Genetic organization of cellobiose transport system in Bacillus stearothermophilus

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Genetic organization of cellobiose transport system in Bacillus stearothermophilus
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
    Abstract
        Page viii
        Page ix
    Chapter 1. Introduction
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    Chapter 2. Review of literature
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    Chapter 3. Isolation of thermophilic cellulolytic Bacillus strains
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    Chapter 4. Cloning and sequencing of cellobiose-specific permease of phosphoenolpyruvate-dependent phosphotransferase system from Bacillus stearothermophilus
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    Chapter 5. Cloning and sequencing of the phosphoenolpyruvate-dependent phosphotransferase genes (Ptshit) from Bacillus stearothermophilus and hyperexpression in Escherichia coli
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    Chapter 6. Summary and conclusions
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    Literature cited
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    Biographical sketch
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Full Text











GENETIC ORGANIZATION OF CELLOBIOSE TRANSPORT SYSTEM
IN BACILLUS STEAROTHERMOPHILUS



















By

XIAOKUANG LAI


















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

1994














ACKNOWLEDGEMENTS


I would like to thank Dr. Lonnie O'Neal Ingram, my major

advisor, for his guidance, support, motivation, and patience,

for my completion of this dissertation. I am extremely

grateful to the members of my supervisory committee, Dr. James

A. Lindsay, Dr. Francis C. Davis, Dr. John E. Gander, and Dr.

Keelnatham T. Shanmugam, for their contributions to my

research and to the preparation and review of this manuscript.

I must also thank David Beall, Joy Doran, and Lorraine Yomano

for their friendship and help during the course of my

research.

I wish also to express my deep gratitude to Dr. Richard

Hartmann and Mrs. Remy Hartmann for their love and care.

Thanks are due to my family for their encouragement and

support. Finally, I would like to thank my loving wife,

Dongping, and beautiful daughter, Diana, for making my life

special.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . . . . . . . . . .

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

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

ABSTRACT . . . . . . . . . . .

CHAPTERS
1 INTRODUCTION . . . . . . . . .

2 REVIEW OF LITERATURE . . . . . . .
Thermophilic Microorganisms . . . . .
Thermnophilic Bacillus . . . . . . .
Bacterial Cellulase System . . . . .
Sugar Transport Systems in Bacteria . . .
Phosphoenolpyruvate-Dependent
Phosphotransferase System . . . . .

3 ISOLATION OF THERMOPHILIC CELLULOLYTIC
BACILLUS STRAINS . . . . . . . .
Materials and Methods . . . . . .
Results . . . . . . . . . .
Discussion . . . . . . . . .

4 CLONING AND SEQUENCING OF CELLOBIOSE-SPECIFIC
PERMEASE OF PHOSPHOENOLPYRUVATE-DEPENDENT
PHOSPHOTRANSFERASE SYSTEM FROM BACILLUS
STEAROTHERMOPHILUS . . . . . . .
Materials and Methods . . . . . .
Results . . . . . . . . . .
Discussion . . . . . . . . .

5 CLONING AND SEQUENCING OF THE
PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE
GENES (PTSHIT) FROM BACILLUS STEAROTHERMOPHILUS
AND HYPEREXPRESSION IN ESCHERICHIA COLI . .
Materials and Methods . . . . . .
Results . . . . . . . . .
Discussion . . . . . . .. .....

iii


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15
18
19

20


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42
44
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55
56
60
89




93
94
97
124










CHAPTERS Page

6 SUMMARY AND CONCLUSIONS . . . . . .. 128
LITERATURE CITED . . . . . . . . .. 135

BIOGRAPHICAL SKETCH . . . . . . . . .. 149














LIST OF TABLES


Table Page

1 Differential properties of thermophilc Bacillus 16

2 Sequenced El and HPr genes of PTS . . . .. 24

3 Differential properties of 55C and 65C
isolates . . . . . . . . . .. 45

4 Comparison of amino acid sequences in carboxyl
terminal portion of alanine racemases from
XL-65-6 and other organisms . . . . .. 47

5 Enzymatic activities expressed by isolates
capable of growing at 65C or higher . . .. 48

6 Enzymatic activities expressed by isolates
capable of growing at 55C or lower . . .. 50

7 Strains and plasmids used in this study ... . 57

8 Comparison of predicted amino acid sequences for
celA, B, and D and cel'R from
B. stearothermophilus to homologous PTS
polypeptides from other organisms . . . .. 70

9 Characteristics of the cel operon from
B. stearothermophilus XL-65-6 . . . . .. 74

10 Codon usage of cel operon . . . . . .. 78

11 E. coli strains and plasmids used in this study 95

12 Comparison of predicted amino acid sequences of
ptsH and ptsl from B. stearothermophilus to those
from other bacteria . . . . . . . i

13 Characteristics of ptsHIT opern . . . .. 119














LIST OF FIGURES


Figure Page

1 Organization of ptsH and ptsl in
representative organisms . . . . . .. 28

2 Schematic depicition of representative PTS
permeases and their energy-coupling proteins
found in enteric bacteria, B. subtills, and
R. capsulatus . . . . . . . . .. 32

3 Restriction map of the B. stearothermophilus
DNA fragment contained in pLOI902 . . . .. 61

4 Nucleotide sequence of DNA fragment from
B. stearothermophilus contained in pLOI902 . . 64

5 Comparison of deduced amino acid sequences of
cel operon genes from B. stearothermophilus and
E. coli . . . . . . . . . .. 72

6 Southern hybridization analysis of cel operon . 81

7 Hydropathy plots of PTS enzymes from different
organisms . . . . . . . . . .. 83

8 Comparision of helical wheel plots of terminal
regions from PTS enzymes . . . . . .. 86

9 Diagram illustrating the features of recombinant
plasmids containing B. stearothermophilus DNA . 99

10 Southern hybridization analysis of ptsHIT operon 102

11 Nucleotide sequence of B. stearothermophilus
DNA fragment in pLOI1801 . . . . . .. .104

12 Comparision of deduced amino acid sequences
of ptsH from B. stearothermophilus and other
Gram positive bacteria . . . . . . .. .112

13 Comparison of nucleotide sequence of ptsH from
B. stearothermophilus and from other Gram positive
bacteria . . . . . . . . . .. 116

vi










Figure Page

14 SDS-PAGE of protein extracts from JLT2
recombinants . . . . . . . . .. 120

15 Model for cellobiose transport and metabolism
in B. stearothermophilus . . . . . .. .132














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 ORGANIZATION OF CELLOBIOSE TRANSPORT SYSTEM
IN BACILLUS STEAROTHERMOPHILUS

By

Xiaokuang Lai

August 1994

Chairperson: Lonnie O'Neal Ingram
Major Department: Microbiology and Cell Science

A large collection of Gram positive, spore-forming,

thermophilic, cellulolytic bacilli were isolated from diverse

natural sources. These organisms were characterized and fell

into two groups. One group which grew at 55C or lower was

identified as Bacillus coagulans. The other group which grew

at over 65C was identified as Bacillus stearothermophilus.

Both groups of organisms represent a rich source of bacterial

genes encoding enzymes for the depolymerization of cellulose

to cellobiose and glucose and for the depolymerization of

other polysaccharides.

Little was previously known about the mechanisms of

cellobiose transport and metabolism in Gram positive bacteria.

This was extensively investigated in one of the B.

stearothermophilus isolates, strain XL-65-6. The cellobiose

transport system in this strain consisted of a multicomponents

viii








phosphoenolpyruvate-dependent phosphotransferase system (PTS).

The three genes encoding the cellobiose-specific permease and

the gene encoding a proposed phospho-B-glucosidase were cloned

and sequenced. These were found to be organized as an operon

which is present as a single copy on the B. stearothermophilus

chromosome. The celB product forms the major membrane-

spanning channel (EIICcet) for this transport system. Products

of celA and celD are cytoplasmic components of enzyme II

(EIIBc'1 and EIIACet) The ceIC gene encodes the glycohydrolase

which serves as a cleavage enzyme. It is likely that this

enzyme is phospho-B-D-glucopyranosidase. These genes were

functionally expressed in recombinant Escherichia coll.

Genes (ptsH and ptsl) encoding the PTS general proteins,

HPr and enzyme I, were also cloned and sequenced from strain

XL-65-6. These two genes together with a third, ptsT, were

also organized as an operon. This ptsHIT operon exists as a

single copy in the B. stearothermophilus chromosome. Deduced

amino acid sequences of B. stearothermophilus ptsHl and ptsl

are most similar to those from B. subtilis although they

exhibit a high degree of similarity with homologues from many

other bacteria. No gene similar to ptsT was found in the

current GenBank data base. The exact function of ptsT remains

undetermined.

Results from these studies provided the basis for a model

of cellobiose transport and metabolism in B.

stearothermophilus.














CHAPTER 1
INTRODUCTION




Thermophiles are a fascinating group of microorganisms

which have received considerable interest in recent years

because of their potential for biotechnological applications.

Research on thermophiles is expanding into many different

fields including biochemistry, enzymology, genetics, and

molecular biology. The biotechnological interest will further

stimulate basic research on this group of microorganism and

increase our understanding of thermophiles in the future

(Kristjansson and Stetter, 1992).

The genus Bacillus is one of the earliest bacteria to be

described. Species within this genus have been important as

models for studying biological problems, including

differentiation, secretion, DNA replication, DNA repair,

chemotaxis, genetic transformation, and translation apparatus

(Skepecky and Hemphill, 1992). They have also been utilized

in a wide range of industrial processes due to metabolic

diversity and low reported incidence of pathogenicity

(Harwood, 1989).

Interest in cellulose decomposition and in the

microorganisms that produce enzyme systems for cellulose








2

depolymerization have been stimulated by the perceived need to

exploit cellulose as a renewable source of energy and chemical

feedstocks, and to improve the efficiency of digestion of

fodder by ruminants (Coughlan and Mayer, 1992). Cellulose is

the most abundant carbohydrate in nature. It has been

estimated that 7 x 1011 metric tons of cellulose exist in

primary sources at any one time, and that approximately 4 x

1010 tons are replenished each year by photosynthesis

(Coughlan, 1985). Unlike fossil fuels, cellulose is clearly

a vast and renewable resource. In addition to primary

sources, cellulose also represents approximately 50% of the

dry weight of many secondary sources of biomass such as

agricultural, forest, industrial, and domestic waste. Huge

amounts of these materials are also generated each year

(Coughlan and Mayer, 1992).

Cellobiose is one of the primary soluble extracellular

products of microbial cellulose degradation. Despite the

abundance of cellobiose in the environment, little is known

about its entry into cells and its intracellular metabolism.

Many Gram positive and negative bacteria have been reported to

contain cell-associated 8-glucosidases (Coughlan and Mayer,

1992). Cellulomonas species have cellobiose phosphorylases

which are presumed to be involved in intracellular cellobiose

metabolism (Coughlan and Mayer, 1992). In E. coli, two

cryptic operons have been described at a molecular level which

provide the potential to transport cellobiose into the cell by








3

a phosphoenolpyruvate-dependent phosphotransferase system

(PTS) and to cleave cellobiose into monomer (Hall and Xu,

1992; Parker and Hall, 1990). Both operons have different

gene organizations but potentially encode a cellobiose-

specific enzyme II complex and a phospho-B-glucosidase. This

is the only organism for which cellobiose uptake has been

defined. But this organism typically does not have a

functional cellobiose transport system.

Different organisms often transport the same sugars by

alternative mechanisms. For example, E. coli and Bacillus

species use non-PTS to take up lactose into the cell while

Staphylococcus and Streptococcus species initiate the

metabolism of this sugar by the PTS (Saier and Yamada, 1987).

However, no information is available about the uptake of

cellobiose in any Gram positive bacteria.

PTS is a complex system which is used to transport a

variety of sugars in different bacteria. This system consists

of several interacting proteins, including cytoplasmic and

membrane proteins (Postma et al., 1993). In addition to its

primary function of carbohydrate translocation, the PTS also

has a variety of other important physiological functions in

bacterial cells. One such function is to regulate the order

of sugar utilization when mixtures of sugars are present. For

example, in media containing sugars transported by the PTS

(PTS-sugar) and sugars transported by other system (non-PTS

sugar), PTS-sugars are utilized prior to induction of the








4
catabolic systems for remaining sugars (diauxic growth). The

PTS can also function as a signal transduction system through

which bacteria respond to the presence of PTS-sugars by

positive chemotaxis. The PTS may also serve as a link between

carbon and nitrogen metabolism in Gram positive bacteria. One

domain of the B. subtills levR gene product, a protein

involved in the regulation of PTS levanase operon, is

homologous to a part of NifA and NtrC proteins, which are

activators of a54-dependent transcription (Ddbarbouill6 et al.,

1991). a54 is required for the transcription of a number of

genes important for glutamate production, especially under the

conditions of nitrogen limitation.

The primary aim of this dissertation is to investigate

the genetic organization of cellobiose uptake system in a

thermophilic species of Bacillus. The specific objectives

were as follows:

1. To isolate and identify thermophilic cellulolytic Bacillus

strains from nature.

2. To characterize cellobiose uptake genes at a molecular

level from one isolate.

3. To construct cellobiose-utilizing strains of E. coli by

introducing the Bacillus genes into E. coli.














CHAPTER 2
REVIEW OF LITERATURE


Thermophilic Microoranisms


Definition of Thermophilic Microorganisms

Temperature is one of the most important variables in our

environment. The classification of living organisms based on

their relation to temperature has therefore always been

considered as one of the most basic elements of biological

systematics. Microorganisms are frequently divided into three

broad classes on the basis of temperature ranges for growth,

that is psychrophiles, mesophiles, and thermophiles (Neidhardt

et al., 1990). Brock (1986) has suggested a definition of a

thermophile boundary at 55C to 60C based on two main

arguments: first, temperatures lower than 50C are widespread

on earth, whereas temperatures greater than 55C to 60C are

much rarer in nature. Second, 60C is the upper temperature

limit for eucaryotic life. However, bacteria that grow at

50C or above are traditionally called thermophiles

(Kristjansson and Stetter, 1992; Neidhardt et al., 1990).

The upper temperatures for different thermophilic

bacteria vary considerably. In general, nonphotosynthetic

organisms are able to grow at higher temperatures than

photosynthetic forms; structurally less complex organisms can










grow at higher temperatures than more complex organisms. The

upper growth temperature for cyanobacteria and other

phototrophic bacteria is 70C to 73C, for organotrophic

bacteria is over 90C, while archaebacteria can grow at over

100C. However, not all organisms from these groups are

thermophiles. Usually only a few species or genera are able

to function successfully near the upper temperature limits

(Brock and Madigan, 1991).

General Physiological Characteristics of Thermophiles

Thermophilic bacteria exhibit a wide range of

capabilities for energy and nutrition metabolism: phototrophy

and chemotrophy, autotrophy and heterotrophy, aerobiosis and

anaerobiosis. Examples of thermophiles capable of

phototrophic growth include 16 cyanobacteria species and some

purple and green bacteria (Brock and Madigan, 1991). Although

these organisms use light as an energy source, most

thermophiles are chemotrophs, using either inorganic or

organic chemicals as energy sources. Thermophiles that grow

autotrophically include many of the methanogens, Bacillus

schlegelii and Clostridium thermoautotrophicum. However, like

the majority of thermophilic bacteria, these organisms also

grow heterotrophically (Cato et al., 1986; Claus and Berkeley,

1986). Many thermophiles, including most of the thermophilic

members of the genera Bacillus, Clostridium, Thermomicrobium,

and Thermus only grow heterotrophically. Heterotrophic

thermophiles utilize a wide range of carbon sources for








7

growth, including carbohydrates, protein, lipids, organic

acids, alcohols, aromatic compounds as well as many other

compounds (Sundaram, 1986). Thermophilic Bacillus strains

have been isolated which are able to degrade methanol

(Dijkhuizen et al., 1988; Al-Awadhi et al., 1989), ethanol

(Al-Awadhi et al., 1989), phenol, and cresol (Buswell and

Twomey, 1975; Buswell, 1975). Examples of aerobic

thermophiles include the genus Thermus (Williams, 1992), and

thermophilic species in the genus Bacillus (Claus and

Berkeley, 1986). For these organisms, oxygen is involved in

the energy-generating step, serving as a terminal electron

acceptor. In contrast to this group, some thermophiles, such

as thermophilic species in the genus Clostridium, are

anaerobes. These organisms produce energy through anaerobic

fermentative pathways.

Adaptation of Thermophiles to Hiah Temperature

Considerable information has been obtained about the

ecology, physiology, metabolic capacities, and biochemical and

physical properties of cellular components of thermophiles.

It is clear that the ability to grow at high temperature

cannot be explained by a single mechanism but, rather, that

thermophiles appear to use a variety of mechanisms (Welker,

1993).

The content of guanine and cytosine (G+C) often

correlates with maximum growth temperature, although

Clostridium species are an exception (Welker, 1976). The DNA










of thermophiles showed a consistently higher G+C content than

that of DNA from mesophiles. Thermal melting profiles

demonstrated that DNA from thermophiles had higher melting

temperatures than DNA from mesophiles. The greater stability

to thermally-induced strand separation is attributable to the

more extensive hydrogen bonding that occurs with a higher G+C

content.

Stability of the cellular membrane is a determinant of

upper temperature limit for growth of organisms (Sundaram,

1986). The structure of cell membrane of thermophilic

bacteria, like those of other biological membranes, conforms

to the model of a lipid bilayer. However, the lipids in

thermophiles tend to have a greater abundance of fatty acids

with higher melting points than do the lipids of mesophiles.

A significant property of the phospholipid bilayer of the

bacterial membrane is that it can undergo thermotropic,

reversible transition between an ordered, rigid gel or solid

phase and a more fluid liquid-crystalline phase. The change

from gel to liquid-crystalline phase involves the cooperative

melting of the hydrocarbon chains in the interior of the

bilayer, and this phase transition behavior may set the

minimal and maximal temperatures for growth (Sundaram, 1986).

The stability of proteins also determine the maximal growth

temperature (Sundaram, 1986). A large number of proteins from

thermophiles have been isolated in pure form. Generally,

these proteins are more thermostable than their homologous








9

counterparts from mesophiles (Sundaram, 1986). Amelunxen and

Lins (1968) compared the thermostability of 11 enzymes from

the thermophile Bacillus stearothermophilus and the mesophile

Bacillus cereus. They found that nine of these enzymes from

B. stearothermophilus were more thermostable than the

corresponding enzymes from B. cereus. However, pyruvate

kinase and glutamate-oxaloacetate transaminase from both

bacteria exhibited similar rates of inactivation at 70C.

Thermophilic adaptation has been correlated with

preferential alterations of the amino acid composition in

terms of 'traffic rules' governing gross amino acid

compositions of mesophilic and thermophilic proteins. Mozhaev

and Martinek (1984) concluded that many thermophilic proteins

are deficient in polar amino acids, mostly serine and

threonine. Substitution of the inner serine and threonine for

nonpolar amino acid residues was proposed as a mechanism to

stabilize proteins to high temperature. DiRuggiero et al.

(1993) reported that thermostable glutamate dehydrogenase

(GDH) from hyperthermophilic Archaeon ES4 (optimal growth

temperature 98C and maximum growth temperature 110C) had a

relatively high hydrophobicity and a low number of sulfur-

containing residues compared with mesophilic GDHs. However,

after statistically comparing dihydrofolate reductases from 22

different organisms, BShm and Jaenicke (1994) concluded that

simple 'traffic rules' were insufficient to explain the








10

mechanisms of thermophilic adaptation or to predict the

stabilization or destabilization of proteins.

Indeed proteins from thermophiles are rather similar to

their mesophilic counterparts in most respects, including

size, subunit structure, gross higher order structural

parameters, and modulation of activity by metal ions and other

effectors (Amelunxen and Murdock, 1978). Attempts to transfer

heat stability and heat liability by mixing cell-free extracts

from thermophiles and mesophiles failed to change the original

properties of individual enzymes (Amelunxen and Lins, 1968;

Koffler and Gale, 1957). These results established that the

functioning of proteins at high growth temperatures of

thermophiles is not merely dependent on the presence of

stabilizing factors in the cellular environment of the

thermophile and that intrinsic structural stability of

proteins is the more important basis for thermophily

(Sundaram, 1986).

Amelunxen and Murdock (1978) have suggested that the

structural stabilization of thermophilic proteins is primarily

achieved through extra hydrogen bonds, ionic interactions and

apolar interactions in certain critical parts of the protein

molecules. After comparing the structures of triosephosphate

isomerase from thermophilic bacteria, Moraxella sp., and

mesophilic chicken, Rentier-Delrue et al. (1993) concluded

that the adaptability of this enzyme to high temperature








11
appeared to be favored by better stabilizing residues for the

helix dipole as well as better helix-forming residues.

Also, there is evidence that thermostability of a protein

can be altered by a single amino acid change. The

thermostability of E. coli ribonuclease HI was increased by

replacement of single amino acid residues (Ishikawa et al.

1993). Crystal structures of these mutant proteins revealed

that only changes in the local structure around each mutation

site are essential for the increase in thermostability. After

His65 was replaced by Pro, the mutant protein was stabilized

because of a decrease in the entropy of the unfolded state,

without a change in the native backbone structure. After

Lys95 was replaced by Gly, the left-handed backbone structure

in the typical 3:5 type loop was eliminated. The mutant

protein with a replacement of Lys95 by Asn was stabilized due

to formation of a hydrogen bond between the side-chain N6-atom

of the asparagine residue and the main-chain carboxyl oxygen

within the same residue.

Inherent structural stability alone may not be sufficient

to ensure the survival and functioning of proteins in

thermophilic cells. Thermophilic proteins display a rather

broad spectrum of stability in vitro, suggesting that they are

not all equally stable. Many enzymes from thermophiles are

stabilized in vitro by supplements, such as substrates, metal

ions, allosteric effectors, and electrolytes, all of which are

present in the growing bacterial cells (Sundaram, 1986).








12
Glutamine synthetase from Bacillus caldolytics was

demonstrated to be stabilized by the binding of metal ions and

substrate (Merkler et al., 1988). In vitro stability of

glyceraldehyde-3-phosphate dehydrogenase from Bacillus

coagulans was enhanced by increasing the ionic strength to 1.8

M with many neutral salts (McLinden et al., 1986). Calcium

ions confer stability to several extracellular enzymes, such

as a-amylase (Yutani, 1976), thermolysin (Tajima et al.,

1976), and proteases (Sidler and Zuber, 1977).

Insights into the molecular mechanisms of thermophily

have been hampered because little is known about the genetic

and molecular basis of thermophily. This gap is directly

related to lack of a genetic exchange systems for the

manipulation and genetic analysis of most thermophilic species

(Welker, 1993). Lindsay and Creaser (1975) reported that

after a mesophilic B. subtilis strain was transformed using

DNA isolated from a obligately thermophilic B. caldolyticus

strain, the resulting recombinant of B. subtilis strain was

able to grow above 70C. These investigators suggested that

thermophily was controlled by a small number of genes acting

at the ribosomal or tRNA level affecting translation resulting

in expression of more thermostable enzyme variants. An a-

glucosidase purified from the high-temperature-growth

transformant had been confirmed to have changes in the amino

acid composition. Although this enzyme had much higher

optimum growth temperature than its counterpart in its








13

mesophilic parent, many physiological characteristics remained

unchanged (Krohn and Lindsay, 1991). Similar results have

been reported by Droffner and Yamamoto (1985). Most recently,

St&hl (1991) isolated 3 plasmids from thermophilic B.

stearothermophilus, which could be involved in thermophily

since strains cured of these plasmids were no longer obligate

thermophiles. Furthermore, when these plasmids were

transformed into a mesophilic B. subtilis strain, the

resulting transformants shifted their optimum growth

temperature about 10C higher, and about 6C higher for the

maximum growth temperature. Based on these experiments, StAhl

concluded that Lindsay and Creaser's results (1975) might be

due to the presence in the donor strain of plasmids involved

in thermophily.

Evolution of Thermophilic Microorganisms

Thermophilic bacteria are distributed among all main

groups in bacterial genetic tree. However, it is interesting

to note that they are usually the oldest types in their

respective groups (for example Thermotoga, Thermomicrobium,

Chloroflexus, and Thermous), and even the oldest of the

bacterial kingdom. These results suggest that thermnophily

arose very early in the evolution of bacteria. Indeed, the

ancestral bacterium might have been a thermophile

(Kristjansson and Stetter, 1992).

AppDlications of Thermoohilic Microorganisms

Thermnophiles offer some major advantages for








14

biotechnology. The most attractive attribute of thermophiles,

from a biotechnological point of view, is that they produce

enzymes capable of catalyzing biochemical reactions at

temperatures markedly higher than those of conventional

organisms. The main advantages of increased temperature are

generally higher reaction rates, higher solubility of most

chemicals, and increased fluidity and diffusion rates. High

temperature also prevents contamination from mesophilic

microorganisms. In addition, many enzymes from thermophiles

are more stable at conventional temperatures, thus prolonging

the shelf life of commercial products (Brock, 1986). Interest

in thermostable enzymes from thermophiles has grown. The

variety of thermostable enzymes used in industrial application

has been steadily increasing, mainly as replacements for

thermolabile enzymes in existing processes. For example,

thermostable amylase from B. licheniformis and B.

stearothermophilus have replaced thermolabile amylase from B.

subtilis (Zamost et al., 1991). The main applications for

thermostable enzymes have been starch liquification using

amylases and proteases for food processing and detergents

(Zamost et al., 1991). Other thermostable enzymes with a wide

range of characteristics remain to be exploited and applied.

The application of thermophiles is not limited to industrial

enzyme processes. There exist many other potential

applications, such as fermentation, waste treatment, and

microbial leaching (Brock, 1986).










Thermophilic Bacillus

In Bergey's Manual of Systematic Bacteriology (Claus and

Berkeley, 1986), the thermophilic, aerobic or facultative

anaerobic, spore-forming, Gram positive rods are placed in the

species B. acidocaldarius, B. schlegelii, B.

stearothermophilus, B. coagulans. and B. licheniformis. The

first three species are able to grow at 65C or higher, and

the later two are able to grow at 55C. The differential

properties of these organisms are listed in Table 1.

B. acidocaldarius was first isolated by Darland and Brock

(1971) from hot springs in Yellowstone and from fumaroles in

Hawaii. This organism appears to be restricted to thermal

springs and the surrounding soils (Priest, 1989). It has a

temperature range between 45C and 70C and a pH range between

pH 2 and pH 6 (Darland and Brock, 1971). One of the unique

features of B. acidocaldarius is its fatty acid profile. Like

other Bacillus spp., it possesses MK-7 as a major menaquinone

component, but in addition it also possesses the unique fatty

acid w-cyclohexane (Minnikin and Goodfellow, 1981).

B. schlegelii is a thermophilic hydrogen-oxidizing

organism. It was isolated by Aragno (1978) from the

superficial layer of the sediment of a small eutrophic lake in

Switzerland and was described by Schenk and Aragno (1979). It

is an obligate aerobe. The Gram reaction is variable, but

cell wall structure of this organism is typical of a Gram

positive bacteria (Aragno, 1992). The optimal temperature for










Table 1. Differential Properties of Thermophilic Bacillus



Properties' Bco Bli Bac Bsc Bst

Growth at
30C + + -
55C + + + + +
650C + + +

Growth in
5% NaCl + ND d
7% NaCl + ND -

Acid from
D-Glucose + + + +

Hydrolysis of
Casein d + ND d
Gelatin + ND +
Starch + + + +

Growth at
pH 6.8 + + + +

Autotrophic with
H2 + CO2 or CO + -


Source: Claus and Berkeley, 1986

Note: Bco: Bacillus coagulans
Bli: Bacillus licheniformis
Bac: Bacillus acidocaldarius
Bsc: Bacillus schlegelii
Bst: Bacillus stearothermophilus
S+: 90% or more strains are positive
-: 90% or more strains are negative
d: 11-89% of strains are positive
ND: no data available








17

growth is about 70C, with no growth at 37C or 80C. The

optimal pH for growth is between pH 6 to pH 7 (Schenk and

Aragno, 1979). B. schlegelii is facultatively

chemolithotrophic using either H2 as the electron donor and CO2

as the carbon source or CO to serve both requirements (Claus

and Berkeley, 1986).

B. stearothermophilus is a highly heterogeneous species.

Wolf and Sharp (1981) subdivided this species into three

groups, while Bartolomeo et al. (1991) clustered this species

into four groups on the basis of morphological, physiological,

and biochemical characteristics as well as the DNA base

compositions of 133 thermophilic Bacillus strains.

B. coagulans is an important food spoilage agent involved

in the coagulation of canned milk and flat souring of

carbohydrate containing canned foods through the production of

high concentration of L-(+)-lactic acid (Frazier, 1958). It

was originally isolated from spoiled canned milk by Hammer in

1915. This organism is considered to be a facultative

thermophile, growing well at 45C to 55C with some strains

growing up to 65C (Wolf and Sharp, 1981). Early studies

indicated considerable heterogeneity among members of this

species with variations in morphology of both cells and spores

(Smith et al., 1952; Sei et al., 1978). Wolf and Barker

(1968) divided this species into two distinct physiological

types. On the basis of a more recent study by Bartolomeo et








18

al. (1991), this species was further divided into three

groups.

Although some investigators have proposed species names

for certain thermophilic Bacillus strains, These strains have

not been recognized as species in Bergey's Manual (Claus and

Berkeley, 1986).

Bacterial Cellulase System

A complete cellulase system is defined as that which can

catalyze extensive hydrolysis of crystalline cellulose

(Coughlan and Mayer, 1992). Such systems require the

concerted action of several different types of enzymes, among

which mainly include: (a) endoglucanases (EC 3.2.1.4, CMCase),

(b) cellobiohydrolases (EC 3.2.1.91, exoglucanase), and (c) B-

glucosidases (EC 3.2.1.21). The endoglucanase cleaves B-

glucosidic bonds randomly in the internal region of the

cellulose molecules. Typically, these enzymes are inactive

against crystalline cellulose although they may yield a small

amount of reducing sugar by hydrolysis of the amorphous

regions of crystalline substrates. This group of enzymes,

generally, is active against acid-swollen amorphous cellulose,

soluble derivatives of cellulose such as carboxyl methyl

cellulose (CMC), and cellooligosaccharides.

Cellobiohydrolases attack cellulose molecules stepwise from

the nonreducing ends, liberating soluble cellobiose units.

These enzymes can act, albeit slowly, against crystalline

substrates such as Avicel. They are also active against








19

amorphous celluloses and cellooligosaccharides, but are

inactive against cellobiose or substituted soluble celluloses

such as CMC. The B-glucosidases hydrolyze cellobiose to

glucose and catalyze the removal of glucose from the low

molecular weight cellooligosaccharides, but they are inactive

against crystalline or amorphous cellulose (Coughlan and

Mayer, 1992; Wood and Bhat, 1988). In addition to the above

three major types of cellulases, many other types of enzymes,

such as phospho-B-glucosidase, cellobiose phosphorylase,

cellodextrin phosphorylase, may also play roles in various

organisms (Coughlan and Mayer, 1992; El Hassouni et al.,

1992).

At least 35 bacterial genera have been reported to

contain cellulolytic species, many of which can grow on

cellulose and produce enzymes that catalyze the degradation of

soluble derivatives of cellulose or the amorphous regions of

otherwise crystalline cellulose. However, very few bacteria

synthesize the complete enzyme system needed for the extensive

hydrolysis of the crystalline cellulose in nature (Coughan and

Mayer, 1992).

Sugar Transnort Systems in Bacteria

Bacteria employ various transport systems for uptake of

different sugars. In E. coli, almost all of the transport

mechanisms (including facilitated diffusion, shock-sensitive

active transport, and secondary active transport) participate

in the uptake of one or more sugars or sugar alcohols








20

(Neidhardt et al., 1990). Glycerol enters E. coli by

facilitated diffusion with the aid of a glycerol-specific

transmembrane protein. This mechanism does not concentrate

the substrate within the cytoplasm. Maltose enters E. coll by

a shock-sensitive system. This system is made up of four

distinct proteins, a binding protein, two transmembrane

proteins, and an energy-transducing protein. When maltose

enters the periplasm, it binds tightly to the binding protein;

the binding protein then passes maltose to sites on the

transmembrane proteins; the energy-transducing protein

hydrolyze a high-energy phosphate bond of ATP, the energy of

which changes the conformation of transmembrane proteins such

that the bound maltose is released on the membrane's inner

surface. Lactose enters E. coli by a proton symport and

melibiose by a sodium symport. Symport is the transport of

two substrates simultaneously in the same direction by a

single carrier: if one of these substrates flows down its

concentration gradient, the other flows with it. Glucose

enters E. coli by a phosphoenolpyruvate-dependent

phosphotransferase system (PTS), which will be described in

more detail in next section. Interestingly, the uptake of a

particular sugar does not follow a pattern: for example

lactose is taken into E. coli by proton symport but into

Staphylococcus aureus by a PTS.

Phosphoenolpvruvate-Devendent Phosphotransferase System

Bacteria take up many sugars into the cell by a complex








21

system, phosphoenolpyruvate-dependent phosphotransferase

system (PTS). This system consists of several interacting

proteins and has a variety of physiological functions in

bacterial cells in addition to sugar transport (Postma et al.,

1993).

The PTS was discovered in cell extracts of E. coli by

Kundig et al. (1964) as a system which catalyzes the

phosphorylation of a number of carbohydrates with PEP as the

phosphoryl donor. This system consists of two general energy-

coupling proteins, enzyme I (El) and HPr, as well as a sugar-

specific permease, commonly referred as the enzyme II complex

(EII) (Saier and Reizer, 1992). The general reaction scheme

of the system is illustrated as following (Postma et al.,

1993; Saier and Reizer, 1992):

P-enolpyruvate + El P-EI + pyruvate (1)

P-EI + HPr P-HPr + El (2)

P-HPr + EIIA(domain or protein) P-EIIA + HPr (3)

P-EIIA + EIIB (domain or protein) P-EIIB + EIIA (4)

P-EIIB + EIIC (domain or protein) P-EIIB + EIIA (5)

P-EIIC + carbohydrate(.) -. EIIC + carbohydrate-P(i,) (6)


In most cases, El and HPr are soluble, cytoplasmic

proteins that participate in the phosphorylation of all PTS

carbohydrates in a given organism, and thus are referred to as

general PTS proteins (Postma et al., 1993).










Enzyme I

El has been purified from a variety of organisms (Postma

et al., 1993). The monomer molecular weight ranges from about

64,000 in E. coli and S. typhimurium to about 85,000 in

Staphylococcus aureus. However, evidence suggests that

autophosphorylation of El (reaction I) requires a dimeric form

of the protein (Kukuruzinska et al., 1982; Misset et al.,

1980; Weigel et al., 1982). The Els from most organisms

consist of identical monomers that self-associate to form

dimers. The exception is the El from Mycoplasma capricolum,

which is a tetramer composed of three different subunits

(Meadow et al., 1990). Phosphorylation occurs at the N-3

position of a histidyl residue of El during

autophosphorylation with PEP (Alpert et al., 1985; Weigel et

al., 1982). Studies on S. typhimurium El shows that this

enzyme requires Mg2*, Mn2*, or other divalent cations for its

autophosphorylation by PEP but not for the transfer of the

phosphoryl group to HPr (Weigel et al., 1982).

The kinetics of the reaction of El have been well studied

and the data have extensively been reviewed (Postma et al.,

1993; Meadow et al., 1990). According to the studies on Els

from enteric bacteria, El associates at room temperature to

form an active dimer of identical subunits, which is

phosphorylated on each subunit in the presence of Mg2+ and PEP.

The P-EI then dissociates, especially at lower temperatures,

to the monomers which then phosphorylate HPr. Reassociation










of dephosphorylated subunits is apparently the rate-limiting

step for this whole cycle. This enzyme exhibits a ping-pong

Bi Bi mechanism and catalyzes the isotope exchange reactions

expected for an enzyme that forms a covalent intermediate.

The apparent K. values range from 0.2 to 0.6 mM for PEP and 4

to 30 AM for HPr.

The gene encoding El (ptsI) has been cloned and sequenced

from 3 Gram negative organisms, E. coli (Saffen et al., 1987),

S. typhimurium (LiCalsi et al., 1991), and Alcaligenes

eutrophus (Pries et al., 1991), and from 4 Gram positive

organisms, Bacillus subtilis (Reizer et al., 1993),

Staphylococcus carnosus (Kohlbrecher et al., 1992),

Streptococcus mutans (Boyd et al., 1994), and Streptococcus

salivarius (Gagnon et al., 1992) (Table 2). In all cases,

there is a high degree of sequence similarity around and

including the phosphorylated histidine residue (Postma et al.,

1993). The deduced amino acid sequences of Els from E. coli

and S. typhimurium differ in only 16 residues, consistent with

the fact that these proteins are functionally interchangeable

in these organisms (Postma et al., 1993). However, these Els

and those from Gram positive bacteria often substitute poorly

for one another in PEP-dependent phosphorylation of

heterologous HPr (Postma et al., 1993). Rhodobacter

capsulatus produces a multiphosphoryl transfer protein which

contains the functional and structural equivalents of EIIA,

HPr, and El in a single polypeptide chain (Wu et al., 1990).







Table 2. Sequenced El and HPr genes of the PTS


Proteins No. amino acids M, References


RPr
Bacillus subtilis
Enterococcus faecalis
Staphylococcus carnosus
Staphylococcus aureus
Streptococcus mutans
Streptococcus salivarius

Alcaligenes eutrophus
Escherichia coli
Klebsiella pneumoniae
Mycoplasma capricolum
Salmonella typhimurium

Psuedo-HPr
E. coll
S. typhimurium

Enzyme I
B. subtilis
S. carnosus
S. mutans
S. salivarius

A. eutrophus
E. coli
S. typhimurium

Multiphoaphoryl Transfer Protein
Rhodobacter capsulatus


9,121
9,449
9,511
9,367
8,936
8,901

9,476
9,109
9,119
9,418
9,109


Gonzy-Treboul et al., 1989
Deutscher et al., 1986
Eisermnann et al., 1991
Reizer et al., 1988
Boyd et al., 1994
Gagnon et al., 1993

Pries et al., 1991
Saffen et al., 1987
Titgemeyer et al, 1990
Zhu et al., 1993
Byrne et al., 1988


39,647 Richterich et al., 1994
39,593 Geerse et al., 1989


63,076
63,369
63,354
62,948


Reizer et al., 1993
Kohlbrecher et al., 1992
Boyd et al., 1994
Gagnon et al., 1992


65,207 Pries et al., 1991
63,489 Saffen et al., 1987
63,368 LiCalsi et al., 1991


86,393 Wu et al., 1990


827








25

In addition, two other enzymes, pyruvate phosphate dikinase

(Pocalyko et al., 1990; Wu et al., 1990) and PEP synthase

(Niersbach et al., 1992), were found to have regions of

sequence which are similar to El. These included the region

containing the phosphorylated histidine residue.

Histidine-Containinq Protein (HPrl

HPr, the second general protein involved in the

phosphoryl transfer reaction sequence, is a small monomeric

protein with a molecular weight of 9,000 to 10,000. Its small

size and heat stability have aided its purification from a

variety of organisms (Postma et al., 1993). The phosphoryl

group from P-EI is transferred to the N-l position of a

histidine residue (His15) in HPr. All HPrs so far studied from

Gram positive bacteria can also be phosphorylated by an ATP-

dependent HPr kinase at a serine residue (Ser6) as part of an

important regulatory modification (Meadow et al., 1990; Reizer

et al., 1993). This regulatory phosphorylation also occurs in

HPr from M. capricolum, an unusual bacterium (Zhu et al.,

1993). The region around Ser46 is conserved in all sequenced

HPrs of Gram positive bacteria. The Ser"-phosphorylated HPr

is not able to transfer its phosphate group to a sugar, and

the PEP-dependent phosphorylation at His15 of a Ser46-

phosphorylated HPr is 600-fold slower than the phosphorylation

of a non-phosphorylated HPr (Reizer et al., 1993). In B.

subtilis, this regulatory phosphorylation is involved in

catabolite repression (Deutscher et al., 1994; Reizer et al.,










1993). Ser6 is also present in HPrs from Gram negative

bacteria but is not phosphorylated, and the corresponding HPr

kinase is absent in these organisms. The region surrounding

Ser" in Gram negative organisms shows little sequence identity

with those from Gram positive organisms (Meadow et al., 1990;

Reizer et al., 1993).

The three-dimensional structures of HPr from both Gram

negative and Gram positive bacteria have been investigated by

several different methods: two-dimensional nuclear magnetic

resonance (NMR) and three-dimensional NMR of HPr in solution

and X-ray crystallography (Postma et al., 1993). The

structure of the E. coli HPr determined by NMR studies

consists of four B-strands that form a single, pleated

antiparallel B-sheet on one face of the protein and three a-

helices that lie in a plane parallel to the B-sheet.

According to the X-ray structure of the E. coli HPr, however,

the four B-strands exist in two pairs removed from each other

and a-helices lie in different planes and orientations,

whereas the region containing the phosphorylated His residue

is the same in both studies (Postma et al., 1993). The B.

subtilis HPr has also been investigated by X-ray

crystallography and NMR. The overall topology of three

dimensional structure of B. subtilis HPr derived from NMR and

X-ray crystallography is very similar. The secondary-

structure folding topology of this protein is the classical

topology which has been described as an open-face B-sandwich.








27

It is formed by four antiparallel B-strands with three apposed

a-helices packed on top of the B-strands. Both His15 and Ser46

are located on the surface of the protein with their side

chains capping the N-termini of the first and second a-

helices, respectively (Chen et al., 1993).

The primary amino acid sequences of HPr from 11 bacteria

have been determined (Table 2). Those from E. coli and S.

typhimurium are identical (Byrne et al., 1988), and only one

conservative exchange from K. pneumoniae (Titgemeyer et al.,

1990). All HPrs share a striking similarity around the active

site His residue, and all have this active site His residue at

position 15. All HPrs also have an Arg residue at position 17

(Meadow et al., 1990). HPrs from Gram positive bacteria,

although very similar to each other, do differ considerably in

amino acid sequences from the Gram negative bacterial HPrs

outside of the region containing the phosphorylated His

residue (Postma et al., 1993).

Genetic Organization of ptsH and VtsI

The arrangements of ptsH and ptsI on genome are the same

in most of bacteria with the exception of M. capricolum (Fig

1). In most bacterial species characterized thus far, these

two genes are always adjacent to each other and form an operon

with ptsH on the 5' end. The M. capricolum ptsH gene,

however, is part of a monocistronic operon that is situated

between two ORFs unrelated to the function of PTS (Zhu et al.,

1993). The genes on two flanking regions of ptsHI operon are






















Figure i. Organization of ptsH and ptsI in representative organisms







ps3G b pHI psl



cysK 3W8bp IpisH I| I Crr


S.t yph&uum cysK 383bp p&sHI


S. canesus





M.capric&um


ptsl Ierr


270b1 11,00I


B. subilis


E. co


-rDI piH oIfl


270 bp I sH 1


*looa ^n








30
vary in different organisms. In some organisms, these regions

contain PTS related genes, and in other organisms, genes

unrelated to PTS functions are present in these regions. For

example, the upstream region of B. subtillis ptsH contains the

gene encoding glucose permease (ptsG) (Zagorec and Postma,

1992), but in E. coil and S. typhimurium, this region encodes

O-acetylserine (thiol)-lyase A (cysK), an enzyme involved in

the synthesis of L-cysteine from O-acetyl-L-serine and sulfide

(Byrne et al., 1988).

Enzyme II Complexes

The sugar-specific PTS permeases (EII) are the most

diverse PTS proteins (Meadow et al., 1990). They may consist

of one, two, three, or four distinct polypeptide chains (Saier

and Reizer, 1992). EII proteins found within a single species

often show little sequence similarity (Meadow et al., 1990).

However, regardless of the number of polypeptide chains and

the orders of these chains, most Ells share several conserved

features. First, they generally have similar molecular

weights of about 68,000, corresponding to about 635 total

amino acyl residues (Saier et al., 1988). Second, there are

always at least three well-recognized functional domains: a

hydrophobic transmembrane domain which binds and transports

the sugar substrate (EIIC), a hydrophilic enzyme III-like

domain which possesses the first phosphorylation site (always

a histidyl residue) (EIIA), and a second hydrophilic protein

or protein domain which possesses the second phosphorylation










site (either a cystidyl residue or a histidyl residue) (EIIB)

(Fig. 2) (Saier and Reizer, 1992). The activity of EII may be

controlled by HPr Ser46 phosphorylation via the ATP-dependent

kinase in Gram positive bacteria (Ye et al., 1994b).

Regulation of PTS Protein Synthesis

The synthesis of PTS proteins is highly regulated. In

enteric bacteria, expression of the ptsH, ptsl and crr

increases about threefold during growth on PTS sugars,

especially glucose, when compared to growth on lactate (Mattoo

and Waygood, 1983; Stock et al., 1982). Anaerobic growth

conditions also favor higher expression (Lengeler and

Steinberger, 1978). Transcription of the ptsHI operon is also

enhanced by unphosphorylated EIICBGIC (De Reuse and Danchin,

1988; De Reuse and Danchin, 1991). These three genes (ptsH,

ptsl, crr) constitute an operon, but three major mRNA species

have been found, composed of those including entire three

genes, ptsH only, and crr only, respectively (De Reuse and

Danchin, 1988). In B. subtilis, the expression of sucrose-

specific EII of PTS is induced by sucrose (Fouet et al.,

1989).

The expression of ptsHI operon of E. coil is under very

complex regulation. It is controlled by two contradictory

regulatory mechanisms, the glucose-mediated and the CAP-cAMP-

mediated activations (De Reuse et al., 1992; Ryu and Garges,

1994) which may involve three, possibly five, promoters

(Postma et al., 1993; Ryu and Garges, 1994). Three of these































Figure 2. Schematic depiction of representative PTS
permeases and their energy-coupling proteins around in
enteric bacteria (A to D and F), B. subtilis (E), and R.
capsulatus (G) (Saier and Reizer, 1993).









33








Out / In
(A) Mannitol Mannitol-1 P

B IA
tic
P P
P GIUcos-6.P
(B) Glucose





S ID
Celoblog| CobioM.6-P

ic) i a PEP

P P
Clic""'/. /
11mn 10IIB A
(D) Mannoa, 1 awm-fl-P
I P P
ic, IIB ~-

(E) Fructose I10 Fm
| | ~FructOsa-6-P f
\ructo \IDTPI
r110^FM ^ -IIB 11W -- -


(F) Frluctos Fructos..-1 -P
\ \ IMTPI



(G) p--ma --naP








34

promoters (P0, P1, and Px) are located upstream from ptsH,

separated by about 100 bp. A fourth (P2), the major promoter

for crr, is located within the 3'-terminal end of ptsl. A

fifth transcription start site, possibly involved in

antitermination, is located downstream from ptsH and is

directed against PO and PI. In the PO-Px-Pl promoter region,

there are two sequences showing similarity with the consensus

of CAP-binding sites, one of which, CAPa, is located near P0,

and the other, CAPb, is located in the -35 region of P0. P0

is the major promoter in the presence of the CAP-cAMP complex

and P1 is the major promoter in its absence. In the absence

of CAP, P0 is occluded. In the presence of CAP, binding of

CAP to the P0 site is increased by PI. P2 is not activated by

the CAP-cAMP complex. Most recently, Ryu and Garges (1994)

showed that P0 and P1 exhibit a switching mechanism in vitro,

where, depending upon the presence or absence of CAP-cAMP,

transcription is initiated from different start sites (POa and

POb, Pla and Plb). In vivo POa is the major start site for

P0, and POb, a site 3 bases upstream from POa, is never

detected, although POb is active in vitro when linear DNA is

used and CAP-cAMP is present. There is no P0 expression when

CAP or cAMP are absent in the cell unless glucose is present.

In vivo, most transcription from P1 is from Pla unless glucose

is present. Glucose causes a switch to Plb, a site 7 bases

downstream from Pla. But in both events, the overall

expression of Pla and Plb do not change much, meaning that








35
there is very little CAP-cAMP activation of this promoter in

vivo. Apparently, some barrier to Pib initiation can be

relieved by the addition of glucose. Px is recognized by a32

RNA polymerase, which is absent unless CAP-cAMP or glucose are

present. All of these results indicate that the two different

regulatory mechanisms (one through CAP-cAMP, the other through

glucose) are working together for fine control of ptsHI

operon.

Transcription antitermination is another way to regulate

the expression of PTS proteins. One such example is the

expression of bgl operon in decryptified E. coli strains. A

model on this antitermination mechanism has been proposed

(Postma et al., 1993). This operon is preceded by a

constitutive promoter and consists of three genes, bgIG, bglF,

and bglB. These genes encode antiterminator, EIIB9', and

phospho-B-glucosidase, respectively. The first gene, bgIG, is

flanked on both sides by two terminators tl and t2. The

transcription of the operon is terminated by tl and t2 in the

absence of inducer, B-glucosides. The antiterminator, BglG,

can be phosphorylated in a reversible reaction by P-EII1gL.

The phosphorylated BglG is inactive. In the presence of B-

glucosides, the uptake of B-glucosides causes the phosphoryl

group on P-EII691 to preferentially transfer to the substrate.

This drain causes the dephosphorylation of P-BglG and

subsequently become an active antiterminator. The active BglG

binds to specific RNA sequences located upstream of tl and t2,








36
prevents termination, and makes the transcription of bgl

operon possible. In B. subtilis, expression of sacPA operon

which encodes a EIIBCscr (sacP) and a sucrase (sacA) is also

controlled by a proposed antiterminator, SacT. This

antiterminator is homologous to the E. coll BglG, but requires

El and HPr for activity (Arnaud et al., 1992).

Many other mechanisms exist to regulate the expression of

PTS proteins which include regulation through classical

regulator-operator pairs, such as the expression of nag genes

from E. coli and K. pneumoniae (Postma et al., 1993).

Metabolic Regulation of Non-PTS Sugars by PTS

In addition to the primary function in sugar transport,

the PTS has an important role in the regulation of metabolism

of non-PTS sugars. It has been observed in enteric bacteria

that mutants defective in El and HPr are not only unable to

utilize PTS carbohydrates as the sole source of carbon for

growth, but also unable to grow on a number of non-PTS sugars,

including lactose, maltose, melibiose, glycerol, rhamnose,

xylose, and Krebs cycle intermediates. This defect can be

relieved by the addition of cAMP or by the introduction of a

crr mutation (Postma et al., 1993). A model for this type of

PTS-mediated regulation in enteric bacteria has been proposed

(Postma et al., 1993). The central regulatory molecule is

EIIAGIC, the product of crr. EIIAic can exist in two states:

phosphorylated EIIAGIC (P-EIIAGt') and nonphosphorylated EIIAGIC.

The phosphorylation state of EIIAGC is determined by the








37

balance between phosphorylation via P-HPr and

dephosphorylation via EIICBGIC in the presence of its

substrate. EIIGtC and P-EIIO'c interact with and regulate

different proteins. EIIGLc binds to and inhibits several

enzymes essential in carbohydrate metabolism, for example, the

lactose and melibiose carriers. But the binding of EIIGtC

occurs only when a substrate of the target protein is present.

The direct result of binding is inhibition of uptake and of

subsequent metabolism of these carbon sources. P-EIIAGtc is an

activator of adenylate cyclase, the enzyme for the synthesis

of cAMP. cAMP plays a central role in gene expression in

enteric bacteria. Together with CRP, cAMP is involved in the

(generally) positive regulation of a number of catabolic

genes. The inhibition of transport and metabolism of non-PTS

carbohydrates can be brought about by any PTS carbohydrate

since all PTS carbohydrates can dephosphorylate P-EIIAGtc,

either directly via EIICBGiC (if the PTS carbohydrate is

glucose) or indirectly (transport and phosphorylation of all

other PTS sugars via their respective EII complexes result in

dephosphorylation of P-HPr). Since the phosphorylation of

EIIAGc by P-HPr is a reversible process, the dephosphorylation

of P-HPr by other EII complexes will dephosphorylate P-EIIAGLc.

Recently, van der Vlag et al. (1994) demonstrated a

quantitative relationship between the amount of EIIAG'c and the

regulation of glycerol and maltose metabolism in S.

typhimurium. The inhibition of glycerol and maltose uptake by









the addition of a PTS sugar was sigmoidally related to the

amount of EIIAGiC. For complete inhibition of glycerol uptake,

a minimal ratio of about 3.6 mol of EIIAGtc to 1 mol of

glycerol kinase was required while a ratio of about 18 mol of

EIIAGiC to 1 mol of MalK protein (part of maltose transport

system and the target of EIIAGtc) was required for complete

inhibition of maltose uptake. Varying the level of EIIAGtc did

not affect the growth rate on glycerol, the rate of glycerol

uptake, or the synthesis of glycerol kinase. In contrast,

the growth rate on maltose, the rate of maltose uptake, and

the synthesis of maltose-binding protein increased two- to

fivefold with increasing levels of EIIAGIC. The synthesis of

MalK protein was not affected by varying the levels of EIIAGtc.

Many Gram positive organisms also show a distinct

"glucose effect" (Meadow et al., 1990). Several mechanisms

exist whereby expression of catabolic genes can be regulated

in Gram positive bacteria. In B. subtilis, catabolite-

repressible genes are controlled by multiple mechanisms rather

than by a single global regulatory system, and these

mechanisms are known to differ from those operating in enteric

bacteria (Stewart, 1993). Deutscher et al. (1994) has

demonstrated that phosphorylation of HPr at Ser" by the ATP-

dependent kinase is directly or indirectly involved in

catabolite repression in B. subtilis. After Ser46 in HPr of

a B. subtlis strain was replaced with a nonphosphorylatable

alanyl residue, the catabolite repressive effects of glucose








39
on gluconate kinase, glucitol dehydrogenase, and the mannitol-

specific catabolic enzymes were abolished, but this mutation

had no effect on the uptake either of the PTS substrates,

glucose and mannitol, or of the non-PTS substrates, gluconate

and glucitol. A comparable result was obtained in S.

salivarius (Vadeboncoeur et al., 1994). The replacement of

Met48 in HPr with a valine resulted in the inhibition of

phosphorylation of HPr on Ser46 by the ATP-dependent kinase but

did not prevent phosphorylation of HPr by enzyme I or the

phosphorylation of EII complexes by HPr (His-P). However, the

mutant had pleiotropic properties, including reduced growth on

non-PTS sugars. In Lactobacillus brevis, glucose and lactose

are transported via a proton symport. The activities of

glucose and lactose permeases in this organism are also

regulated by the phosphorylation of HPr on Ser46 (Ye et al.,

1994a).

Other Functions of PTS

PTS also has many other functions, including regulation

of gluconeogenesis and other processes, interaction with

acetate kinase, nitrogen regulation (Postma et al., 1993).

The PTS is also a signal transduction system through which

bacteria respond by positive chemotaxis to the presence of PTS

carbohydrates. In PTS-dependent chemotaxis, stimulation

corresponds to uptake and phosphorylation of a substrate

through an EII. No methyl-accepting chemotaxis proteins

(CheA, CheY, CheW, and CheZ) are involved in this process,








40

since mutants lacking the methyl-accepting chemotaxis proteins

retained normal PTS-dependent chemotaxis. Cells lacking a

specific EII do not show chemotaxis toward the corresponding

substrate. In contrast, mutants lacking El and HPr do not

show a chemotaxis response to any PTS carbohydrate, even when

the corresponding Ells are present.














CHAPTER 3
ISOLATION OF THERMOPHILIC CELLULOLYTIC BACILLUS STRAINS




Most thermophilic bacteria are generally isolated from

hot springs, solfatatas or geothermally heated soils. But

thermophilic species of Bacillus may be isolated from a wide

range of both thermophilic and mesophilic environments. They

comprise the first group of thermophilic microorganisms to be

described and characterized in detail. The first published

reference is attributed to Miquel in 1918 who described an

organism growing at 70C and that had been isolated from the

river Seine in Paris (Sharp et al., 1992).

The genus Bacillus is one of the most diverse and

commercially useful groups of microorganisms. Our

understanding of the genetics and physiology of one of

representatives, B. subtilis, is second only to that of EB.

coli (Harwood, 1989). Representatives of this genus are

widely distributed in soils, air, and water where they are

involved in a range of chemical transformations. The

metabolic diversity and low reported incidence of

pathogenicity have led to representatives of this group of

organisms being used in a wide range of industrial processes.

Strains of Bacillus are used for the production of four main








42

types of products: (1) enzymes, (2) antibiotics, (3) fine

biochemicals including flavor enhancers and food supplements,

and (4) insecticides. It is the enzyme market that currently

accounts for the main commercial importance of members of this

genus. In particular they are the major source of hydrolyzing

enzymes for the food and detergent markets.

Complete biodegradation of crystalline cellulose requires

the concerted action of at least three types of enzymes which

include cellobiase (see Review of Literature). Various

methods have been developed for the screening of

microorganisms which are able to degrade crystalline cellulose

(Kluepfel, 1988).

The objective of this study was to isolate and identify

thermophilic Bacillus strains which have cellulolytic

activities and other hydrolase activities.

Materials and Methods

Sources of Samples

102 natural samples were screened for the isolation of

thermophilic cellulolytic Bacillus strains. These samples

included animal manures, composts, foods, pond water, sea

water, soils, etc.

Isolation of Thermophilic Bacillus Strains with Putative
Cellulolvtic Activity

Approximately, 5 grams of environmental samples were

combined with 5 ml of nutrient broth (3 g beef extract, 5 g

peptone per liter) and mixed vigorously by votexing for








43
approximately 5 minutes. After a brief period to allow the

settling of large particles, 1 ml of this suspension was

combined with 1 ml of 100% ethanol and shaken gently for 1

hour at room temperature (22C) to enrich for endospores by

inactivating vegetative cells (Koransky et al., 1978).

Dilutions of treated samples were spread on nutrient agar

plates and incubated overnight at 55C or 65C. Single

colonies were transferred to master plates (nutrient agar) and

to nutrient agar plates containing 0.25% powdered cellulose

(Solka-Floc SW40; James River Corp., Berlin, N.H.). Putative

cellulolytic isolates were identified by the presence of a

clear zone or a partially cleared halo after 4 to 7 days of

incubation at the temperatures from which they were isolated.

Testing for Hvdrolases and Taxonomic Traits

The presence of complete cellulase activity was tested by

using nutrient agar plates containing 0.25% cellulose as

described above. Endoglucanase activity was tested by the

Congo red method with nutrient agar plates containing 0.2%

carboxymethyl cellulose (CMC) (Kluepfel, 1988). Putative

cellobiohydrolase and B-glucosidase activities were tested on

nutrient agar plates containing 10 mg of 4-methylumbelliferyl-

B-D-cellobiopyranoside (MUC) or 4-methylumbelliferyl-B-D

glucopyranoside (MUG) per liter (Wood and Bhat, 1988).

Mannosidase, arabinosidase, and xylosidase activities were

screened in a similar manner with the respective umbelliferyl

derivatives. The presence of pectin-degrading enzymes was








44
tested by a modification of the method described by Brown et

al. (1991) in which sodium polypectate was used instead of

alginate. Taxonomic tests were performed as described in

Bergey's manual (Claus and Berkeley, 1986).

Results

Thermophiles were abundant in all samples screened (2,000

to 1,500,000 CFUs per gram sample). All strains which were

highly cellulolytic (clear zones on cellulose agar) were

provisionally identified as actinomycete based on cell

morphology and growth habit. However, many other isolates

were surrounded by a partially cleared halo and were presumed

to contain incomplete cellulase digestion systems.

A total of 168 representative clones which produced

partially cleared halos on cellulose plates were selected for

further study (112 were from the 55C selection and 56 were

from 65C). All strains were aerobic, endospore-forming, Gram

positive rods. Among the 112 strains from the 55C selection,

only 28 could grow at 65C. In contrast, strains selected at

65C also grew at 55C and 75C. More detailed taxonomic

studies were conducted with 2 representative strains from the

55C selection and 5 from the 65C selection (Table 3).

Closely related organisms may have very similar proteins.

In a separate project (Lai and Ingram unpublished data), a

plasmid containing the genomic DNA of strain XL-65-6 was

cloned and partially sequenced. A search of GenBank data base

revealed that the deduced amino acid sequence of this insert






Table 3. Differential Properties of 55C and 65C Isolates


55C Isolates 65C Isolates
Properties XL-55-60 XL-55-84 XL-65-1 XL-65-6 XL-65-11 XL-65-25 XL-65-56

Growth at
30C + + -
40C + + + + + + +
55C + + + + + + +
65C + + + + +
75C + + + + +


Growth in
2% NaCI
5% NaCl
7% NaCI


Growth at
pH 5.7 +
pH 6.8 +

Acid from
Glucose +
Xylose

Hydrolysis of
Casein +
Gelatin +
Starch +

Catalase +

pH in VP-broth 6.3


6.3 6.3 6.3


6.3 6.3


6.3








46

was highly homologous with the carboxyl terminal end of

alanine racemase from B. stearothermophilus, B. subtilis, E.

coli, and S. typhimurium (Table 4). It is interesting to note

that the deduced amino acid sequence of this insert shares

91.4% identity and 96.2% similarity with the B.

stearothermophilus enzyme, but shares relatively low homology

with the enzymes from other organisms.

These organisms represent a rich source of enzymes for

the depolymerization of carbohydrates (Table 5 and Table 6).

All but 21 of the 168 isolates exhibited activity on one or

more cellulolytic substrates (MUC, MUG, CMC), further

validating the selection procedure. No CMCase activity was

found in the isolates selected at 65C or those selected at

55C but able to grow at 65C when tested at 65C. More than

half of the isolates which were selected at 55C and unable to

grow at 65C exhibited endoglucanase activity. About one-

third of the 65C-growing isolates did, however, display weak

CMCase activity when incubated at 55C. All isolates

contained multiple activities associated with the hydrolysis

of cellulose and hemicellulose as well as other polymers, as

illustrated by the representative strains (Table 5 and Table

6).

The profile of types of cellulase and hemicellulase

activities in 65C- and 55C-growing strains are fairly

different. Comparing the cellulase activities, MUG activity

is the most abundant activity in 65C-growing strains. The







Table 4. Comparison of amino acid sequences in carboxyl terminal portion of alanine
racemases from XL-65-6 and other organisms



% Identity / % SimilarityO
Organisms XL-65-6 Reference

Bacillus stearothermophilus 91.4 / 96.2 Tanizawa et al., 1988
Bacillus subtilis 60.2 / 75.8 Ferrari et al., 1985
Escherichia coli 40.7 / 61.1 Lobocka et al., 1992
Salmonella typhimurium 30.3 / 57.0 Galakatos et al., 1986

8 The results were from the comparison of 186 amino acids at the carboxyl terminal end
of XL-65-6 alanine racemase with corresponding region and number of amino acids of
alanine racemases from other organisms.






Table 5. Enzymatic Activities Expressed by Isolates Capable of Growing at 65C or Higher

Enzymes and
Substratesa No. Strains % Total XL-65-1 XL-65-6 XL-65-11 XL-65-25 XL-65-56

Cellulases
CMCC 24 28.6 + +
MUC 41 48.8 + + + + +
MUG 59 70.2 + + + + +
CMC/MUC 11 13.1 + +
CMC/MUG 20 23.8 + +
MUC/MUG 38 45.2 + + + + +
CMC/MUC/MUG 7 8.3 + +

Hemicellulases
MUA 20 23.8 + + + +
MUM 65 77.3 + + + + +
MUX 27 32.1 + + +
MUA/MUM 17 20.2 + + + +
MUA/MUX 16 19.0 + + +
MUM/MUX 20 23.8 + + +
MUA/MUM/MUX 12 14.3 + + +

Other enzymes
Milk 24 28.6 + + +
Pullulan 18 21.4 + + -
Starch 58 69.0 + + + +
Sodium
polypectated 39 46.4 + + +


CMC: carboxymethyl cellulose
MUC: 4-methylumbelliferyl-B-D-cellobiopyranoside
MUG: 4-methylumbelliferyl-B-D-glucopyranoside
MUA: 4-methylumbelliferyl-a-L-arabinofuranoside
MUM: 4-methylumbelliferyl-B-D-mannopyranoside






Table 5 -- continued

MUX: 4-methylumbelliferyl-B-D-xyloside
b A total of 84 strains were isolated and tested.
c Results were from tests at 55C incubation. None were active at 65C.
d No attempt was made to distinguish hydrolase from lyase activities.








Table 6. Enzymatic Activities Expressed by Isolates Capable of Growing at 55C or Lower

Enzymes and
Substratesa No. Strains % Total XL-55-60 XL-55-84


Cellulases
CMC
MUC
MUG
CMC/MUC
CMC/MUG
MUC/MUG
CMC/MUC/MUG

Hemicellulases
MUA
MUM
MUX
MUA/MUM
MUA/MUX
MUM/MUX
MUA/MUM/MUX

Other enzymes
Milk
Pullulan
Starch
Sodium polypectatec


69.0
80.9
76.2
58.3
56.0
75.0
54.8


61.9
19.0
52.4
15.5
47.6
9.5
9.5


58.3
6.0
42.9
20.2


I The abreviations of substrates see Table 5
b A total of 84 strains were isolated and tested
c No attempt was made to distinguish hydrolase from


lyase activities.








51

percentage of strains with this activity (70.2%) is

predominantly higher than those with CMC or MUC activities

(28.6% and 48.8%, respectively). Only 8.3% of the strains

exhibited all three cellulase activities. In 55C-growing

strains, all three cellulase activities are fairly common.

Approximately 80% of the strains exhibited MUC activity, 76%

exhibited MUG activity, and 69% exhibited CMC activity. Over

half of these strains contained all of the three activities.

For the three hemicellulase activities tested

(arabinosidase, mannosidase, and xylosidase), these two groups

of bacteria also exhibited different profiles. In 65C-

growing strains, MUM activity was the most common activity

(77%) with MUA and MUX activities far less common (23% and

32%, respectively). In contrast, MUM activity was the least

common activity in 55C-growing stains. Only 19% of the

strains had this activity, while more than half of the strains

had MUA and MUX activities (61% and 52%, respectively).

Strains which exhibited all three hemicellulase activities

were rare in both groups of strains, with 14% for 65C-growing

strains and 9% for 55C-growing strains.

Other hydrolytic activities are also common in all the

isolates. Approximately 69% of the 65C-growing strains

exhibited amylase activity and 58% of the 55C-growing strains

had caseinase activity.

Strain XL-65-6, which was isolated from rotting wood by

Lake Alice (Gainesville, Florida), was selected for further








52
study on cellobiose utilization at a molecular level since it

had high activities on both MUC and MUG, as well as on MUA and

MUM (Table 5).

Discussion

The objective of this study was to isolate thermophilic

cellulolytic strains of Bacillus. The isolation method used,

combining ethanol pre-treatment, high incubation temperature,

and nutrient agar medium, appeared to be fairly selective for

thermophilic Bacillus species from various habitats. All

colonies isolated were either Bacillus species or

actinomycetes. Bacillus species constituted the majority of

these isolates. Since most of actinomycete also form spores

and many are thermophiles (Brock and Madigan, 1991), it was

not surprising that members of this group were also recovered

with Bacillus species although the number of actinomycete was

much lower.

Representative strains were classified according to

Bergey's Manual (Claus and Berkeley, 1986). All strains had

the distinctive characteristics of Bacillus species aerobic,

endospore-forming, Gram positive rods.

The Bacillus species which can grow at 65C or higher

include B. acidocaldarius, B. schlegelii, and B.

stearothermophilus. But B. acidocaldarius can grow only under

acidic condition (below pH 6) while the 65C isolates from

this study grew well at pH 6.8 (Table 3). B. schlegelii does

not produce acid from glucose and xylose, and lacks the








53

ability to hydrolyze gelatin and starch. Most of the 65C

isolates produced acid by fermenting these two sugars and

hydrolyzed gelatin and starch. In contrast, most of the

traits of the isolates fit those of B. stearothermophilus.

Furthermore, one 65C isolate, XL-65-6, had very similar

alanine racemase to that from B. stearothermophilus. Thus,

these 65C isolates were identified as B. stearothermophilus.

Among the Bacillus species, only B. coagulans and B.

licheniformis strains are capable of growing at temperatures

ranging from 30C to 55C. B. licheniformis are capable of

growing at high NaCl concentration (up to 7%), while B.

coagulans cannot grow at 5% of NaCl. Both strain XL-50-60 and

XL-50-84 grew at 30C and 55C, but not at 65C, and neither

grow in the presence of 2% NaCl. Thus, the strains which were

isolated at 55C and unable to grow at 65C are most similar

to B. coagulans. It is likely that the strains which were

isolated at 55C and able to grow at 65C were strains of B.

stearothermophilus.

Many isolates did not exhibit all properties listed in

Bergey's Manual. For example, 11% 89% of B.

stearothermophilus strains can grow at 5% NaCl. however, all

of our isolates failed to grow in 2% NaCl. As mentioned in

the Literature Review section, both B. coagulans and B.

stearothermophilus are highly heterogeneous species. The

positive traits in Bergey's Manual indicate only that more

than 90% of strains are positive. The occurrence of some










traits which are different from the standard strains in

Bergey's Manual is thus not surprising.

The activities on MUC and MUG, as well as those of MUA

and MUM appear to be linked. Among B. stearothermophilus

strains, 45.2% had both MUC and MUG activities. This

percentage is very close to that showing MUC activity alone

(48.8%). The percentage of strains with CMC and MUC or MUG

activity was different from those with only one activity. The

percentage of B. coagulans strains expressing both MUC and MUG

activities was 75.0%, similar to MUG activity alone (76.2%).

Those containing CMC activity were also quite different.

There are at least three possible explanations for these

results. First, genetic linkage, where in B.

stearothermophilus and B. coagulans MUG and MUC activity are

linked. Second, ambiguity of the substrates, MUC and MUG.

The chromogenic substrate MUC has often been used to measure

cellobiohydrolase activity, but potential confounding

activities from B-glucosidases are well known (Coughlan,

1988). Thus, both substrates may be cleaved by a single

enzyme. Third, multiple activities, where one enzyme truly

has both exoglucanase and 8-glucosidase activity. A

comparable situation may occur for MUA and MUM activity.














CHAPTER 4
CLONING AND SEQUENCING OF CELLOBIOSE-SPECIFIC PERMEASE OF
PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE
SYSTEM FROM BACILLUS STEAROTHERMOPHILUS





Despite the potential abundance of cellobiose in the

environment, comparatively little is known about the uptake

systems which initiate metabolism in bacteria. The best

characterized cel operon is cryptic and must be activated in

laboratory strains of E. coil (Parker and Hall, 1990).

Functional genes for cellobiose utilization have been reported

in some recent isolates of E. coli from human and animal

manures (Hall and Faunce, 1987). Cellobiose is also

metabolized by other enteric bacteria such as Klebsiella sp.

(A1-Zaag, 1989) and Erwinia sp. (El Hassouni et al., 1992).

Considering the similarities between these enteric genera, it

is likely that all use a cellobiose PTS to initiate

metabolism. Many Gram positive and Gram negative bacteria

have been reported to contain cell-associated B-glucosidases

(Coughlan and Mayer, 1992). Gram positive bacteria such as

Cellulomonas uda and C. favigena contain cellobiose

phosphorylases which are presumed to be involved in

intracellular cellobiose metabolism (Coughlan and Mayer,

1992).








56

The objective of this study was the cloning and

sequencing of Bacillus stearothermophllus genes which confer

the ability to hydrolyze the cellobiose model substrates, 4-

methylumbelliferyl-B-D-cellobiopyranoside (MUC) and 4-

methylumbelliferyl-B-D-glucopyranoside (MUG).

Materials and Methods


Bacterial Strains. Plasmids. and Growth Conditions

Bacterial strains and plasmids used in this study are

listed in Table 7. Strains of B. stearothermophilus were

grown in Difco Nutrient Broth (or agar) at 65C. Strains of

E. coli were grown at 37C in Luria broth (or agar) (Atlas and

Parks, 1993). Ampicillin (50 Mg/ml) and tetracycline (10

Ag/ml) were added to media after sterilization as appropriate

for selection of recombinant E. coll.

DNA Manipulation

Procedures used for the preparation of E. coli plasmids,

assembly of recombinant DNA, and the transformation of E. coli

were as described previously (Sambrook et al., 1989).

Digestions with restriction enzymes were carried out as

recommended by the manufacturers. Genomic DNA was isolated

from B. stearotherimophilus strain XL-65-6 by the standard

method (Cutting and Vander Horn, 1990).

Clonina of B. stearothermophilus cel Operon

Initial testing indicated that DNA isolated from XL-65-6

was not efficiently digested by restriction enzyme Sau3AI but

was digested into small fragments by MboI. Poor digestion by






Table 7. Strains and plasmids used in this study


Strain or plasmid Genetic characteristics Sources

Bacteria
B. stearothermophilus Prototroph this study
E. coli
DH5a F AlacZMI5 deoR recAl endAl supE44
hsdRl7(rK, m- ) )- thi-1 gyrA96 relAl BRL"
XLl-Blue F'::TnlO (tetr)lacqA (lacZ)M15 recAl
endAl gyrA96 thi hsdR17(r,-, m+) supE44
relAl Stratageneb
MM6 1acI22 dctB3 ptsI2 relAl thi-1 spoTi CGSCc

Plasmids
pUCI8 bla lacI'Z' BRL"
pBluescript II KS- bla lacZ Stratageneb
pBluescript SK" bla lacZ Stratageneb
pLOI902 pUCI8 containing cel operon This study
pLOI905 pUCIS containing cel operon This study
in opposite orientation

" Bethesda Research Laboratories, Gaithersburg, MD
b Stratagene, La Jolla, CA
c E. coli Genetic Stock Center, Yale University, New Haven, CT








58

Sau3AI is presumed to result from the presence of 5-

methylcytosine which is tolerated by MboI (Roberts and

Macelis, 1992). Fragments of 4-6 kilobase pairs (kbp) were

isolated from partial MboI digestions of chromosomal DNA by

agarose gel electrophoresis. These were ligated into the

BamHI site of pUCl8 and transformed into E. coli strain DH5a.

Plasmid DNA was isolated from the pooled colonies

(approximately 3000 clones) and served as an amplified

library. Secondary transformants of DH5a were screened for

MUC and MUG activity. Clones containing both activities were

subsequently found to contain the cel operon.

Southern hybridization

Samples of genomic DNA from B. stearothermnophilus XL-65-6

were digested with five restriction enzymes (BssHII, EcoRI,

HindIII, PstI, and Styl), respectively. After agarose gel

electrophoresis, fragments were transferred to Zeta-Probe GT

membranes (Bio-Rad Laboratories, Richmond, CA) and probed with

a 1.1 kbp Styl fragment from within the B. stearothermophilus

cel operon. This probe was labeled with digitoxigenin (Genius

System, Boehringer Mannheim Biochemicals, Indianapolis, IN)

and used as recommended by the manufacturer. Genomic DNA from

E. coli DH5a was digested with PstI and served as a control.

DNA sequencing and sequence analysis

The 5 kbp fragment in pLOI902 was subcloned into

pBluescript II KS" and pBluescript SK" prior to sequencing.

Two series of nested deletions were generated by using the








59

Erase-a-Base System (Promega Corp., Madison, WI) to allow

sequencing of the entire fragment in both directions. The

Magic Miniprep DNA Purification System (Promega Corp.,

Madison, WI) was used to prepare double-stranded plasmid DNA.

Sequencing was performed by the dideoxynucleotide-chain

termination method of Sanger et al. (1977) using fluorescent-

labeled primers (forward, 5'-CACGACGTTGTAAAACGAC-3'; forward,

5' -GGTTTTCCCAGTCACGACG-3'; reverse, 5'-ATAACAATTTCACACAGGA-3')

(LI-COR, Inc., Lincoln, NE) and the Sequenase 7-deaza-dGTP DNA

Sequencing kit (United States Biochemical Corp., Cleveland,

OH). Regions of compression were resolved by using either

dITP (United States Biochemical Corp., Cleveland, OH) or the

TaqTrack Deaza Sequencing System (Promega Corp., Madison, WI).

Sequencing was carried out on a LI-COR model 4000 DNA

sequencer and image analyzer using 7% acrylamide gels (Long

Ranger Gel Concentrate, AT Biochem, Malvern, PA).

Nucleotide and deduced amino acid sequences were

manipulated and analyzed with the Genetics Computer Group

Sequence Analysis Software Package (University of Wisconsin,

Genetics Computer Group, Madison, WI) (Devereux et al., 1984)

and the National Center for Biotechnology Information using

the BLAST network service (Benson et al., 1993). Amino acid

sequences were aligned by using the Clustal V program

(Higgins et al., 1992). The nucleotide sequence data has been

submitted to GenBank and assigned the accession number U07818.










Results

Clonina of Genes Encoding MUC and MUG Activities (cel Operon)

The plasmid library was screened for activities

hydrolyzing MUC and MUG after transformation into DH5a.

Approximately 50 positive clones were identified from the

50,000 colonies tested. Those with weak activities were

unstable. Nineteen clones were found which exhibited high

activities for both substrates. These activities were present

together in all clones. Digestions with restriction enzymes

indicated that all active clones contained the same 5 kbp DNA

fragment and were siblings. One of these, denoted pLOI902,

was selected for further study.

A restriction map of pLOI902 is shown in Figure 3. The

minimal coding region for the activities hydrolyzing MUC and

MUG was localized by testing subclones. Subclones pLOI911g,

pLOI913, and pLOI9l5 were constructed by deleting HindIII,

SmaI, and NsiI fragments, respectively. All but pLOI9l5 were

inactive. This subclone retained both activities. Subclones

pLOI922, pLOI923, pLOI954, and pLOI955 were constructed by

deleting terminal regions with exonuclease III. MUC and MUG

activities remained after the deletion of ca. 400 bp adjacent

to the lac promoter (pLOI922) or 1,500 bp from the opposite

end (pLOI954). However, further deletion of 375 bp from

pLOI922 or 264 bp from pLOI954 resulted in the concomitant

loss of both activities (pLOI923 and pLOI955, respectively).

Thus a rather large region of DNA (approximately 3.2 kbp)























Figure 3. Restriction map of the B. stearothermophilus DNA fragment contained in
pLOI902. Subclones pLOI902, pLOI905, pLOI91l, pLOI913, pLOI915, and pLOI969 are
derivatives of pUC18. Subclones pLOI922 and pLOI923 are derivatives of pBluescript
II KS. Subclones pLOI954 and pLOI955 are derivatives of pBluescript SK. Genes
of cel operon are labled A, B, C, and D. The incomplete regulatory gene is labeled
'R. The proposed terminator is marked as a solid ball. Approximate positions of
frame-shift mutations are indicated by triangles in pLOI981 and pLOI982. These
mutations were introduced by Klenow treatment and religation after digestion with
BstXI and ApaI, respectively. Derivates of pUC18 are aligned beneath restriction
sites used for construction; others are aligned beneath their respective sequence
positions. The activities of MUC and MUG are indicated at the right.







I Kbp
0 1 2 3 4Kbp
r Native-P--- p 5
P 3 n B B "-LacZ-P
|l mZ a ^ l^ |

PLO1902 -
P^91 'R A B C D " +'t
pLOI9Ul
pLOI913 -- -
pLOB15 --- -- -
PLO1954 1432- """"
pLOI955 16 + ++
pLO1922 "
PLO1923 4606 + + +
pLOB19 1432 ... -4231 -
PLO1982 1432 .....- "
LacZ-P- "
Native-P-
pLO1905 +
'R- A B C D" """
p101969 1696-"
-s -


m
m








63

appeared to be required for the expression of MUC and MUG

activities in E. coli DH5a. Both activities were expressed or

lost concurrently in all subclones. Active clones

consistently hydrolyzed MUG more rapidly than MUC.

Experiments were conducted to determine the direction of

transcription of the genes encoding MUC and MUG activities in

pLOI902. The presence or absence of inducer (isopropyl-thio-

B-D galactopyranoside or IPTG) did not alter the level of

expression in XLl-blue. Attempts to subclone this fragment

into plasmid pUCl8 in the opposite orientation were

unsuccessful using DH5a as a host, but clones were readily

obtained using XLl-Blue. The resulting plasmid (pLOI905) was

lethal when transformed back into DH5a. The growth of XLl-

Blue harboring pLOIg905 was strongly inhibited by the presence

of inducer (IPTG), consistent with partial control by the

resident lacIl in the F' element. Thus, in the original

clone, pLOI902, the direction of transcription for the eel

operon is opposite to that of the lac promoter.

Analysis of DNA Seauence

The entire 5,026 bp DNA fragment in pLOI902 was sequenced

in both directions (Fig. 3 and 4). Four complete open reading

frames were found in the region required for the two

activities, bordered by incomplete genes at both ends. All

appear to be transcribed in the same direction. The four

central genes appear to be part of an operon (bases 1541

through 4306) with a potential promoter at the 5' end and a





























Figure 4. Nucleotide sequence of the 5 kbp DNA fragment
from B. stearothermophilus contained in pLOI902. The
deduced amino acid sequences for the cel operon and the
incomplete regulatory gene are listed below the first
nucleotide of the corresponding codon. A putative
promoter region for the cel operon is underlined with the
-35 and -10 regions labeled. The sequence for the 3'
terminus of 16s rRNA from B. stearothermophilus (Van
Charldorp et al., 1981) is shown above the potential
ribosomal binding region (underlined and labeled RB) from
the cel genes. Unmatched bases are in lower case. Genes
within the cel operon are marked at their start codons.
Stop codons are indicated by asterisks. The proposed
terminator is marked with inverted arrows which
correspond to a proposed stem-loop structure.








65

1 TCCCGACGTCCTTGAGTTGACCGAAGCGATGGTCGTCTATGCGGAGAAAAAACTTGGGCG
P D V L E L T E A M V V Y A E K K L G R
61 CCGCCTCGCCGAGAAGGTGATGTATGCGCTCGCCATGCACATTCAAACGGCGATCAACCG
R L A E K V M Y A L A M H I Q T A I N R
121 CCTGCGGGCCGGGACGATCGTTTCCCATCCGAAACTGAATGAAGTTCGCGCCGCCTATAA
L R A G T I V S H P K L N E V R A A Y K
181 GCAGGAATTCGCCGTCGCGCTCGACTGCCTTCAGCTGATGGAAGAAAGGACGAATATCGA
Q E F A V A L D C L Q L M E E R T N I D
241 CTTCCCGATCGATGAAGCGGGATTTTTGACGATGTTTTTTGCGTTCCACGAGGAGCAAGC
F P I D E A G F L T M F F A F H E E Q A
301 GGAAGAGCGGGAGGAACGGGTGGCGATCGTGGTTGTGATGCATGGCAACGGTGTGGCTTC
E E R E E R V A I V V V M H G N G V A S
361 GGCGATGGCGGACGTCGTGAATCAGCTGTTGGCCGCCGCGTGTGTGCATGCGGTCGATAT
A M A D V V N Q L L A A A C V H A V D M
421 GCCGCTTGATGCCGACCCGAAACGAATTTACGAGCAGGTGAAAGCGGTGCTACAGCCCGT
P L D A D P K R I Y E Q V K A V L Q P V
481 CGCATCGAAAAAAGGGGCGCTGTTGCTTGTTGACATGGGATCGCTCGTGTCGTTTTCGAA
A S K K G A L L L V D M G S L V S F S N
541 CTTTTTGGAAAAAGAGCTCGCTGTTCCGGTGCGGGTGATTTCCGCTGCCAGCACGCCGCA
F L E K E L A V P V R V I S A A S T P H
601 CGTGCTCGAAGCGGCCCGCAAAGCGATGCTCGGCTATGCGCTTCAGGAAATTTACGAAGA
V L E A A R K A M L G Y A L Q E I Y E E
661 AGTGAAAGCCGCAGCGCCGTTTTACATTCGCGGGCCGCTTTGGGAGGAGGAGGCTGCGGA
V K A A A P F Y I R G P L W E E E A A E
721 GCAGGACATGCTGGCGATCGTCACCGCCTGTTTTACTGGAAAAGGAAGCGCGCTGGCGCT
Q D M L A I V T A C F T G K G S A L A L
781 AAAGCATATATTGGAAACGTATTTGCAGCTCGATGAGCGCATGTGGCGCATTATTCCCAT
K H I L E T Y L Q L D E R M W R I I P I
841 TCAAATGGCAGATGCGGAAGAAGCGCGGCAAACCTTGTCCAACGTCGCGAAACATTTCCG
Q M A D A E E A R Q T L S N V A K H F R
901 CATCGTCTGTATCGTCAGCCATCTTTGCCTTGATGAGCGAATCCCGCACTTTTCTCTTGA
I V C I V S H L C L D E R I P H F S L E
961 GGACGTGCTTAGCTTGAGGGCGATGAAAGAAATCCAAGCGTTAGCCGACGTTGAGGAAAT
D V L S L R A M K E I Q A L A D V E E I
1021 TCATATGCATATGGCTAGAGAGCTGACCAATCATCTTCGCCACCTCGCACCGGCGCGGGC
H M H M A R E L T N H L R H L A P A R A
1081 CATTCCCGCCATTCGCGCTGCGCTGGCGGCGATTGGTCGGGAACTCGGCCTTGAAGCGGA
I P A I R A A L A A I G R E L G L E A D
1141 TGGCCGCGATCTGGTGGGGCTTGTCTTTCATCTTTGCTGCTTGCTCGATCGCCTGCTGTC
G R D L V G L V F H L C C L L D R L L S
1201 CGGAGAGAGAACGAGCGGTGACCGAGGAGCAGCGAAAGCTTGCGGGCATGAAGATGAAGA
G E R T S G D R G A A K A C G H E D E D
1261 TGGCGCACTGTACAGGGCGGTCAAGGAGGGGTTGTTTCCGCTTGAACAGCAGTACGGTGT
G A L Y R A V K E G L F P L E Q Q Y G V
1321 CTGCATCGATGAAGATGAACTGTGTCATATCGTTCACTTTTTTCGCTCCCTGCAAGGAAA
C I D E D E L C H I V H F F R S L Q G K
1381 ACGAGATGGATAAGGTGCGATAATTGGAAACGCTTCCATTACACAGTTTCCCTCCGTTAA
R D G *
1441 CACACAACCGTTCCATCCGCTTCAGCGCTGTCTTTTCGGCGTTGGCATGGAATTTGCTTG
UUUCCUCC -35
1501 AACAATAATGAATCATTGCAAAAAAAGGAGGAGGAAGCGGATGAACATTTTGCTCATTTG
-10 RBS M N I L L I C
CelA










1561 CGCTGCCGGCATGTCGACGAGTTTGCTTGTGACGAAGATGAAGGAGGCGGCAAAGCAAAA
A A G M S T S L L V T K M K E A A K Q K
1621 AGGGATCGAGGCGAACATTTGGGCTGTGTCAGCTGATGAAGCGAAAAGTCATCTCGACCA
G I E A N I W A V S A D E A K S H L D Q
1681 GGCGGATGTTGTGCTGATTGGTCCGCAAATCCGTTATAAGCTCGCCGCCTTCAAAAAAGA
A D V V L I G P Q I R Y K L A A F K K E
1741 GGGAGAGGCGCGCGGCATTCCGGTTGACGTCATCAATCCAGCTGACTATGGGCGGGTCAA
G E A R G I P V D V I N P A D Y G R V N
UUUucUCCaC
1801 CGGCGCCGGCGTGTTGGACTTTGCGCTGCGGTTAAAAAAATAAACAGGGGGTAGAAGCGG
G A G V L D F A L R L K K RBS
1861 ATGGACCGGTTTATTCGGATGTTGGAAGACCGCGTGATGCCTGTCGCCGGCAAGATTGCC
M D R F I R M L E D R V M P V A G K I A
CelB ->
1921 GAACAGCGCCATTTGCAGGCGATTCGTGACGGGATTATTTTGTCGATGCCATTGTTGATT
E Q R H L Q A I R D G I I L S M P L L I
1981 ATCGGGTCTTTATTTTTAATCGTTGGCTTTTTGCCGATTCCCGGTTACAACGAATGGATG
I G S L F L I V G F L P I P G Y N E W M
2041 GCGAAATGGTTCGGCGAGCATTGGCTTGATAAGCTTCTCTATCCGGTTGGAGCGACGTTC
A K W F G E H W L D K L L Y P V G A T F
2101 GATATTATGGCGCTTGTTGTCAGCTTCGGAGTCGCCTATCGGCTGGCGGAAAAGTATAAA
D I M A L V V S F G V A Y R L A E K Y K
2161 GTGGATGCGCTGTCCGCCGGCGCGATTTCACTGGCTGCTTTTTTGCTCGCAACCCCGTAT
V D A L S A G A I S L A A F L L A T P Y
2221 CAAGTGCCGTTCACGCCGGAAGGAGCGAAAGAAACCATTATGGTCAGCGGTGGCATCCCG
Q V P F T P E G A K E T I M V S G G I P
2281 GTGCAATGGGTCGGCAGCAAGGGGTTGTTTGTTGCCATGATTTTGGCGATTGTGTCAACC
V Q W V G S K G L F V A M I L A I V S T
2341 GAAATTTACCGGAAAATCATTCAAAAAAATATTGTCATTAAGCTGCCGGACGGGGTGCCG
E I Y R K I I Q K N I V I K L P D G V P
2401 CCTGCTGTGGCCCGCTCCTTTGTTGCTTTGATCCCGGGAGCCGCCGTTCTCGTCGTTGTC
P A V A R S F V A L I P G A A V L V V V
2461 TGGGTAGCCCGGCTGATTTTGGAAATGACACCGTTTGAAAGTTTCCATAACATTGTATCT
W V A R L I L E M T P F E S F H N I V S
2521 GTCCTCCTGAACAAACCGCTCAGTGTGCTCGGCGGCAGTGTATTTGGCGCCATTGTCGCC
V L L N K P L S V L G G S V F G A I V A
2581 GTGCTGCTTGTCCAGCTGCTATGGTCGACCGGACTGCACGGAGCGGCCATCGTCGGCGGG
V L L V Q L L W S T G L H G A A I V G G
2641 GTCATGGGGCCGATCTGGCTGTCGCTGATGGATGAAAATCGAATGGTGTTCCAGCAAAAT
V M G P I W L S L M D E N R M V F Q Q N
2701 CCGAATGCCGAACTGCCCAACGTCATTACGCAACAGTTTTTTGATCTTTGGATTTACATC
P N A E L P N V I T Q Q F F D L W I Y I
2761 GGCGGTTCAGGAGCGACATTGGCGTTGGCGTTGACGATGATGTTTCGGGCCCGCAGCCGG
G G S G A T L A L A L T M M F R A R S R
2821 CAGCTGAAAAGCTTAGGTCGGCTGGCGATCGCGCCCGGCATTTTCAATATTAATGAGCCG
Q L K S L G R L A I A P G I F N I N E P
2881 ATCACGTTCGGCATGCCGATCGTCATGAACCCATTGCTGATCATTCCGTTCATTCTCGTG
I T F G M P I V M N P L L I I P F I L V
2941 CCTGTCGTGCTTGTGGTTGTTTCCTACGCCGCGATGGCGACCGGGCTTGTCGCCAAACCA
P V V L V V V S Y A A M A T G L V A K P
3001 AGCGGGGTGGCCGTGCCATGGACGACACCGATCGTGATCAGCGGCTATTTAGCGACGGGA
S G V A V P W T T P I V I S G Y L A T G










3061 GGCAAAATTTCCGGGAGTATTTTGCAAATCGTTAACTTCTTCATCGCGTTTGCCATCTAC
G K I S G S I L Q I V N F F I A F A I Y
3121 TATCCATTTTTCTCGATTTGGGACAAACAAAAAGCGGCCGAAGAGCAGGCCGATCCAACG
Y P F F S I W D K Q K A A E E Q A D P T
UUUCCUC
3181 ATTTCAAGCGGAGCGGGGGCAACGCACTCGCTGTAAAGGAGAGGAACGAAATGCCGCGCT
I S S G A G A T H S L RBS M P R Y
celc -
3241 ATTGCATCGTCAACGCCGATGATTTCGGTTACTCGAAAGGGGTCAACTACGGGATTTTGG
C I V N A D D F G Y S K G V N Y G I L E
3301 AAGCGTTTCAGAACGGTGTCGTCACGTCGGCGACGCTGATGGCGAATATGCCAGCGGCCG
A F Q N G V V T S A T L M A N M P A A E
3361 AACACGCCGCCCGGCTGGCGAAGGACCATCCGGAACTCGGCGTTGGCATTCATTTTGTGC
H A A R L A K D H P E L G V G I H F V L
3421 TGACGTGCGGCCGGCCGCTGGCCGATGTTCCATCGCTGGTGAACGAGAATGGGGAGTTTC
T C G R P L A D V P S L V N E N G E F P
3481 CGCGGCGCGGGGAGGCGCTTGTCGGCGCTAGGCGCGGCGATATCGAGCGGGAGCTTTGCG
R R G E A L V G A R R G D I E R E L C A
3541 CCCAATTGGAGCGTTTTTTCTCGTTCGGGCTCACTCCGACGCATATTGACAGCCATCATC
Q L E R F F S F G L T P T H I D S H H H
3601 ACGTTCATGAGCACCCGAATGTGTTTCCGGTTGTGGAACAATTGGCCGAACGCTATCGGC
V H E H P N V F P V V E Q L A E R Y R L
3661 TGCCGATCCGCCCGGTGCGGACGGCACGGCCGCATCGGCTGCCGACCGTCGACGTCTTTT
P I R P V R T A R P H R L P T V D V F F
3721 TTCCGGATTTTTACGGCGATGGACTGACGAAAGACCGTTTTATCTCGCTGATCGACCGAA
P D F Y G D G L T K D R F I S L I D R I
3781 TTGGCGACGGGCAGACGGCGGAAGTGATGTGCCACCCGGCGTATATCGATGTTCCGCTCG
G D G Q T A E V M C H P A Y I D V P L A
3841 CGTCAGGAAGTTCCTATTGTCAACAACGAGTCGAAGAGCTGGCGGTGCTGACCGACCCAA
S G S S Y C Q Q R V E E L A V L T D P T
3901 CGCTCGTTGCCGAGATGGCCGAGCGCGGTGTTCAGCTGATCACGTACCGCGAATTCTATA
L V A E M A E R G V Q L I T Y R E F Y K
UCCUCcaCU
3961 AACTATAGGA9AGG9CGTTATGCAAACGTATGAACAAACTGTATTCCAACTGATTCTTCA
L RBS M Q T Y E Q T V F Q L I L H
CelD -
4021 TGGCGGAAACGGCCGCAGTTATGCAATGGAGGCGATTACGGCGGCGAAAAAAGGGGAATT
G G N G R S Y A M E A I T A A K K G E F
4081 TGCCGAGGCGCGCAGGCTGCTTGAACAGGCGGGAGCAGAACTGCAGGCGGCCCATGGGCT
A E A R R L L E Q A G A E L Q A A H G L
4141 GCAGACCGCGTTGCTGCAACAAGAAGCGAGCGGCGGGCAGCCGGTGGTGACGCTTCTGAT
Q T A L L Q Q E A S G G Q P V V T L L M
4201 GGTGCATGCCCAAGATCATTTAATGACGGCGATCACGGTCAAGGATTTAGCCGCTGAATT
V H A Q D H L M T A I T V K D L A A E F
4261 CGTGGAGCTGTATGAAGCGCTCAAGCGGCAAACAACCGAATCATAATTGTCGTCGGCCGG
V E L Y E A L K R Q T T E S *
4321 CTTGTTGACCGGGTGAGGATGATTCGCTTCAACGGGAATGAACCCAAGGCGGGCGGCAAC

4381 GGGCTGTTCTAGCGACCGCCCGCTTTGTTGATTTTACAATAATTTTATTATGTGAACATA

4441 AGGGCGCCAGCCGTTCAGCCCTCCCGGCTGCCGCCGGTCTCGGCTGAACGGAGAAGGAAA
4501 ACTCCGCTTGTTTGCGTCACATATCGTGGCTTGTGTTGTGCTTTTGCCGCGTGTGATTGC









68

4561 GGTTCTTCACTTTTTTCGAACCGTCAAGCGGCTCCGGCTGCCCAGGATTGTCGCCTTTTG
4621 GGGCGTTTTTGCGCATATCTTTGCCGTCGTTTTTGTTCGTCATTTGTTGTCCCTCCTTTG
4681 TCCGTTAGCATGCGGCGTTGTGCTTTTGCTTATTCGGGGCATAATTTGCGCCGGTTTGAA
4741 ACGATAAGTAGTGGATGACCGCGATGACTGCGAAGGAGGAACGAGCCATGGTTTATCATA
4801 AAACGAAGCAAGACGCGTTTCAGGCGCGCGCAAAAGGCGACGATGGAGGCAAAAGAGTGG
4861 CATGACCATCTCGTCCGCGATCAAGCCGATTACGGCCATCAGTTAGCCCATTTGCGGCAG
4921 GAAGTGAACGAAGCGTTTGCACAAATTGAAAACGCGCTTGAAGTCGCCTCGGAAACGCAG
4981 CGCATGCAGCTTGAGAAATTCCGCAGCGATTTGCAGGCGATCCCCGG








69
possible rho-independent terminator at the 3' end (Rosenberg

and Court, 1979). The coding regions for all four genes

utilize ATG codons to initiate transcription. The potential

ribosomal-binding sequences for celA and ceIC provide a better

match with the 3' terminus of 16s rRNA from B.

stearothermophilus (Van Charldorp et al., 1981). It is

interesting to note that the potential ribosomal binding

sequences in celB, ceIC, and celD are overlapped by stop

codons from the preceding genes, and may provide translational

coupling. A similar phenomenon was known in B. subtilis

sucrose-specific PTS (Fouet et al., 1987), in which the stop

codon for enzyme II overlapped the start codon for sucrose-6-

phosphate-hydrolase.

Identification of Cloned Genes by Homolocgy

An initial search of the DNA data base revealed strong

homology between the translated sequences of the cloned genes

and previously described genes encoding sugar-specific

proteins in PTS systems (Table 8). The highest degree of

similarity was observed with the E. coil cel operon (Table 8

and Fig. 5). Since MUC and MUG can be considered analogues of

cellobiose, the sequenced genes from B. stearothermophilus XL-

65-6 were designated as a cel operon, the first PTS cellobiose

system to be characterized at a molecular level in a Gram

positive organism. Table 9 summarizes some of the properties

of these four genes. The gene products from celA and celB










Table 8. Comparison of predicted amino acid sequences for
celA, B, and D and ceIR' from B. stearothermophilus to
homologous PTS polypeptides from other organisms.


%Identity %Similaritv)
Proteins Bs-CelA8 Bs-CelB8 Bs-CelDa Bs-'CelRa

EIIBC
Ec-CelAb 42.9 (59.2)
LI-LacEc 23.2 (50.5)J 30.6 (58.2)k
Sa-LacEd 21.9 (51.0)1 29.4 (57.7)'
Lc-LacEe 16.0 (48.0)j 29.1 (55.9)k
Ec-CelBb 32.9 (60.1)

EIIA
Ec-CelCb 41.6 (67.3)
L1-LacFc 41.8 (66.0)
Sa-LacF'd 38.4 (62.6)
LC-FIIIe 36.5 (58.9)

Reculatory proteins
Bsu-LevR+ 29.5 (50.1)
Bsu-SacY9 27.2 (57.0)
Bsu-SacTh 30.0 (58.0)
Ech-ArbG' 30.2 (54.2)


a Bs-CelA, Bs-CelB, and Bs-CelD refers to polypeptides for
gene celA, celB, and celD of cel operon from B.
stearothermophilus. Bs-'CelR refers to the incomplete
regulatory protein.
b Ec-CelA, Ec-CelB, and Ec-CelC refer to EIIBCeL, EIICC"et, and
EIIAc*t of glucoside-specific PTS enzyme II of cel operon
from E. coli (Parker and Hall, 1990; Reizer et al., 1990).
c Ll-LacE and LI-LacF refer to lactose-specific PTS operon
EIIBCLac and EIIALOC from Lactococcus lactis, respectively (de
Vos et al., 1990).
d Sa-LacE and Sa-LacF refer to lactose-specific PTS EIIBCL
and EIIALac of lac-operon from Staphyloccus aureus,
respectively (Breidt et al., 1987).
e Lc-LacE and Lc-FIII refer to lactose-specific PTS EIIBCLac
and EIIALac from Lactobacillus case respectively, (Alfred
and Chassy, 1988; Alfred and Chassy, 1990).
f Bsu-LevR refers to transcriptional regulator of PTS levanase
operon of B. subtilis (Debarbouille et al., 1991). The
result of comparison with Bs-'celR were from the comparison
of amino acid 2 to 459 with amino acid 475 to 935 of levR
gene product.
g Bsu-SacY refers to antiterminator of PTS levansucrase gene
of B. subtilis (Zukowski et al., 1990). The result of
comparison with Bs-'celR were from the comparison of amino








71
Table 8 -- continued

acid 2 to 115 with amino acid 68 to 181 of sacY gene
product.
h Bsu-SacT refers to transcriptional antiterminator of PTS
sucrase operon of B. subtills (Debarbouille et al., 1990).
The result of comparison with Bs-'CelR were from the
comparison of amino acid 2 to 101 with amino acid 66 to 165
of sacT gene product.
SEch-ArbG refers to antitermninator of PTS phospho-B-
glucosidase operon of Erwinia chrysanthemi (El Hassouni,
1992). The result of comparison with Bs-'celR were from the
comparison of amino acid 2 to 97 with amino acid 69 to 164
of arbG gene product.
J These results are comparisons of Bs-CelA with carboxyl
terminal portions of LI-LacE, Sa-LacE, and Lc-LacE,
respectively.
k These results are comparisons of Bs-CelB with amino terminal
portions of LI-LacE, Sa-LacE, and Lc-LacE, respectively.























Figure 5. Comparison of deduced amino acid sequences of cel operon genes from B.
stearothermophilus and E. coli. Identical residues are indicated by asterisks.
Conserved residues with similar properties are indicated by dots. The proposed
phosphorylation sites are overlined. Gaps (dashes) have been introduced to
optimize alignment.





A. EfIB
Bs-CelA M--NILLICAAGMSTSLLVTKMKEAAKQKGIEANIWAVSADEAKSHLDQADVVLIGPQIRYKLAAFKKE 67
Ec-CelA MEKKHIYLFCSAGMSTSLLVSKMRAQAEKYEVPVIIEAFPETLAGEKGQNADVVLLGPQIAYMLPEIQRL 70
.* ,.*. *********.***o o * o .*****.**** *.
Bs-CelA GEARGIPVDVINPADYGRVNGAGVLDFALR-LKK--- 100
Ec-CelA LPNK--PVEVIDSLLYGKVDGLGVLKAAVAAIKKAAAN 106
**.**.. **.*.* *** *. .**

B. BIIC
Bs-CelB MDRFIRMLEDRVMPVAGKIAEQRHLQAIRDGIILSMPLLIIGSLFLIVG--FLPIP--GYNEWMAKWFGE 66
Ec-CelB MSNVIASLEKVLLPFAVKIGKQPHVNAIKNGFIRLMPLTLAGAMFVLINNVFLSFGEGSFFYSLGIRLDA 70
** ..* **. *..**..*.* *** *..*... **..
Bs-CelB HWLDKL--LYPVGA--- TFDIMALVVSFGVAYRLAEKYKVDALSAGAISLAAFLLATPYQVPFTPEGA 129
Ec-CelB STIETLNGLKGIGGNVYNGTLGIMSLMAPFFIGMALAEERKVDALAAGLLSVAAFMTVTPYSVG ------ 134
.* .*. *..**.*. .* .. *** *****.** *.***. *** *
Bs-CelB KETIMVSGGIPVQWVGSKGLFVAMILAIVSTEIYRKIIQKNIVIKLPDGVPPAVARSFVALIPGAAVLVV 199
Ec-CelB -EAYAVGA----NWLGGANIISGIIIGLVVAEMFTFIVRRNWVIKLPDSVPASVSRSFSAF--NSRLYYS 197
*. *.* .*.*. .. ..*...* .*.. *...* ******.**..*.*** *.
Bs-CelB VWVARLILEMTPFES-FHNIVSVLLNKPLSVLGGSVFGAIVAVLLVQLLWSTGLHGAAIVGGVMGPIWLS 268
Ec-CelB FRDGDYCLALNTWGTNFHQIIMDTISTPLASLGSVWLAYVI--LSTALVLRIHAACADRTGQRHYDAW-A 264
**.*. .. **. ** ....* *. *
Bs-CelB LMDENRMVFQQNPNAELPNVITQQFFDLW-------- IYIGGSGATLALALTMMFRARSRQLKSLGRLAI 330
Ec-CelB L--ENIATYQQYGSVE-AALAAGKTFHIWAKPMLDSFIFLGGSGATLGLILAIFIASRRADYRQVAKLAL 331
** .** . o *..* *..*******.* *.. .* . o..**.
Bs-CelB APGIFNINEPITFGMPIVMNPLLIIPFILVPVVLVVVSYAAMATGLVAKPSGVAVPWTTPIVISGYLATG 400
Ec-CelB PSGIFQINEPILFGLPIIMNPVMFIPLYWYNRILAAITLAAYYMGIIPPVTNIA-PWTMPTGLGAFFNTN 400
..***.***** **.**.***...**. .* ** .. .* *** *.... *
Bs-CelB GKISGSILQIVNFFIAFAIYYPFFSIWDKQKAAEEQADPTISSGAGATHSL 451
Ec-CelB GTSPHC--------------------WSHSSTLASQR -------- 417
.* *

C. EIIA
Bs-CelD M -------------QTYEQTVFQLILHGGNGRSYAMEAITAAKKGEFAEARRLLEQAGAELQAAHGLQTA 57
Ec-CelC MMDLDNIPDTQTEAEELEEVVMGLIINSGQARSLAYAALKQANRGDFAAAKAMMDQSRMALNEAHLVQTK 70
*. **...*..** *. *..*.** *. ...*. *. ** .**
Bs-CelD LLQQEASGGQPVVTLLMVHAQDHLMTAITVKDLAAEFVELYEALKRQTTES 108
Ec-CelC LIEGDAGEGKMKVSLVLVHAQDHLMTSMLARELITELIELHEKLK --- A 106
*.o .*. *.*..*********.. ..* .*..** ** .








Table 9. Characteristics of the cel operom from B. stearothermophllus XL-65-6


No. of Total No. of Predicted
Genes PTS Component Nucleotides %GC Amino Acids Mr Charges pI



celA EIIBCeL 303 53 100 10,737 +3 9.8
celB EIICce' 1,356 53 451 48,805 +4 10.0
celC Putative
cleavage enzyme 738 59 245 27,423 -8 6.1
celD EIIAce' 327 55 108 11,785 -5 5.2








75

have high pI values and positive charges while those from ceIC

and celD have low pI values and negative charges.

The translated amino acid sequence for B.

stearothermophilus celA shares 43% identity and 59% similarity

with that of celA from E. coli K12 (Parker and Hall, 1990)

which encodes a part of enzyme II (EIIB) (Reizer et al., 1990)

(Table 8 and Fig. 5A). In many organisms, the corresponding

domains encoded by celA and celB are fused into a single

polypeptide (EIIBC) (Saier and Reizer, 1992). The translated

B. stearothermophilus celA sequence is homologous to the

carboxyl terminal portion (EIIB domain) of the lactose-

specific enzyme II proteins from Lactococcus lactis,

Staphylococcus aureus, and Lactobacillus case (Table 8).

Cys7 is surrounded by a highly conserved region in all

homologues examined. This Cys residue has been proposed as

the site of phosphorylation in L. lactis (Alfred and Chassy,

1990). The celA gene is predicted to encode a protein with Mr

10,737 (Table 9).

The ceiB gene begins 17 bases downstream from celA and is

preceded by a potential ribosomal-binding site which overlaps

the last 2 bases of the celA stop codon. The celB gene is

predicted to encode a protein with Mr 48,805. The deduced

amino acid sequence for ce1B is most similar to that of the

membrane-spanning polypeptide in the enzyme II complex (EIIC)

for cellobiose from E. coli (celB) (Parker and Hall, 1990),

exhibiting 33% identity and 60% similarity (Table 8 and Fig.










5B). The translated sequence for B. stearothermophilus eel B

is also similar to the amino terminal portion of genes

encoding PTS enzyme II proteins from other organisms (Table

8).

The celC gene begins 14 bases downstream from the celB

stop codon and also exhibits an overlap between the ribosomal-

binding region and the preceding stop codon. In contrast to

the other genes in the eel operon, no genes were found in the

current data base with similarity to the translated sequence

for celC. However, the carboxyl-terminal portion of this

protein exhibited partial homology (22% identity, 53%

similarity) with the carboxyl portion of E. coli celF encoding

the phospho-B-glucosidase. Thus the celC gene is presumed to

encode a cleavage enzyme for cellobiose-phosphate.

The celD gene encodes a small protein containing 108

amino acids. Again, the proposed ribosomal binding site

overlaps the stop codon of the preceding gene. The translated

amino acid sequence for celD exhibits 41.6% identity and 67.3%

similarity to that of E. coli celC which encodes enzyme III

(EIIA) (Table 8 and Fig. 5C). Table 8 summarizes the homology

between celD and related genes from other organisms. A

histidine residue (His76) is located in a region which is

highly conserved among different organisms. This residue has

been proposed as the site of phosphorylation for the lactose-

specific enzyme III in L. easel (Alfred and Chassy, 1988;

Hengstenberg et al., 1989).










An incomplete open reading frame (Fig. 3 and 4) was found

upstream from the cel operon which contained 463 codons and is

transcribed in the same direction as the cel operon. This

gene was provisionally identified as encoding a regulatory

protein (ceIR) based on homology to regulatory proteins in

other PTS systems (Table 8). The translated sequence for the

incomplete celR gene is most similar to that of B. subtilis

levR (de Vos et al., 1990), the transcriptional regulator for

the PTS levanase operon. Although a putative promoter for

celABCD was readily evident in the intercistronic sequence, it

is unclear whether celR is part of the cel operon or

transcribed separately. No stem-loop structure resembling a

rho-independent terminator was found in the intergenic region

and it is possible that all five genes may be transcribed at

the some level from a common promoter.

A portion of the amino terminus of a possible sixth gene

was found downstream from the cel operon but was not

identified by homology searches of the current DNA data base.

Codon Usage

Codon usage in the cel genes was compared to that for the

genes encoding phosphoglycerate kinase (Davies et al., 1991)

and alcohol dehydrogenase T (Sakoda and Imanaka, 1992) from B.

stearothermophilus (Table 10). Many codons (ie. AGA, TGA,

TAG, ATA, and TGT) were rarely used. Either a G or C was

usually present in the wobble positions resulting in G+C

contents of 52.8 to 54.9 for celAB and celD, respectively.











Table 10. Codon Usage of cel Operon


Mole %


AmAcid Codon celA

Gly GGG 2.00
GGA 1.04
GGT 1.04
GGC 4.00

Glu GAG 4.00
GAA 1.00

Asp GAT 1.98
GAC 4.02

Val GTG 4.00
GTA 0.00
GTT 2.00
GTC 2.00

Ala GCG 6.00
GCA 1.05
GCT 4.05
GCC 4.05

Arg AGG 0.00
AGA 0.00
CGG 2.00
CGA 0.00
CGT 1.00
CGC 1.00

Ser AGT 2.00
AGC 0.00
TCG 1.00
TCA 1.00
TCT 0.00
TCC 0.00

Lys AAG 4.00
AAA 6.00

Asn AAT 1.00
AAC 3.00

Met ATG 3.00


celB

2.19
2.02
0.93
3.29

0.67
2.66

1.34
1.10

3.57
0.71
2.24
3.77

4.69
0.42
0.94
4.48

0.00
0.00
1.77
0.22
0.22
0.90

0.90
1.80
1.14
0.90
0.42
0.90

1.10
1.38

1.33
1.33

3.77


celC

2.57
0.88
0.88
3.01

4.33
3.01

3.03
2.69

2.87
0.45
2.42
3.23

4.11
0.34
0.77
3.34

0.41
0.00
3.67
1.22
0.82
2.04

0.37
0.37
1.84
0.73
0.00
0.37

0.54
1.09

1.23
1.63

2.04


celD

2.18
1.85
0.00
2.81

2.75
7.44

1.85
0.00

3.72
0.94
0.00
0.94

9.29
1.89
0.94
3.78

0.93
0.00
0.93
0.00
0.00
1.85

0.92
0.92
0.00
0.92
0.00
0.00

1.85
1.85

0.00
0.93

3.70


gap adhT

0.62 1.82
1.17 1.82
0.28 4.70
4.81 2.35

1.17 0.61
5.70 6.21

2.09 3.58
3.88 1.46

4.18 2.35
0.36 5.09
2.03 1.44
5.37 4.18

5.70 1.82
1.16 3.53
1.16 2.93
3.61 1.82

0.00 0.00
0.00 0.00
0.31 0.00
0.31 0.00
0.31 1.18
3.58 1.50

0.00 0.00
0.59 0.29
3.87 0.59
0.00 0.88
0.32 0.29
0.59 0.59

0.89 1.16
5.97 7.14

0.60 0.59
5.37 1.78

2.39 0.89











Table 10 -- continued


Mole %


AmAcid Codon celA

lie ATA 0.00
ATT 5.04
ATC 3.04
Thr ACG 2.00
ACA 0.00
ACT 0.00
ACC 0.00

Trp TGG 1.00

End TGA 0.00
TAG 0.00
TAA 1.00

Cys TGT 0.00
TGC 1.00

Tyr TAT 2.00
TAC 0.00

Leu TTG 3.00
TTA 1.00
CTG 2.00
CTA 0.00
CTT 1.00
CTC 3.00

Phe TTT 1.00
TTC 1.00

Gin CAG 0.99
CAA 2.01

His CAT 1.00
CAC 0.00

Pro CCG 2.01
CCA 0.99
CCT 0.00
CCC 0.00


celB

0.00
6.34
3.77
2.00
0.68
0.00
1.09

2.22

0.00
0.00
1.00

0.00
0.00

1.34
1.10

3.51
0.90
3.51
0.23
1.58
1.58

2.88
2.66

1.55
1.55

0.67
0.44

3.79
1.35
0.64


celC

0.36
1.89
2.25
3.28
0.00
0.83
0.83

0.00

0.00
0.00
1.00

0.67
1.37

2.09
1.58

1.56
0.00
4.67
0.45
0.90
1.71

3.98
1.33

0.94
1.91

2.60
1.89

5.76
1.18


celD

0.00
1.86
0.92
4.67
0.92
0.92
1.83

0.00

0.00
0.00
1.00

0.00
0.00

2.78
0.00

0.91
1.81
6.48
0.00
2.72
0.91

0.92
1.86

3.67
6.52

3.70
0.00

0.93
0.00


gap

0.00
2.09
3.88
3.57
0.62
0.00
1.47

0.60

0.00
0.00
1.00

0.00
0.60

0.60
1.79

5.64
0.56
0.89
0.00
0.32
0.56

0.60
0.80

0.00
0.90

2.10
0.59

3.28
0.00


adhT

0.54
3.56
1.78
0.29
2.68
0.29
0.00

1.19

0.00
0.00
1.00

0.59
2.08

2.09
1.18

0.31
3.86
0.31
0.00
2.39
0.93

1.18
0.89

0.00
3.56

2.67
0.00

1.46
0.91


0.00 0.00 0.00 2.67


0.64 0.00 0.00 0.00 0.00








80
The G+C content of ceiC is unusually high (59.1%) and differs

significantly from other genes in this operon.

Southern hybridization analysis

Southern analysis confirmed that the cel genes originated

from B. stearothermophilus XL-65-6. A Styl fragment from the

internal region of the cel operon was used as a probe. This

probe did not hybridize to DNA fragments from E. coli DH5a and

bound single regions in digestions of XL-65-6 genomic DNA,

consistent with the cel operon being present as a single

chromosomal copy (Fig. 6).

Hydropathy analysis

Analyses of the deduced amino acid sequences by the

method of Kyte and Doolittle (1982) revealed a hydrophilic or

slightly hydrophobic character for the products of celA, celC,

and celD (Fig. 7). Further analysis for membrane spanning

domains by the method of Klein et al. (1985) predicted a

single domain in the celA product which may serve as a

membrane anchor. No membrane-spanning domains were predicted

in the ceiC and celD products. Hydropathy profiles for the

celA and celD encoded polypeptides exhibit a remarkable degree

of conservation when compared to homologous PTS enzymes from

other organisms despite differences in the transported sugars

(Fig. 7). The ceIB gene product is very hydrophobic with 9 to

11 predicted membrane-spanning domains. The hydropathy plot

for this protein is also very similar to the celB homologues.

Reizer et al. (1990) previously noted that the N-terminal































Figure 6. Southern hybridization analysis of cel operon.
Lane assignment: Lane 1 to lane 5: style, pstI, HindlII,
EcoRI, and BssHII digestions of B. stearothermophilus XL-
65-6 genomic DNA; Lane 6: ptsl digestion of E. coli DH5a
genomic DNA; Lane 7: A marker.





















mc' .,'


1 2 3 4 5 6 7
























Figure 7. Hydropathy plots of PTS enzymes from different organisms. Bs-CelB and
Ec-CelB, Bs-CelA and Ec-CelA refer to EIIC and EIIB from B. stearothermophilus (Bs)
and E. coli (Ec), respectively. Sa-LacE refers to EIIBC from Staphylococcus
aureus. Bs-CelD, Ec-CelC, and Sa-LacF denote EIIA from the 3 listed organisms.

















A. Bs-CelB 1 .,.-.'. ... Bsi-CeIA 1- B. Bs-CelD I ---

Ec-CeIB 3. Ec.. -CCIA E c-CelC I

Sa-LacE I ---........ ----- Sa-LacF ----- -- --I










region of E. coli celB gene product and the C-terminal region

of the E. coli ceIC translation product can be folded into

helical wheel plots to reveal distinctly hydrophobic and

hydrophilic faces. A similar analysis of the corresponding

regions in the translated sequences for B. stearothermophilus

celB and celD confirmed that these patterns were also

maintained in this Gram positive organism (Fig. 8). When

folded, the first 16 amino acids at the amino terminus of the

B. stearothermophilus celB encoded protein produced a highly

hydrophobic face which may be involved in the formation of the

transport complex but is not predicted to span the membrane

(Klein et al., 1985). Similarly, the carboxyl terminal

region of the celD gene product (close to the histidinyl

residue of the proposed phosphorylation site) can also be

folded into an a-helix in which most hydrophobic residues are

segregated on one side and may function in the assembly of the

active enzyme II complex.

Functional analysis of genes in the B. stearothermophllus cel
oberon in E. coli

The B. stearothermophilus cel genes were constitutively

expressed in strain DH5a(pLOI902). Recombinant cells

hydrolysed MUC, MUG, and p-nitrophenyl-glucopyranoside (pNPG),

and formed slight pink colonies when growing on cellobiose-

MacConkey agar, indicating poor utilization of cellobiose.

All activities were concurrently lost as a result of deletions

in the carboxyl terminal region of celD and frame-shift

mutations in ceiB (Fig. 3). The full insert was subcloned in






















Figure 8. Comparisons of helical wheel plots of terminal regions from PTS enzymes.
Hydrophobic residues are indicated by capital letters and hydrophilic residues by
small letters. Hydrophobic and hydrophilic phases are divided by the line. Bs-
CelB, Ec-CelB, and Sa-LacE are folded from the first 16 amino acid residues at
amino terminal ends EIIC. Bs-CelD, Ec-CelC, and Sa-LacF are folded from the
carboxyl terminal ends of EIIA, from amino acid 86 to 104, 97 to 115, and 86 to
103, respectively.










A. LMV


Bs CdB


Bs CelD


Ec -CeIB


Lie k

Ec CeIC


Sa -LacE


Sa LacF








88

the reverse orientation to provide transcription from the lac

promoter. Deletions in the amino terminal region of celA also

resulted in a loss of activity (Fig. 3). A variety of

approaches were tried to isolate mutations in the ceIC gene

without success. Thus a requirement for three of the four

genes within the cel operon has been demonstrated while the

fourth gene, ceIC, is presumed to encode a cleavage enzyme for

cellobiose-phosphate.

Cells of strain DH5a(pLOI902) exhibited strong MUC, MUG

and pNPG activities when incubated at 37C. However, little

activity was detected when cells were incubated at 55C

despite the thermal tolerance of B. stearothermophilus.

Previous studies had demonstrated that the general proteins

(El and HPr) of the E. coil PTS complement sugar-specific

components from other Gram positive bacteria (de Vos et al.,

1990; Fouet et al., 1987). The loss of activity at 55C could

result from thermal inactivation of essential host-supplied

components (enzyme I). To test this hypothesis, a ptsI mutant

of E. coil, MM6 (Fraenkel et al., 1964), was transformed with

pLOI902. The resulting recombinant was inactive at both

temperatures.

The requirement for host El (and presumably also HPr) for

activity at both temperatures indicates that the

phosphorylation of chromogenic substrates may be essential

prior to cleavage.










Discussion


The chromogenic substrate MUC has often been used to

measure exocellobiohydrolase activity, although potential

confounding activities from B-glucosidases are well known

(Coughlan, 1988). However, the discovery that MUC and MUG

activities could be used as a marker for the cloning of a

cellobiose PTS operon from a Gram positive bacterium, B.

stearothermophilus, was rather unexpected.

The organization of the PTS has been described by many

researchers (Saier and Reizer, 1992). This system consists of

two general energy-coupling proteins, enzyme I and HPr, as

well as a sugar-specific permease, commonly referred as the

enzyme II complex. Although the enzyme II complex may consist

of 1, 2, 3, or 4 distinct polypeptide chains, each complex

contains at least three functional domains: a hydrophobic

trans-membrane domain which binds and transports the sugar, a

closely associated hydrophilic domain which possesses the

first phosphorylation site, and a second hydrophilic domain

containing an additional phosphorylation site. In B.

stearothermophilus, celB encodes the membrane-spanning

polypeptide which forms the transmembrane channel. The celA

product is less hydrophobic and contains a hydrophobic tail

which may serve as a membrane anchor. The celA product is

predicted to have a high pI, similar to that of the celB

product. Since celA and celB encode domains which form a

single polypeptide in many organisms (Saier and Reizer, 1992),








90
these two gene products can be assumed to interact closely in

the enzyme II complex. The celD product is hydrophilic

although a hydrophobic surface appears to be present near the

carboxyl terminus. This gene product is predicted to have a

low pI which may allow the formation of ionic interactions

with both the celA and celB encoded polypeptides. The ceiC

product is also very hydrophilic and is proposed to encode an

enzyme for the cleavage of cellobiose-phosphate, based on

homology with E. coli celF. All attempts to mutate this gene

were unsuccessful.

In recombinant E. coli expressing MUC and MUG activities,

enzyme I (and presumably HPr) must be supplied by the host and

complement the PTS genes from B. stearothermophilus. Other

examples of functional complementation with heterologous PTS

systems from Gram positive organisms have been reported

previously in recombinant E. coli (de Vos et al., 1990; Fouet

et al., 1987).

MUC, MUG, and pNPG are analogues of cellobiose and

require hydrolysis to release the respective chromogens.

Since a functional E. coli enzyme I (ptsl) was required for

hydrolysis, phosphorylation can be presumed to be essential.

In the simplest case, the phosphorylated intracellular product

must be hydrolysed once to release the chromogen from MUG or

twice to release the chromogen from MUC. However, it is

possible that the transported substrate is cleaved by a

phosphorylase as proposed for Cellulomonas uda and C. favigena








91
(Coughlan and Mayer, 1992). A phosphorylase would produce 4-

methylumbelliferyl-phosphate or p-nitrophenyl-phosphate as

products which must be cleaved by a host phosphatase to

release the chromogen. The precise nature of the reaction

catalyzed by the proposed cleavage enzyme (celC) is still

unknown.

The PTS cellobiose-specific proteins from the Gram

positive thermophile B. stearothermophilus (celA, celB, and

celD) appear remarkably similar to the corresponding genes

from the Gram negative mesophile, E. coli celA, celB, and celC

(Parker and Hall, 1990) and may be derived from a common

ancestor. Indeed, the sugar-specific proteins from most

organisms which have been examined exhibit a high degree of

homology despite differences in the transported substrates

(Saier and Reizer, 1992). However, the proposed phospho-B-

glucosidases from these two organisms (ceIC in B.

stearothermophilus and celD in E. coli, respectively) exhibit

only modest homology to each other and little homology to

other glycohydrolases, consistent with an independent origin.

The independent origin of ceiC is further supported by the

high G+C composition of this gene, considerably higher than

that of other sequenced genes from B. stearothermophilus. The

discovery of a PTS cel operon in B. stearothermophilus which

is similar to that in E. coli (Parker and Hall, 1990) is

consistent with the PTS being the prevalent route for

cellobiose uptake in bacteria.