene product
was only required for anaerobic induction in one of the double mutants
tested, strain SE1657. The activity produced by this culture was
reduced 3-fold from parental values, but only in LB medium. The chlD
and narL mutations had no apparent effect on hyd (hyp) operon
expression. Additionally, nickel supplementation (250 pM) had no
influence which is in contrast to the repressive effect in strain SE-
2009, the hyd mutant which is not located in this 58-59 min region. It
would be interesting to test whether a hyd (58 min) mutation, presumed
to be necessary for nickel processing, would alleviate the nickel
repression observed in strain SE-2009. Potentially, a protein-bound
form of nickel would exert this negative effect on transcription.
These results are comparable to the DNA sequence data which


74
FORMATE (mM)
Figure 7. Effect of formate concentration on the levels of p-
galactosidase activity produced by a ^{fhlB'-'lacZf) pfl double mutant,
strain MJ-9, in the presence of plasmids pSE-133 and pSE133-2
[fhlA::Tn5). Cultures were grown for 4 hr in LB medium with appropriate
concentrations of formate under anaerobic conditions before the assay.


99
Table 8. Effect of media composition on the expression
of Q(fhlA'-'lacZ*) in an fhlA mutant, strain SE-2007
Medium
p-Galactosidase
Activity3
Luria Broth
1,800
+ Formate (0.5%)
1,300
o

fH
i
CO
o
+
1,700
+ N02" (1.0%)
1,200
+ Glucose (0.3%)
1,200
+ Glucose + buffer13
2,000
+ Fumarate (0.5%)
1,700
All cultures were grown anaerobically at 37C in Luria Broth
supplements.
Expressed as nanomoles of o-nitrophenol produced per minute per milligram
of protein.
b0.1M Phosphate buffer at pH 7.0.


55
MC4100, measured either as BV or fumarate reduction. The hydrogenase
activity of this strain, measured as tritium exchange was close to 100%
of the parent. Strain M9s produced elevated levels of FDH-N (6.6-fold)
and lower levels of fumarate reductase (28%) as compared with its
parent, strain MC4100. These values which are comparable to the
phenotype described by Pecher et al. (171) are quite distinct from the
properties of strain SE-2011 (Table 3).
Strain SE-2011 is normal for nitrate respiration which requires
active FDH-N, but has not been tested for the recently described third
FDH isoenzyme which is presumed to be a major component of formate
oxidase, expressed both aerobically and anaerobically (192). The level
of fumarate reductase activity in strain SE-2011 was also lower (less
that 20% of the parent value). This deficiency is probably the reason
for the growth characteristics of strain SE-2011. The aerobic and
anaerobic growth of this mutant was comparable to that of the parent,
strain MC4100, in LB supplemented with different sugars. However,
strain SE-2011 failed to grow in glucose-minimal medium and to produce
succinate as a fermentation product. Since succinate is a necessary
precursor for biosynthesis, this would account for the poor growth of
the organism in defined medium. As will be discussed later, the same
phenotype was observed when this mutation was transduced into strain
MC4100 or other lac deletion mutants of E. coli (strains CSH26 and
BW545), indicating that the pleiotropic effect is due to a single gene
defect in these genetic backgrounds. The altered gene is termed fhlB


48
Table 2. Plasmids used in this study
Plasmid
Relevant Genotype or Phenotype
Reference
pRBH
Cmr, 14 kb CZoI-flomHI fragment with
the complete hyd-17 gene cluster from
MC4100 in pACYC184
A. Bock (27)
pSE-111
Apr, 14.7 kb SauSM fragment with the
complete hyd [hyp) and partial hyc
operon in pBR322
Laboratory
collection
(185)
pSE-125
Tcr, 2.8 kb Sail fragment from pSE-22 with
partial hypC and fhlA genes and complete
hypDF [hydFB) genes in pBR322
Laboratory
collection
(187)
pSE-125-1
Tcr, 0.8 kb Sall-Clal fragment from
pSE-125 with partial hypCD [hydXF) genes
in pBR322
This study
pSE-128
Apr, 6.5 kb SoZI-Sou3Al fragment from
pSE-22 with partial hypC [hydX) gene
and complete hypDF [hydFB) and fhlA
genes in pBR322
Laboratory
collection
(185)
pSE-130
Apr, 4.7 kb Pst I fragment with partial
hypA and fhlA genes and complete hypBCDF
(hydFXFB) genes in pBR322
Laboratory
collection
(186)
pSE-132
Apr, 2.0 kb Kpnl-Sall fragment from Laboratory
pSE-125 with partial hypD [hydF) and fhlA collection
genes and complete hypF [hydB) gene in pUC19
(187)
pSE-133
Apr, 3.8 kb CZoI fragment with partial
hydF gene and complete hydB and fhlA genes
Laboratory
collection
(187)
pSE-133-1
As pSE-133, hydB::lr\5
This study
pSE-133-2
As pSE-133, fhlA::Tr\5
This study
pSE-137
Apr, 1.0 kb Kpnl-Sall fragment from
with partial hypCD [hydXF) genes in pUC19
Laboratory
collection
(138)
pSE-190
Apr, 1.3 kb SoZI-Pstl fragment from
pSE-111 with partial fhlA gene in pUC19
Laboratory
collection
(188)


69
activities to 75% and 100% of wild type levels, respectively. This
suggests that the mutation is more complex than anticipated. Further
analysis is currently being done to determine the extent of the hyb
operon on the Sau3M fragment which is carried by plasmid pSE1213.
Also, the exact location of the fhlB mutation in strain SE-2011 is under
investigation using polymerase chain reaction (PCR) procedures combined
with ${fhlB'-'lacZ+) cloning methods.


122
formate dehydrogenase gene (fdhF) of Escherichia coli. Mol.
Microbiol. 3:187-195.
23. Birkmann, A., H. Hennecke, and A. Bock. 1989. Construction of
chimeric promoter regions by exchange of the upstream regulatory
sequences from fdhF and nif genes. Mol. Microbiol. 3:697-703.
24. Birkmann, A., R. G. Sawers, and A. Bock. 1987. Involvement of the
ntrA gene product in the anaerobic metabolism of Escherichia coli.
Mol. Gen. Genet. 210:535-542.
25. Birkmann, A., F. Zinoni, G. Sawers, and A. Bock. 1987. Factors
affecting transcriptional regulation of the formate-hydrogen-lyase
pathway of Escherichia coli. Arch. Microbiol. 148:44-51.
26. Bock, A., K. Forchhammer, J. Heider, W. Leinfelder, G. Sawers, B.
Veprek, and F. Zinoni. 1991. Selenocysteine: the 21st amino acid.
Mol. Microbiol. 5:515-520
27. Bohm, R., M. Sauter, and A. Bock. 1990. Nucleotide sequence and
expression of an operon in Escherichia coli coding for formate
hydrogenlyase components. Mol. Microbiol. 4:231-243.
28. Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72:248-254.
29. Bremer, E., T. J. Silhavy, J. M. Weisemann, and G. M. Weinstock.
1984. Lambda pZocMu: a transposable derivative of bacteriophage
lambda for creating lacZ protein fusions in a single step. J.
Bacteriol. 158:1084-1093.
30. Brosius, J., and J. R. Lupski. 1987. Plasmids for the selection
and analysis of prokaryotic promoters. Meth. Enzymol. 153:54-68.
31. Buchanan-Wollaston, V., M. C. Cannon, J. L. Beynon, and F. C.
Cannon. 1981. Role of the nifA gene product in the regulation of
nif expression in Klebsiella pneumoniae. Nature 294:776-778.
32. Campbell, J. H., J. A. Lengyel, and J. Langridge. 1973. Evolution
of a second gene for p-galactosidase in Escherichia coli. Proc.
Natl. Acad. Sci. USA 70:1841-1845.
33. Casse, F. 1970. Mapping of the gene chlB-controlling membrane
bound nitrate reductase and formic hydrogenlyase activities in
Escherichia coli K12. Biochem. Biophys. Res. Commun. 39:429-436.


79
Analysis of the fhlA Gene
Primary structure of the fhlA gene. Previous experiments
identified the fhlA gene product as a putative regulatory element of
both fdhF and hyd-17 genes (185, 187, 196). The results presented above
(Table 6, Fig. 6-8) also show that the FHL-A protein is a needed
regulatory element for the fhlB operon. Because of these observations,
the DNA sequence of the fhlA gene was determined to identify the
characteristics of the gene and its product. The fhlA gene (coding
region position 2421 to 4497; Fig. 9) codes for a protein of 692 amino
acids with an anhydrous molecular weight of 78,467 Da which is
comparable to the apparent molecular weight of 78,000 Da obtained by
other experiments (144, 185). This protein did not contain any
significant hydrophobic region indicating that the primary location of
this protein is the cytoplasm. Eight base pairs from the end of
translational stop codon (position 4497), the coding region is followed
by an inverted repeat (underlined in Fig. 9; positions 4508 to 4517 and
4524 to 4533) which can produce a 10 base pairs stem and a 6 bases
(positions 4518 to 4523) loop structure. This region is followed by a
stretch of 6 thymine residues at a distance of 14 bases (positions 4548
to 4553). Five more thymine residues can be found 9 bases from the
first set of thymines (positions 4563 to 4567). This segment of DNA, in
appropriate configuration may function as a p-independent transcription
termination site. The 5'-end of the putative coding region is preceded


68
Table 5. Plasmid complementation analysis of fhlB mutant,
strain SE-2011
Specific activity
Strain/plasmid
FHLa
HUPb
(H2 to BV) (H2
HUPC
to fumarate)
BW545
100%
694
44
SE-2011
UDd
UD
UD
SE-2011/pRBH
100%
+e
NDf
SE2011/pSE1213
10%
1,017
63
SE201l/33pBR
75%
616
91
Expressed as % wild type levels of H2 produced.
Expressed as nanomoles of BV reduced per minute per milligram of cell
protein.
^Expressed as nanomoles of H2 consumed per minute per milligram of cell
rotein.
UD, Undetectable.
eMainly HYD-3 activity.
fND, Not determined.


44
and incubated for 2 hr standing at 37C. Cells were sedimented (3,500 x
g; 5 min; 25C) and resuspended in 0.4 ml of 0.1 M CaCl2.2H20. DNA
(about 50 ng) was added to 0.2 ml of cell suspension and incubated on
ice for 20 min. This was transferred to 42C for 2 min and then
returned to ice for 10 min. Fresh LB medium (1.0 ml) was added, and
cells were incubated at 37C for 1 to 2 hr and then plated on applicable
selection medium. If more cells were needed, the overnight culture was
inoculated into 10 ml LB medium and aerobically grown at 30C for 1.5 to
2 hr. This was transferred to 37C for 30 min and then harvested (3,500
x g; 5 min; 25C). After washing with 0.1 M NaCl, the cells were
resuspended in equal volume of 0.1 M CaCl2.2H20 and incubated for 20 min
at 25C. This was then pelleted and resuspended in CaCl2 solution at
approximately one-fifth the original volume. Similar procedures were
then followed to that described above.
For high efficiency transformation, competent cells were prepared
by a modified procedure and stored at -70C (Sankar, personal
communication). An overnight culture was used to inoculate 50 ml of
fresh LB medium. This was grown to early-log phase (2 x 108 CFU/ml)
aerobically at 37C. The cells were centrifuged (3,000 x g; 5 min;
4C), washed in 0.1 M cold MgCl2.6H20, resuspended in equal volume of
0.1 M cold CaCl2.2H20, and incubated on ice 20 min. Treated cells were
then pelleted (3,000 x g; 5 min; 4C) and resuspended in one-tenth the
starting volume with 0.1 M CaCl2 containing 15% glycerol. This was
aliquoted and stored at -70C for later use. Cells were thawed slowly


LIST OF GENE SYMBOLS
All the genes listed below are from Escherichia coli unless otherwise
indicated.
Gene Alternate gene symbols; phenotype affected
symbol
ant See hyc; anaerobic electron transport
arcA Aerobic regulatory control, putative DNA binding
protein of Arc modulon
arcB Aerobic regulatory control, histidine-protein-
kinase of Arc modulon
chi A Synthesis of the pterin component of MPT
chlB "Association factor-FA"; synthesis of functional MPT
chlC See narGHJI
chlD Peripheral protein of molybdate binding-protein-dependent
transport system
chlE Synthesis of the pterin component of MPT
chlF See fdhGHI
chlG Molybdate-restorable nitrate reductase activity
chlJ Integral membrane protein of molybdate binding-protein-
dependent transport
cyd Cytochrome d\ high-affinity oxidase
cyo Cytochrome o; low-affinity oxidase
dctD C-4 dicarboxylate transport; Rhizobium leguminosarum
dmsABC DMSO and TMAO reductase activity
xi


CONCLUSION
Genetic regulation of the FHL system encompasses a number of
factors including anaerobiosis, nitrate, formate, low pH, molybdate,
nickel and selenium. Genetic and physiological analysis of the fhlB
operon can provide answers to the molecular mechanism by which these
various components influence the levels of FHL activity in the cell.
This study demonstrates that the fhlB operon is anaerobically inducible
and requires o54, FHL-A, formate and molybdate for expression.
Sigma-54 is an alternate sigma factor required for fhlB operon
expression and is encoded by the rpoN (ntrA, glnF) gene. By monitoring
the expression of the rpoN gene using lac gene fusions, it has been
demonstrated that this sigma factor is constitutively expressed in £.
coliy K. pneumoniae and R. meliloti (34, 54, 154, 182). Whereas, the
majority of alternate sigma factors which have been analyzed are
inducible (as is the case for sporulation in B. subtilis; ref. 129,
142). This implies an interesting mechanism of transcriptional
regulation at o54-dependent promoters. Currently it is presumed that
transcriptional control is mediated predominately by modulation of the
activator protein (receiver) and not by altering the levels of the o54-
subunit (for review, see 90, 131). Therefore, identification and
116


107
required for formate inducibility of the hyd [hyp) operon, as is the
case for fhlB operon expression, the increase would be absent in strains
SE-2001 and SE-2002. Plasmid pSE-133, a Clal insert retaining the
partial hydF and complete hydB, fhlA genes, complements the fhlA mutants
(SE-1174 and SE2007). Even though the hyd [hyp) promoter(s) are absent
in this plasmid, it is conceivable that the cloned genes are transcribed
from a pBR-322 vector promoter.
Effect of multiple copies of the fhlA gene on fhlA and hyd [hyp)
transcription. In order to study these possibilities, the effect of
multiple copies of the fhlA gene on its own expression and transcription
of the hyd [hyp) operon were monitored using plasmid pSE-133 (Fig. 14).
The p-galactosidase activity of strains SE-2007 plasmid pSE-133 was
comparable, irregardless of the presence of high copy fhlA+ or medium
composition (Table 9). Interestingly, the fhlA gene is induced at very
early stages of the organism's anaerobic growth to approximately 500 U.
Then during late-log phase, a second induction occurs at a lower rate to
a maximum value of about 1,500 U. The FHL-A protein would be available
initially to activate the FHL pathway as formate is produced by the
organism. In later stages of growth, formate levels would be higher (pH
lower) and therefore, FHL-A protein would be necessary for maximum
activation of the FHL system. However, transcription of the fhlA gene
does not require formate as an inducer (Table 9).
From the data presented (Fig. 14), the hyd [hyp) operon expression
differs from the fhlA gene regulation. In the presence of multiple


RESULTS AND DISCUSSION
Physiological Properties of an fhlB Mutant, Strain SE-2011
Biochemical characteristics of an fhlB mutant, strain SE-2011.
Using ApZocMu53 mutagenesis (29), strain SE-2011 was isolated as a lac
operon fusion derivative of strain MC4100 which produced p-galactosidase
activity anaerobically and was deficient in fermentative dihydrogen
production (Fhl-). Upon detailed biochemical analysis, this strain was
found to be affected in the production of hydrogenase, formate
dehydrogenase-H, and fumarate reductase activities (Table 3). The lack
of tritium exchange activity in strain SE-2011 shows that all three HYD
isoenzymes are absent in this strain. As a consequence of this defect,
both FHL, which requires active HYD-3, and hydrogen uptake, mediated by
HYD-2, activities were not detectable in this strain. The FDH-H
activity in strain SE-2011 was less than 10% of the levels observed in
the parent, strain MC4100. On the basis of this property, strain SE-
2011 can be distinguished from all known hyd mutants, which produced
FDH-H activity. Similarly, the SE-2011 phenotype can be readily
distinguished from FDH-H mutants using the hydrogen uptake
characteristics of the other strains. In experiments which are similar
to the ones described in Table 3, strain M9s, a known fdhF mutant (171),
produced 75 to 85% of hydrogen uptake activity of the parent, strain
53


134
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Covarrubias. 1987. Complete nucleotide sequence of the glnALG
operon of Escherichia coli K12. Nucleic Acids Res. 15:2757-2770.
158. Morett E., and M. Buck. 1988. Nif-A dependent in vivo protection
demonstrates that the upstream activator sequence of nif promoters
is a protein binding site. Proc. Natl. Acad. Sci. USA 85:9401-
9405.
159. Newman, M., and J. A. Cole. 1978. The chromosomal location and
pleiotropic effects of mutations of the nirA+ gene of Escherichia
coli K12: the essential role of nirA+ in nitrite reduction and in
other anaerobic redox reactions. J. Gen. Microbiol. 106:1-12.
160. Nicholls, D. G. 1982. Bioenergetics. An Introduction to the
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161. Ninfa, A. J., and B. Magasanik. 1986. Covalent modification of the
glnG product NRI, by the glnL product, NRII, regulates the
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162. Ninfa, A. J., E. G. Ninfa, A. Lupas, A. Stock, B. Magasanik, and
J. Stock. 1988. Crosstalk between bacterial chemotaxis signal
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Ntr regulon: evidence that nitrogen assimilation and chemotaxis
are controlled by a common phosphotransfer mechanism. Proc. Natl.
Acad. Sci. USA 85:5492-5496.
163. Nixon, B. T. C., C. W. Ronson, and F. M. Ausubel. 1986. Two-
component regulatory systems responsive to environmental stimuli
share strongly conserved domains with the nitrogen assimilation
regulatory genes ntrB and ntrC. Proc. Natl Acad. Sci. USA 83:7850-
7854.
164. Nohno, T., S. Noji, S. Taniguchi and T. Saito. 1989. The narX and
narL genes encoding nitrate-sensing regulators of Escherichia coli
are homologous to a family of prokaryotic two-component regulatory
genes. Nucleic Acids Res. 17: 2947-2957.
165. Pabo, C. 0., and R. T. Sauer. 1984. Protein-DNA recognition. Ann.
Rev. Biochem. 53:293-321.


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78. Gray, C. T., J. W. T. Wimpenny, D. E. Hughes, and M. R. Mossmann.
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with shifts. Biochim. Biophys. Acta. 117:22-32.
79. Green, J., M. Trageser, S. Six, G. Unden, and J. R. Guest. 1991.
Characterization of the FNR protein of Escherichia coli, an
iron-binding transcriptional regulator. Proc. R. Soc. Lond. B
244:137-144.
80. Gross, R., B. Arico, and R. Rappuoli. 1989. Families of bacterial
signal-tranducing proteins. Mol. Microbiol. 3:1611-1667.
81. Gunsalus, R. P., L. V. Kalman, and R. R. Stewart. 1989. Nucleotide
sequence of the narL gene that is involved in global regulation of
nitrate controlled respiratory genes of Escherichia coli. Nucleic
Acids Res. 17:1965-1975.
82. Haddock, B. A., and C. W. Jones. 1977. Bacterial respiration.
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83. Hansen, R. G., and U. Henning. 1966. Regulation of pyruvate
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84. Harold, F. M. 1986. The Vital Force: A Study of Bioenergetics. W.
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81
AGGCGATTGCCGATGTGTTCT6T6TCAACGGCGACAGCGAATGGCGCGGCTTAGGCGTGA 1080
AIADVFCVNGDSEWRGLGVI
TTGAATCTTCTGGCGTGCACCTGACGCCGGATTATCAACGATTCGATGCCGAAGCACATT 1140
ESSGVHLTPDYQRFDAEAHF
TCCGCCCGGCACCGCAGCAGGTCTGCGATGACCCGCGCGCGCGTTGTGGTGAGGTATTAA 1200
RPAPQQVCDDPRARCGEVLT
CGGGCAAATGTAAGCCGCATCAATGCCCGCTGTTTGGTAACACCTGTAATCCTCAAACCG 1260
GKCKPHQCPLFGNTCNPQTA
CGTTTGGTGCGCTGATGGTTTCCTCCGAAGGAGCGTGCGCCGCGTGGTATCAGTATCGTC 1320
FGALMVSSEGACAAWYQYRQ
AGCAGGAGAGTGAAGCGTGAATAATATCCAACTCGCCCACGGTAGCGGCGGCCAGGCGAT 1380
Q E S E A *** M
GCAGCAATTAATCAACAGCCTGTTTATGGAAGCCTTTGCCAACCCGTGGCTGGCAGAGCA 1440
QQLINSLFMEAFANPWLAEQ
GGAAGATCAGGCACGTCTTGATCTGGCGCAGCTGGTAGCGGAAGGCGACCGTCTGGCGTT 1500
EDQARLDLAQLVAEGDRLAF
CTCCACCGACAGTTACGTTATTGACCCGCTGTTCTTCCCTGGCGGTAATATCGGCAAGCT 1560
STDSYVIDPLFFPGGNIGKL
GGCGATTTGCGGCACAGCCAATGACGTTGCGGTCAGTGGCGCTATTCCGCGCTATCTCTC 1620
AICGTANDVAVSGAI PRYLS
CTGTGGCTTTATCCTCGAAGAAGGATTGCCGATGGAGACACTGAAAGCCGTAGTGACCAG 1680
CGFI LEEGLPMETLKAVVTS
CATGGCAGAAACCGCCCGCGCGGCAGGCATTGCCATCGTTACTGGCGATACTAAAGTGGT 1740
MAETARAAGIAIVTGDTKVV
GCAGCGCGGCGCGGTAGATAAACTGTTTATCAACACCGCTGGCATGGGCGCAATTCCGGC 1800
QRGAVDKLFINTAGMGAIPA
GAATATTCACTGGGGCGCACAGACGCTAACCGCAGGCGATGTATTGCTGGTGAGCGGTAC 1860
NIHWGAQTLTAGDVLLVSGT
ACTCGGCGACCACGGGGCGACTATCCTTAACCTGCGTGAGCAGCTGGGGCTGGATGGCGA 1920
LGDHGATI LNLREQLGLDGE
ACTGGTCAGCGACTGCGCGGTGCTGACGCCGCTTATTCAGACGCTGCGTGACATTCCCGG 1980
LVSDCAVLTPLIQTLRDIPG
CGTGAAAGCGCTGCGTGATGCCACCCGTGGTGGTGTAAACGCGGTGGTTCATGAGTTCGC 2040
VKALRDATRGGVNAVVHEFA
GGCAGCCTGCGGTTGTGGTATTGAACTTTCAGAAGCGGCACTGCCTGTTAAACCTGCCGT 2100
AACGCGI ELSEAALPVKPAV
GCGTGGCGTTTGCGAATTGCTGGGACTGGACGCCCTGAACTTTGCCAACGAAGGCAAACT 2160
RGVCELLGLDALNFANEGKL
AGTAATAGCTGTTGAACGCAACGCGGCAGAGCAAGTGCTGGCAGCGTTACATTCCCATCC 2220
VIAVERNAAEQVLAALHSHP
ACTGGGGAAAGACGCGGCGCTGATTGGTGAAGTGGTGGAACGTAAAGGTGTTCGTCTTGC 2280
LGKDAALIGEVVERKGVRLA
CGGTCTGTATGGCGTGAAACGAACCCTCGATTTACCACACGCCGAACCGCTTCCGCGTAT 2340
GLYGVKRTLDLPHAEPLPRI
Figure 9continued


31
Table 1. Bacterial strains used in this study
Strain
Relevant Genotype or Phenotype
Source or Reference
E. coli
BW545
h[lacU)169, rpsL
G. Walker (242)
CSH26
ara, A{lac-pro), thi
Laboratory
collection
JRG780
trpA9761, frdAll, trpR72, gal-25,
rpsL195
CGSC #5916
JRG861a
gal, trpA9761, iclR, trpR, rpsL, fnr
J. Guest
LCB898
thr-1, leuB6, pfl-1, thi-1, lacYl,
rpsL175, tonA21
CGSC #6161
LS853
trpA9605, his-85, cya-2, trpR55
CGSC #5381
M2508
Hfr, relAl, spoTl, metBl, melA7
CGSC #4926
M9s
MC4100, 4>{fdhF'-'lacZ+)
A. Bock (171)
MC4100
araD139, 5(argF-lacll)205, ptsF25,
relAl, rpsL150, deoCl, flb5301
CGSC #6152
MJ-2
[fhlB'-lacZ+), hydF102, cysalnlO
PI transduction
(SE-2011 x SE-67-1)
MJ-3
MC4100, (fhlB'-' lacZ+)
PI transduction
(MC4100 x SE-2011)
MJ-4
BW545, *{fhlB'-'lacZ+)
PI transduction
(BW545 x SE-2011)
MJ-5
Q[fhlB'-'lacZ+), rpoN::Tni0
PI transduction
(SE-2011 x YMC18)
MJ-6
[fhlB'-' lacZ+), fnr, zcj-5::JnlO
PI transduction
(SE-2011 x SE1188)
MJ-7
HfhlB'-'lacZ+), narL215::Tr)10
PI transduction
(MJ-4 x RK5278)


16
supercoiling activity in wild type and gyrB (DNA gyrase) mutants. It
was concluded that the respiratory state of the organism influenced DNA
supercoiling. Furthermore, this was mediated by DNA gyrase since,
changes in supercoiling paralleled the [ATP]/[ADP] ratios. This is
analogous to in vitro studies which demonstrated that the [ATP]/[ADP]
ratio influences gyrase-mediated DNA supercoiling (233). Therefore, the
energy status of the organism may be an environmental signal affecting
DNA supercoiling. In general, the state of chromosomal supercoiling is
presumed to be a mechanism of transcriptional control; however,
verification of this hypothesis has been difficult.
Nitrate
Anaerobically, nitrate supplementation induces the nitrate-formate
respiratory system and represses other respiratory and fermentative
pathways of lower redox potential. The narGHJI (nitrate reductase; ref.
36, 62, 137, 216) and fdhGHI (FDH-N; ref. 19) operons, both required for
nitrate respiration, are induced. Whereas, the frdABCD operon (fumarate
reductase; ref. 104, 115), the dmsABC operon (DMSO and TMAO reductase;
ref. 51, 104), the pfl gene (190), the fdhF gene and the hyc operon (25,
171) are repressed in the presence of the high redox potential acceptor,
nitrate.
Current evidence suggests that a two-component regulatory system
mediates transcriptional control of the narGHJI, fdhGHI, frdABCD, and
pfl operons. The NAR-X protein (and possibly NAR-Q) displays sequence
homology to membrane-bound histidine-protein-kinases (sensors) and NAR-L


41
"Eppendorf" micro-centrifuge for 5 min at 4C. Supernatant was
collected and kept on ice for immediate assay using a modified procedure
described by Shaw (200). The reaction mixture was freshly prepared by
dissolving 4 mg of 5,5' dithiobis-2-nitrobenzoic acid (DTNB) in 1.0 ml
Tris-HCl (pH 7.8), adding 0.2 ml of 5 mM acetyl-CoA, and then making the
total volume up to 10 ml. After measuring the rate of change of
absorbance at 412 nm with 900 ul of reaction mixture and 80 pi of
extract, the reaction was started with 20 pi of chloramphenicol (Cm; 5
mM in 70% ethanol) added to a final concentration of 0.1 mM. The
difference in the rate of change at A412nm with and without Cm was
calculated. Protein concentration of the extract was determined using
Coomassie brilliant blue (28). The CAT activity was expressed as
nanomoles of free 5-thio-2-nitrobenzoate produced per min per mg cell
protein.
Genetic and Molecular Biological Experiments
Bacteriophage (PI and lambda) preparation by plate lysis. Host
strain was grown to stationary phase in LB medium for PI infection.
Cells were sedimented by centrifugation (3,500 x g) at 25C and
resuspended in an equal volume of PI adsorption medium (5 mM CaCl2.2H20;
10 mM MgCl2.6H20). Bacteriophage PI (105 to 106 PFU) was added to 0.2
ml of host cells and incubated for 5 min at 25C. Then 3 ml of LCTG
soft agar (5g/L yeast extract; 10 g/L each of NaCl and trypticase
peptone; 2.5 mM CaCl2 2H20; 25 mg/ml thymine; 60 mM glucose; 0.6%


13
the identification and characterization of the Arc system and other
forms of respiratory control, current research forms a much more complex
picture of regulation (105, 106, 212).
Available evidence suggests that this aerobic-anaerobic switch may
involve several genes including arcA, arcB, fnr and pgi (involved in
DNA-supercoiling); mutants deficient in any one of these genes have
pleiotropic phenotypes. The Arc modulon is a two-component sensor-
regulator system (105, 106). Through DNA sequence analysis, ARC-B is
presumed to be a membrane-bound, histidine-protein kinase.
Autophosphorylation of ARC-B occurs in response to dioxygen-limitation
(possibly redox control). The signal is then transmitted to ARC-A by
phosphorylation to produce a transcriptional activator of the cytochrome
d (a high-affinity oxidase) gene (cyd). ARC-A is also a repressor of
"aerobic" enzyme synthesis (i.e. succinate dehydrogenase and cytochrome
o; ref. 68, 105). Cytochrome o, having a lower affinity for dioxygen,
is only synthesized aerobically; where as cytochrome d is stimulated
under microaerobic conditions as the terminal electron carrier (for
review, see 175).
The FNR protein, likewise, acts as both a transcriptional
activator and repressor (for review, see 211, 212). Mutations in the
fnr gene were originally isolated as strains deficient in nitrate
reductase and fumarate reductase (133). It is now known that several
other anaerobically inducible enzymes require FNR for transcription and
include nitrite reductase (both cyt552 and NADH-1inked; ref. 38, 45,


45
on ice and 0.1 ml was transferred to a tube for transformation. DNA
(approximately 50 ng) was added to the competent cells. This was
incubated on ice for 20 min, transferred to 37C for 5 min, and returned
to ice for 2 min. Transformants were preincubated in SOC medium (20 g/L
trypticase peptone; 5 g/L bacto-yeast extract; 10 mM NaCl; 2.5 mM KC1;
10 mM MgCl2.6H20; 10 mM MgS04.7H20; 20 mM glucose) with aeration at 37C
for 1 hr prior to plating on selection medium.
Transposon Tn5 mutagenesis of cloned genes in plasmid DNA. The
two transposon Tn5 derivatives of plasmid pSE-133 were constructed as
described before (188). Plasmid pSE-133 which carries the hydB+ and
fhlA+ genes was described previously (187). Strain MBM7014 (supF) was
utilized as the host strain for plasmid DNA (pSE-133) Tn5 mutagenesis.
A transformant was aerobically grown (250 rpm) in LBM medium (20 ml) to
mid-log phase (3 x 108 CFU/ml) at 37C, the culture was centrifuged
(3,500 x g; 25C), and the pellet was resuspended in 1.0 ml of 10 mM
MgS04.7H20. Cells were infected with NK421 (Tn5) at a M.0.1. of 10 for
30 min at 25C. The infected cells were then vortexed, centrifuged
(3,500 x g; 25C), washed with 5.0 ml of LB medium, and resuspended in
final volume of 10 ml. This was subcultured (2.0 ml inoculum) in 10 ml
fresh LBG medium supplemented with 10 mM sodium citrate and aerobically
grown (250 rpm) at 37C for 30 min and then shifted to 30C for 1 hr.
Kanamycin (50 pg/rol) and ampicillin (100 pg/ml) were added after the hr.
Then the culture was grown to stationary phase (18 hr), and plasmid DNA
was extracted by the alkaline lysis method described below (150).


86
Based on the sequence homology with these proteins, the FHL-A
protein can be divided into 4 regions which are interspersed with
segments of variable length. Region I is unique to FHL-A and includes
the first 353 amino acids. Similarly, NIF-A, FLB-D, LEV-R and TYR-R are
composed of a unique region at the amino-terminus. This singular region
has been thoroughly analyzed in the NIF-A protein which is synthesized
in an active form (21, 31, 63). In K. pneumoniae, the amino-terminus of
the NIF-A protein is presumed to be the site of direct interaction with
NIF-L (58). The NIFL/NIF-A complex is inactive in the presence of 02
and fixed nitrogen (93, 153).
The N-terminal domain of NTR-C and DCT-D, however, demonstrate
significant homology to other pleiotropic control proteins which have
been shown or are presumed to be phosphorylated by a sensor at an
exposed aspartate residue (91, 101, 120, 161, 162, 240). The 5.
typhimurium CHE-Y protein is included in this class of phosphorylated
regulators. The three-dimensional structure of purified CHE-Y protein
has been resolved to 0.27 nm. It was found to be composed of a central
hydrophobic core of five parallel p-sheets surrounded by five a-helices
(221). Comparison of amino acid sequence suggested that this N-terminal
secondary structure is conserved among the phosphorylated receivers.
Stock et al. (222) reported that the highly conserved aspartate and
lysine residues of these proteins are clustered at the carboxy-terminal
end of the p-sheets 1,3 and 5. Furthermore, these conserved residues
are the active site of the transcriptional regulator. Based on the


102
hyd {hyp) operon (59 min) is required for restoration of hydrogenase
activity in these fusion strains. Mutant strain SE-2009 was not
complemented for hydrogenase activity with any of the plasmids tested.
However, 250 pM NiC12 supplementation of the growth medium restored FHL
and HUP (H2 to BV) activities to wild type level. Phage Pl-transduction
experiments confirmed that the hyd mutations in strains SE2001 and SE-
2002 were located between the srl (58 min) and cys (59 min) genes.
However, the lac fusion in strain SE-2009 was not cotransducible within
this region. Mutant strain SE-2009 is phenotypically comparable to
strain HYD723, <\>{hydC'-' lacZ+), described by Wu et al. (239) which maps
at 77.6 min in the E. coli chromosome.
DNA hybridization experiments localized lac gene fusions in
strains SE-2001 and SE-2002 to a 0.9 kb Bgll-Sall fragment which
contains the partial hydE (hypB) coding region (360 amino acids) and
partial coding region of hypC {hydX) gene (20 amino acids) as
determined by Lutz et al. (144; Fig. 13). Neither mutant was suppressed
by nickel supplementation in the growth medium as in the case of point
mutations in hydE gene. This could be explained by the polar effect of
the fusions on the other hyd {hyp) genes whose products are also
essential for production of active hydrogenase.
Genetic regulation of the hyd {hyp) operon. Regulation of
expression of the hyd {hyp) operon was monitored by measuring the levels
of p-galactosidase activity produced by strains SE-2001 and SE-2002.
When cultured under strict aerobic conditions, both strains produced


121
11. Bachmann, B. J. 1990. Linkage map of Escherichia coli, Edition 8.
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K12. J. Bacterid. 163:454-459.
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14. Baker, K. P., and D. H. Boxer. 1991. Regulation of the chlA locus
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34. Castao, I., and F. Bastarrachea. 1984. glnF-lacZ fusions in
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66
chromosomal region (i.e. melAB at 93 min) to the episomal lac DNA
sequences used in the construction of the Hfr (PO-fhlB). Comparable
mapping complications were encountered in another study with strain SE-
1100, a (molR'-' lacZ+) fusion. The molR gene was originally mapped to
66 min using similar Hfr procedures. Phage Pl-mediated transduction
experiments later mapped the molR gene at 17 min in the E. coli
chromosome. Interestingly, a second ancestral gene for p-galactosidase,
the ebgA gene, which is homologous to the lacZ gene was found to map in
this region (68 min; ref. 8, 11, 32, 225, 226, 230).
Stability of the $(fhlB'-' lacZ^_) mutation. The pleiotropic nature
of the fhlB mutation led to the question of whether there was one (or
multiple) chromosomal mutation(s) leading to the observed phenotype in
strain SE-2011. Initially, the same biochemical characteristics as well
as operon expression (p-galactosidase activity) were observed when the
mutation from strain SE-2011 was transduced into various E. coli
strains, including strains MC4100, BW545 and CSH26. Even upon several
years of maintaining the mutant strains at -70C in 20% glycerol, the
original characteristics of strain SE-2011 remained stable. However, in
recent experiments, the majority of transductants which were selected
for Lac+ (Xgal+) and Kmr were no longer analogous to the fhlB mutation
previously described. Only about 1 to 5% of the transductants were
Fhl". Dihydrogen uptake activity of these strains was comparable to
wild type levels of activity. Many of these mutants were also altered
in fhlB operon expression. Basal level anaerobic expression, in the


118
appropriate DNA, in vivo transcription assays have been unsuccessful to
date (A. Bock, personal communication). This suggests that the FHL-A
t
protein alone is a DNA-binding protein (however, purification of FHL-A
as a formate complex cannot be ruled out at this time) but is not in an
active form for transcriptional activation of the FHL pathway. Possibly
another unidentified factor (formate and/or sensor protein) is required
in the reaction.
From the fhlB gene regulation data presented in this study it can
be deduced that formate is required for either (i) direct activation of
FHL-A, (ii) transcription of another unidentified regulatory protein
(sensor) which positively interacts with FHL-A, or (iii) direct
interaction with a protein (sensor) which associates with FHL-A for
transcription of FHL promoters. DNA sequence analysis of the nearby
hypCDE genes did not reveal a typical sensor protein (Fig. 9).
Additionally, transcription of the fdhF gene in the absence of active
FHL-B suggests that the fhlB gene is not this "unidentified sensor
protein." Instead the FHL-B could potentially function as a modulator
of the FHL pathway. Continued research will need to be done in this
area to narrow the possibilities (i.e. formate binding studies with FHL-
A protein, random mutagenesis of FHL-A protein to identify if FHL-A
derivatives are formate independent, identification of a potential
formate "sensor" protein).
The fhlB gene in molR or chlD genetic backgrounds still retained
formate inducibility in the presence of multiple copies of the fhlA+


126
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