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Enzyme biosynthesis in bacteria as a basis for toxicity testing

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Enzyme biosynthesis in bacteria as a basis for toxicity testing
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ENZYME BIOSYNTHESIS IN BACTERIA
AS A BASIS FOR TOXICITY TESTING













By

RONALD JOHN DUTTON


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

1988


O- F I r -io, 0





























Copyright 1988

by

Ronald John Dutton


























To my parents,
Mary and Adam Dutton,
and in memory of their daughter,
Anita Christine Dutton















ACKNOWLEDGEMENTS


The author would like to acknowledge and thank the

chairman of his doctoral committee, Dr. Gabriel Bitton, for

his insight, encouragement, and enthusiasm during the

course of this study, and for his assistance in developing

this dissertation. The author is also grateful to the other

members of his committee, Dr. Thomas L. Crisman, Dr. W.

Lamar Miller, Dr. Seymour S. Block, and Dr. Ben L. Koopman.

Special thanks are extended to Dr. Koopman for providing

considerable advice and editorial guidance.

The author is indebted to Ms. Orna Agami for her

excellent technical assistance.

The author also wishes to thank his fellow students,

in particular, Ms. Judy Awong, for their support during the

course of this study.

This work was supported in part by funds provided by

grant No. CES-8619073 from the National Science Foundation

and grant Nos. WM152 and WM222 from the Florida Department

of Environmental Regulation.

Finally, the author acknowledges with gratitude his

parents, Mary and Adam Dutton, for their encouragement and

assistance, and his wife, Nana, for her love and

understanding.













TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS....................................... iv

ABSTRACT............................................... viii

CHAPTERS

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

2 LITERATURE REVIEW ................................ 5

Part I: Enzyme Biosynthesis...................... 5
Introduction................................... 5
Regulation of Enzyme Synthesis................. 5
Adaptive Enzymes............................... 8
Part II: Enzyme Biosynthesis as a Basis
for Toxicity Testing............................ 11
Inhibition of Enzyme Synthesis................. 11
Inhibition of the Synthesis of Enzymes
Controlled by Different Operons.............. 13
Part III: The Role of the Bacterial
Cell Envelope................................... 13
Introduction.................................... 13
Gram-positive Bacteria.......................... 14
Gram-negative Bacteria......................... 15
Part IV: Cell Envelope Alterations Affecting
Permeability to Toxicants...................... 18
Introduction.................................... 18
Growth Conditions................................ 19
Chemical Treatments.............................. 21
Physical Treatments.............................. 23
Genetic Alterations of the Outer Membrane...... 24
Conclusions...................................... 25

3 ENZYME BIOSYNTHESIS VERSUS ENZYME ACTIVITY
AS A BASIS FOR MICROBIAL TOXICITY TESTING...... 27

Introduction.... .............................. 27
Materials and Methods.............................. 29
Test Bacteria ................................. 29
Test Chemicals and Reagents.................... 30
Test Procedures................................ 31
Data Analysis.................................. 34










Results and-Discussion........................... 35
Comparison of Enzyme Activity and
Enzyme Biosynthesis Assays................... 35
Comparison Enzyme Biosynthesis to other
Toxicity Tests ............................... 38

4 INHIBITION OF 3-GALACTOSIDASE BIOSYNTHESIS IN
ESCHERICHIA COLI: A FUNCTION OF OUTER MEMBRANE
PERMEABILITY TO TOXICANTS...................... 41

Introduction...................................... 41
Materials and Methods.............................. 43
Test Bacteria.................................. 43
Test Chemicals and Reagents.................... 44
3-Galactosidase Biosynthesis Assay Procedures.. 45
Data Analysis.................................. 47
Physical Treatments.............................. 47
Chemical Treatments.............................. 49
Results and Discussion............................. 51
Preliminary Experiments........................ 51
Mutants with Possible Outer Membrane
Alterations.................................. 54
Physical Treatments.............................. 56
Chemical Treatments.............................. 62

5 ASSESSMENT OF 3-GALACTOSIDASE BIOSYNTHESIS:
TOXICITY TESTING IN WATER AND WASTEWATER........ 69

Introduction...................................... 69
Materials and Methods.............................. 71
Sampling and Activated Sludge Treatment......... 71
Test Bacteria .................................. 71
Test Chemicals and Reagents.................... 71
3-Galactosidase Biosynthesis Assay Procedures.. 72
Polymyxin Treatment............................ 74
Microtox and Ceriodaphnia dubia Bioassays...... 74
Expression of Wastewater Toxicity............... 75
Results and Discussion............................ 75
3-Galactosidase versus Microtox and
C.dubia Bioassays: Effect of Selected
Chemicals....... ............................ 75

6 INHIBITION BIOSYNTHESIS OF ENZYMES CONTROLLED BY
DIFFERENT OPERONS: A COMPARISON OF
3-GALACTOSIDASE, a-GLUCOSIDASE AND
TRYPTOPHANASE................................... 80

Introduction...................................... 80
Materials and Methods.............................. 82
Test Bacteria.................................. 82
Test Chemicals ................................ 82
General Assay Protocols......................... 82
Assay for 3-Galactosidase Biosynthesis.......... 83
Assay for Tryptophanase Biosynthesis........... 83










Assay for a-glucosidase Biosynthesis........... 85
Data Analysis....... ........................... 87
Results and Discussion....... .................... 87
Relative Sensitivity of Different Operons to
Toxicants...................................... 87

7 CONCLUSIONS...................................... 92

APPENDICES

A MINIATURIZATION OF 3-GALACTOSIDASE
BIOSYNTHESIS ASSAY.............................. 95

B AN ALTERNATE SUBSTRATE FOR 3-GALACTOSIDASE
DETERMINATION................................. 98

C DEVELOPMENT OF OTHER ENZYME BIOSYNTHESIS
TOXICITY TESTS ................................ 100

REFERENCES..... ......... ............................... 104

BIOGRAPHICAL SKETCH.................................... 116


vii














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


ENZYME BIOSYNTHESIS IN BACTERIA
AS A BASIS FOR TOXICITY TESTING


By

Ronald John Dutton


December 1988



Chairman: Gabriel Bitton
Major Department: Environmental Engineering Sciences

Toxicity assessment of hazardous materials and toxic

wastewaters is greatly facilitated by the availability of

inexpensive short-term toxicity tests. The purpose of

this research was to investigate rapid toxicity assays

based on the inhibition of inducible enzyme biosynthesis in

bacteria.

An assay based on the inhibition of P-galactosidase

biosynthesis was compared to a similar assay based on the

inhibition of 3-galactosidase activity. In both tests,

Escherichia coli were induced to synthesize 3-galactosidase

by exposure to isopropyl-f-thiogalactoside (IPTG). The

induction step preceded contact of the cells with toxic

chemicals in the enzyme activity assay, whereas in the

enzyme biosynthesis test, IPTG was added following


viii









contact of cells with the toxicant. Relative sensitivity

was judged on the basis of responses to heavy metals and

organic toxicants of environmental importance. Comparison

of these results to median inhibitory concentration data

(IC50s) achieved with other microbial systems, Daphnia

bioassay, and fish bioassay indicate that the enzyme

activity test was sensitive to heavy metals, but was

insensitive to organic toxicants. The test based on

inhibition of P-galactosidase biosynthesis was sensitive to

both heavy metals and organic.

It was found that if wild type E. coli were subjected

to physical and chemical treatments known to alter the

outer membrane, the sensitivity of the P-galactosidase bio-

synthesis test to surface active and hydrophobic chemicals

could be significantly increased. The use of E. coli

(EWlb) with an outer membrane mutation (TolC gene),

sensitized further by chemical treatment with polymyxin (2

mg/L), resulted in an improved bioassay for chemical and

wastewater toxicity.

Toxic inhibition of 3-galactosidase and tryptophanase

biosynthesis, both in E. coli, and of a-gaucosidase

biosynthesis in Bacillus licheniformis was also

investigated. The response of a-glucosidase biosynthesis

to selected toxicants was the most sensitive. Disparity in

sensitivity between the biosynthetic systems in E. coli and

B. licheniformis may be a function of differences in cell

permeability to the test chemicals.















CHAPTER 1
INTRODUCTION



Production of synthetic chemicals has greatly

escalated since World War II. It was estimated that there

are approximately 100,000 chemicals in commerce with

about 1000 new ones added annually (McCutcheon, 1980).

It is important to assess the risks posed by these

potentially toxic substances to the environment. Current

concerns include the effects of acid precipitation,

widespread leaching of waste disposal sites, and point

and non-point source discharges to natural waters.

Protection of the environment is mandated by various

federal laws (e.g., Clean Air Act, Clean Water Act,

Resource Conservation and Recovery Act). Assessment of

the risks to the environment involves obtaining

information on (1) the concentration of specific

toxicants, (2) the source and fate of the toxicants, (3)

the potential target species which may be harmed by the

toxic effect, and (4) toxicity data for the pertinent

species (Camougis, 1985).

The "workhorse" in monitoring pollution effects has

been the acute toxicity test (Buikema et al. 1982).

Information generated from toxicity tests can be used to

predict .environmental effects of toxicants; compare










toxicants, test conditions, or test species; and regulate

the environmental discharge of the toxicants.

Toxicity tests employing a variety of organisms are

presently applied to detect toxic chemicals (McFeters et

al. 1983). Toxicity tests using bacteria began to

receive serious consideration after the realization that

a microbial bioassay, for the detection of mutagens (the

Ames Test), proved to be useful in screening for

potential animal carcinogens (Ames et al., 1975; Devoret,

1979).

The basis for the use of microorganisms in toxicity

testing includes the following: (1) they possess the

majority of the same biochemical pathways present in

higher organisms, (2) they exhibit a significantly

organized membrane structure, (3) they play an

instrumental role in nutrient cycling, and (4) they

represent the first level at the base of the food chain.

Endpoints of microbial toxicity tests are based on the

measurement of growth inhibition, oxygen uptake, heat

production, substrate uptake, ATP content,

bioluminescence, and enzyme activity (Bitton, 1983; Liu

and Dutka, 1984; Bitton and Dutka, 1986; Dutka and

Bitton, 1986).

In classical enzyme studies it is conventional to

study the kinetics of enzyme. inhibition with known

metabolic inhibitors. It logically followed that

environmental scientists would apply this principle to










assess the toxicity of environmental toxicants (e.g.,

insecticides versus acetylcholinesterase). Consequently,

short-term enzyme assays (in vitro and in vivo) for

toxicity screening were developed using dehydrogenase,

lipase, luciferase, esterases, carbonic anhydrase,

ribonuclease, urease, acetylcholinesterase, and ATPase,

(Christensen et al., 1982; Bitton, 1983; Obst et al.

1988).

Little attention has been given to the effect of

toxic chemicals on enzyme biosynthesis. This approach per

se should be more sensitive than enzyme inhibition,

because both inhibition of enzyme formation (protein

synthesis) and inhibition of enzyme activity are

measured concurrently. Short-term toxicity screening

tests based on inhibition of bacterial enzyme synthesis

should be feasible because of the rapid kinetics of

enzyme biosynthesis in bacteria.

This dissertation assesses the effectiveness of

enzyme biosynthesis in bacteria as a basis for toxicity

testing. This task was approached in the following

steps: (1) the response of (-galactosidase activity

versus P-galactosidase biosynthesis to selected toxic

chemicals was compared, (2) the inhibition of

P-galactosidase biosynthesis in Escherichia coli was

addressed more fully, with a detailed consideration of

cell permeability to toxicants, (3) the response of

enzyme biosynthesis to toxic wastewaters was examined,







4


and (4) the inhibition of P-galactosidase, tryptophanase,

and a-glucosidase biosynthesis to selected toxic

chemicals was compared.














CHAPTER 2
LITERATURE REVIEW



Part I: Enzyme Biosynthesis

Introduction

Prokaryotic and eukaryotic cells contain complex

regulatory mechanisms that control the concentration of

their enzymes. Enzymes may be synthesized continuously

("constitutive enzymes") or only in response to a specific

stimuli ("adaptive enzymes"). The regulation of enzyme

synthesis is perhaps the most important level of metabolic

control because it determines whether or not a biosynthetic

or catabolic reaction pathway may in fact operate (Walker,

1983).



Regulation of Enzyme Synthesis

Bacteria have adapted to survive and proliferate in

environments with widely fluctuating carbon and nitrogen

sources. The bacterial cells must be able to derive as

much energy as possible from sporadically available

nutrients (Walker, 1983). Catabolic and biosynthetic

pathways mediate these adaptive processes. Bacterial cells

switch all the enzymes in a pathway on or off, and the

genes coding for these enzymes are usually coordinated in a

sequence referred to as an operon.











The functioning of an operon involved in enzyme

synthesis is summarized in the following steps: (1)

inactivation of repressor (e.g., induction), (2) RNA

transcription from DNA template (operon region) forming

precursor mRNA's, (3) maturing/processing of mRNA, (4)

translation of mature mRNA into enzyme molecules at the

ribosomes, and (5) formation of active sites and other

steps necessary for full enzyme activity (activation).

The first definitive model of the genetic regulatory

control of enzyme synthesis was the lactose operon (Jacob

and Monod, 1961). The research culminated in a nobel prize

for the French investigators Jacques Monod, Francois Jacob,

and Andres Lwoff, in 1965. The genetic regulatory control

of the lactose operon is depicted in Figure 2-1.

In response to addition of lactose (or synthetic

lactose analogs), the lactose operon in Escherichia coli

increases the number of 3-galactosidase molecules by a

factor of 1000 within 1-2 hours. This factor is a function

of enzyme synthesis within the cells and the increase in

cells due to growth. In contrast, the induction of

tryptophan oxygenase in rat liver (by tryptophan) leads to

an eightfold increase in enzyme over a period of 6 hours.

These differences in rate and amount are typical of the

responses in bacterial and animal cells (Walker, 1983).


I




























repressor ALLOLACTOSE
protein


P o


120 3500 760 810


genome


polycistronic
mRNA


/3 -galactoside
/3 -galactosidase permease

\ N
\ "-N


galactoside
transacetylase


galactose + glucose < allolactose <- lactose


Sacetyl Co
N -j


LACTOSE


cell membrane


Figure 2-1. The pathway of lactose catabolism and
regulation by the the lactose operon.


-1










Adaptive Enzymes

Adaptive enzymes are classified as induciblee" or

repressiblee" (Braun, 1965; Brock, 1979). Enzyme

induction was defined as "a relative increase in the rate

of synthesis of a specific enzyme resulting from exposure

to a chemical substance" (Cohn et al., 1953). Each

substrate induces the synthesis of the enzymes necessary

for its own catabolism. For example, the presence of the

substrate lactose induces production of B-galactosidase,

which degrades lactose to galactose and glucose.

Enzyme repression is defined as "a relative decrease,

resulting from the exposure of cells to a given substance,

in the rate of synthesis of an specific enzyme" (Vogel,

1957). When a particular substance is absent, the specific

enzyme is derepressed and synthesized. For example, the

absence of phosphate activates (derepresses) synthesis of

alkaline phosphatase. In both enzyme induction and enzyme

derepression, the net result is that a repressor in the

operon region of the geonome is diverted from its

"clogging" function, and enzyme biosynthesis is initiated

(Braun, 1965). Examples of bacterial operons and various

adaptive enzymes are given in Tables 2-1 and 2-2.

Many microbial carbon sources (e.g., lactose,

galactose, arabinose, maltose, and a number of trioses and

pentoses) are catabolized by inducible enzymes.














TABLE 2-1. Some operons in bacteria



Operons Number of Function
genes



lac 3 transport and hydrolysis of 3-galactoside

gal 4 conversion of galactose to glucose-l-
phosphate

hut 4 histidine utilization

leu 4 conversion of a-ketoisovalerate to
leucine

ara 5 transport and utilization of arabinose

bio 5 synthesis of biotin

trp 5 synthesis of tryptophan

arg 8 synthesis of arginine

his 9 synthesis of histidine



Source: adapted after Lehninger (1982) and Walker (1983).






10







TABLE 2-2. Examples of some adaptive enzymes


Enzyme Microorganism Inducer/repressor



P-galactosidase Escherichia coli lactose, IPTG

galactokinase E. coli fucose

tryptophanase E. coli tryptophan

alk. phosphotase E. coli phosphate

a-amylase Bacillus spp. starch

a-glucosidase Bacillus spp. maltose

(-lactamase Bacillus spp. penicillin

glucose isomerase B. coagulans D-xylose

histidase Klebsiella aerogenes uronic acid

pullanase Aerobacter spp. maltose

urease Proteus rettgeri urea

tryptophan Pseudomonas spp. kynurenine
oxygenase



Source: adapted after Demain (1971), Wang et al. (1979),
and Reed (1982).









Inducible catabolic enzymes are expressed only after carbon

sources of a higher energy value have first been depleted,

thus economizing cell energy expenditure. This phenomenon

(e.g., repression of 3-galactosidase biosynthesis in the

presence of glucose) is known as "catabolite repression"

(Brock, 1979).

Enzyme regulation in biosynthetic pathways (for amino

acids, biotin, thiamine, other vitamins) is controlled

primarily by repressible enzymes in analogous systems to

those regulating catabolite operons (Wang et al., 1979;

Walker, 1983). Upon induction catabolic enzymes have

been observed to increase several thousandfold in

activity, while hundredfold changes have been observed in

the specific activity of enzymes in biosynthetic pathways

(Demain, 1971).



Part II: Enzyme Biosynthesis as a Basis
for Toxicity Testing


Inhibition of Enzyme Biosynthesis

In the first studies to examine the effect of

antimicrobial agents on enzyme biosynthesis, Monod (1944;

1947) showed that 2,4-dinitrophenol and chloramphenicol

inhibited 3-galactosidase biosynthesis. Hahn and Wisseman

(1951) later demonstrated that chloramphenicol, aureomycin,

terramycin, and acridine inhibited metabolism of lactose,

arabinose, maltose, and acetate. They attributed this to

inhibition of adaptive enzyme formation. Inhibition of


1









3-galactosidase formation by selected antimicrobial agents

was also demonstrated by Koch (1964) and Nakada and

Magasanik (1964). Kaminski (1963) showed that

staphylococcal penicillinase formation was completely

inhibited by anionic detergents at concentrations (1.0

mg/L) that did not inhibit growth. Kucera et al. (1965)

observed inhibition of staphylococcoal penicillinase

formation by 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T)

at 100 mg/L.

More recently, Naveh et al. (1984) and Ulitzur (1986)

have developed a bioassay for antibiotics that inhibit

protein synthesis, employing a dark mutant luminescent

bacterium (Photobacterium leiognathi). The bacterial

mutant reverts back to luminescence in the presence of DNA-

intercalating agents. The bacteria are exposed to test

samples (potential protein synthesis inhibitors) and then

the luminescence system is induced with a mutagen (e.g.,

proflavin). The relative change in light production is

measured with a photometer.

Cenci et al. (1985) demonstrated that heavy metals

inhibit P-galactosidase synthesis in E. coli. And

Reinhartz et al. (1987) examined the effect of several

common pollutants on the synthesis of P-galactosidase. The

assay was developed as a commercial toxicity test kit using

freeze-dried E. coli (Toxi-Chromotest, Orgenics Ltd.,

Yavne, Israel).


1











Inhibition of the Synthesis of Enzymes Controlled by
Different Operons

The preferential inhibition of enzyme synthesis by

chloramphenicol was examined by Sypherd et al. (1962).

Sublethal amounts of chloramphenicol (0.8g/mL) inhibited

the synthesis of P-galactosidase, galactoside permease,

tryptophanase, and citritase by 68%, 64%, 70% and 58%,

respectively. However, chloramphenicol at this

concentration failed to inhibit D-serine deaminase and any

constitutive enzyme activity (e.g., hexokinase, glucose

dehydrogenase, acid phosphatase, and pyrophosphatase).

Pollock (1963) showed that actinomycin D (0.05ug/mL)

will halt a-glucosidase induction without affecting

penicillinase induction in B.subtilis. Furthermore,

inhibition of alkaline phosphatase, a repressible

periplasmic enzyme, was about five times more sensitive

than 5-galactosidase induction in E. coli exposed to

procaine hydrochloride (Tribhuwan and Pradhan, 1977).



Part III: The Role of the Bacterial Cell Envelope
in Resistance to Toxicants

Introduction

Gram-positive and Gram-negative bacteria all produce

cell walls (with the exception of mycoplasmas) that

surround an inner cytoplasmic membrane. Peptidoglycan or

murein is a common component of these walls, conferring






14


mechanical rigidity to the cells (Nikaido and Vaara, 1985).

All Gram-negative bacteria contain an additional layer, the

outer membrane, composed of phospholipids,

lipopolysaccharide (LPS), and protein (Nakae, 1986). Both

Gram-positive and Gram-negative bacteria may also be

surrounded by globular protein coats or extensive fibrillar

carbohydrate capsules (Costerton and Cheng, 1975). These

capsules may play important roles in pathogenesis or

adhesion and are a potential diffusion barrier to chemicals

(Godfrey and Bryan, 1984).



Gram-positive Bacteria

Peptidoglycan is the major component of the Gram-

positive cell wall, forming a thick fibrous structure with

a mainly negative charge (Costerton and Cheng, 1975). The

cell wall is usually interspersed with covalently linked

teichoic and teichuronic acid polymers that do not form

coherent or continuous structures. The Gram-positive cell

wall with its net negative charge is analogous to an ion

exchange bed that excludes very large molecules and may

adsorb positively charged particles onto structural

polymers (Costerton and Cheng, 1975). The main bulk of the

Gram-positive cell wall probably does not act as barrier to

toxicants since its molecular seiving function is

restricted to molecules larger than 100,000 daltons

(Godfrey and Bryan, 1984). In addition, as Gram-positive

bacteria do not have an outer membrane, they tend to be








more sensitive than Gram-negative bacteria to hydrophobic

compounds such as detergents, dyes, and certain antibiotics

(Koch and Schaechter, 1985).



Gram-negative Bacteria

The outer membrane structure of Gram-negative bacteria

is very complex (Figure 2-2). The cell wall consists of a

thin peptidoglycan layer (0.8nm to 30nm thick) that does

not contain teichoic acids, but is covalently linked to

lipoprotein molecules (Costerton and Cheng, 1975). External

to the cell wall, Gram-negative bacteria have a complex

outer membrane composed of phospholipids, lipopoly-

saccharides (LPS) and proteins.

Most studies conducted on the molecular structure and

permeability of the Gram-negative outer membrane have

focused primarily on enteric bacteria. The resistance of

these organisms to antibiotics has been addressed in

several excellent reviews (Costerton and Cheng, 1975;

Godfrey and Bryan, 1984; Hancock, 1984; Nikaido and Vaara,

1985; Nakae, 1986).

Phospholipids. A phospholipid bilayer is the first

layer external to the peptidoglycan cell wall, and is very

similar structurally to the inner cytoplasmic membrane.

(Nikaido and Vaara, 1985). The phospholipid content of the

outer membrane is much less than in the


j 1
























0










OuterI
membrane V I

memfrarane
LEGEND Perplasmic space
9Protein "^ ^^ ^

L LPoopolyosaccharide a r

0 Liplds Cytoplasm

e ULpoprotein








Figure 2-2. The structure of the Gram-negative cell wall
and membrane layers. (From Godfrey and Bryan,
1984; reprinted by permission; copyright
Academic Press).





17


cytoplasmic membrane. But like its inner counterpart, it

is an effective barrier to hydrophilic compounds (Koch and

Schaechter, 1985).

Proteins. About half the mass of the outer membrane

is made of proteins (Nikaido and Vaara, 1985; Nakae, 1986).

The proteins of the outer membrane are embedded in the

phospholipid bilayer. In E. coli K12 the major proteins

are OmpA, OmpF, and OmpC, and murein lipoprotein. In E.

coli B OmpC is missing (Mizushima, 1987).

The murein lipoprotein covalently anchors the outer

membrane to the underlying cell wall. The other major

membrane proteins (Omp proteins) form water filled channels

called porins. The porins allow small hydrophilic

substances to penetrate through the outer membrane. Certain

minor membrane porin proteins may be important in enhancing

specific solute uptake: LamB (maltose), PhoE (phosphate,

other anions), Tsx (nucleotide), TonA (Fe3+), BtuB (vitamin

B12) (Nakae, 1986). The size of the major porins in

enteric bacteria are about 1.1-1.2 nm, allowing substances

smaller than 600-800 daltons to diffuse through. In

Pseudomonas aeroginosa the porin diameter is about 2 nm

(Nikaido and Vaara, 1985).

Lipolysaccharide (LPS). The LPS forms the external

fringe of the outer membrane and is composed of hydrophilic

oligosaccharide segments and a covalently bonded

hydrophobic segment, called lipid A, that anchors the LPS

to the underlying outermembrne phospholipid (Koch and










Schaechter, 1985; Naikaido and Vaara, 1985; Raetz, 1987).

The LPS constitutes about 20% of the outer membrane by

weight (Nakae, 1986). Adjacent LPS molecules are apparently

stabilized covalently by Mg2+ cross-bridging (Leive, 1974;

Nikaido and Vaara, 1985). It is the LPS, along with the

O-antigen component in enterics, that give Gram-negative

bacteria a hydrophilic coat that effectively excludes

hydrophobic substances (Koch and Schaechter, 1985).

In summary, Gram-negative bacteria have external to

their cell wall a complex outer membrane. The outer

membrane has an external fringe (LPS) that is hydrophilic,

and excludes hydrophobic substances. Inside the LPS, the

outer membrane is composed of a lipoprotein and protein

matrix that create water filled channels called porins. In

enteric bacteria, hydrophilic substances are excluded above

600-700 daltons due to the channel size of the porins. In

addition, Gram-negative bacteria, particularly enteric

bacteria, have evolved an outer membrane defense to

hydrophobic chemicals. The implications of this

permeability barrier in Gram-negative bacteria must be

considered when conducting toxicity tests.



Part IV: Cell Envelope Alterations Affecting
Permeability to Toxicants

Introduction

The composition of the bacterial cell envelope is

affected directly by the conditions of the surrounding






19


environment. The integrity of the cell envelope depends on

nutritional, chemical, physical, and genetic factors.

Manipulation or changes in these parameters can result in

altered cell permeability to antibiotics, dyes, detergents,

substrates, and other chemicals (Leive, 1968; Sinskey and

Silverman, 1970; Unemoto and Macleod, 1975; Nikaido, 1976;

Hitchener and Egan, 1977; Bennett et al., 1981; Huoang et

al. 1983; Mackey, 1983; Hancock and Wong, 1984; Hancock,

1984; Brown and Williams, 1985).



Growth Conditions

Growth conditions can significantly alter the outer

membrane of Gram-negative bacteria (Brown and Williams,

1975; Beuchat, 1978). Ionic strength and osmotic balance

due to magnesium, sodium, and potassium affect the

stability of the outer membrane. Alterations in the outer

membrane resulting from growth in phosphate and magnesium-

limited cultures are associated with increased resistance

to EDTA and polymyxin (Finch and Brown, 1975; Brown and

Melling, 1969), cationic proteins (Finch and Brown, 1978),

and cold shock (Kenward and Brown, 1978). Gilbert and

Brown (1980) found that E. coli cultures became sensitized

to chlorophenol and phenoxyethanol when carbon was

limiting. This was not the case with magnesium or

phosphate-limited cultures.

Pseudomonas fluorescens grown on glucose show higher

resistance to actinomycin D and EDTA than cells grown on










succinate (Walker and Durham, 1975). Oxygen consumption in

E. coli was inhibited in the presence of phenol and

phenoxyethanol when succinate, pyruvate or acetate were

used as substrates, but was stimulated if the substrate was

glucose, mannitol, or lactose (Hugo and Street, 1952).

This was explained partly in terms of intracellular or

membrane associated enzymes for the different substrates

(Hugo, 1967).

Analogous responses to media composition may also

occur in Gram-positive bacteria. Bacillus megaterium

grown in magnesium and carbon-limited media became

sensitized to chlorhexidine and phenoxyethanol with

increasing growth rate, while phosphate-limited cultures

were not (Gilbert and Brown, 1980). Sensitivity was

increased two-fold for phenoxyethanol and up to ten-fold

for chlorhexidine. Protoplasts of these cultures were

sensitized to a similar extent indicating that alterations

in cell wall permeability were involved.

Gram-positive bacteria grown in glycerol to enhance

cell lipid content, or in biotin-deficient media to deplete

lipids, showed altered resistance to antimicrobial agents

(Hugo and Stretton, 1966; Hugo and Franklin, 1968; Hugo and

Davidson, 1973). Lipid "fattened" staphylococci (18% cell

lipid increase) were resistant to phenols with side chains

longer than 4 carbon groups. Uptake studies suggested that

the larger phenols were bound in the cell lipid fraction.










Chemical Treatments

Introduction. Various chemical treatments have been

shown to alter cell permeability (e.g., detergents,

Miozzari et al., 1972, Arnold and Johnson, 1982; solvents,

Beuchat, 1978, Dobrogosz, 1981, Chaudary, 1984;

antibiotics, Nakao et al., 1973; calcium chloride,

Bezinger, 1978; EDTA, Leive, 1968; tris(hydroxymethyl)-

aminomethane (Tris), Irvin et al., 1981; and polycations,

Vaara and Vaara, 1983, Hancock, 1984). An important aspect

of these treatments is the change in permeability and

whether or not this change impairs metabolism. It is

critical to this study that enzyme biosynthesis proceeds

following a particular sensitizing treatment. Several

treatments are discussed in more detail below.

EDTA treatment. Ethylenediaminetetracetate (EDTA)

treatment (10-4M) in Tris buffer results in up to 50%

release in the LPS of E. coli, an increase in permeability

to antibiotics (Leive, 1968; Russel et al., 1973), and an

increase in cell hydophobicity (Mackey, 1983). It was

postulated that EDTA disorganizes the outer membrane by

chelating Ca2+ and Mg2+ in the LPS (Leive, 1974). However,

cells treated with EDTA and subsequently grown in media

were able to repair outer membrane damage in less than one

hour (Scudamore et al., 1979).

Tris treatment. Tris buffer appears to permeabilize

the cell envelope (Irvin et al., 1981). A mutant E. coli









strain treated with Tris buffer for 1.5-2.5 hours released

the periplasmic enzyme alkaline phosphatase and was

sensitized to lysozyme. Tris treatment at 40C, but not at

370C, has been shown to release the cell nucleotide pool

as indicated by increased absorbance of cell supernatant at

260 nm (Leive and Kollin, 1967). The release of the

nucleotide pool induces RNA breakdown and impairs metabolic

processes. Therefore in permeability studies where

metabolic studies are important, washing with Tris should

be at room temperature.

Polycation treatment. Growth in subinhibitory

concentrations of a number of polycationic agents (e.g.,

protamine, lysine, spermine, streptomycin, lysozyme,

amikacin, gentamicin, and polymyxin (Vaara and Vaara, 1983;

Walker and Beveridge, 1987) sensitizes Gram-negative

bacteria to hydrophobic antibiotics (Vaara and Vaara,

1983). Polymyxin B appears to be the most effective

polycationic agent, sensitizing Salmonella typhimurium and

E. coli to detergents and hydrophobic antibiotics (Vaara

and Vaara, 1983).

Polymyxins are antibiotics produced by Bacillus

polymyxa. They are characterized by a high molecular weight

(1000-1200), a heptapeptide ring, a high content of

diaminobutyric acid, and a side chain ending in a

methyloctanoic acid residue (Godfrey and Bryan, 1984).

Unlike many polycations, polymyxin does not release LPS. It

appears that polymyxin causes rodlike projections or blebs










in the outer membrane (Lounatmaa et al., 1976). Polymyxin

interacts with the LPS and its action may be due to

competitive displacement of divalent cations that stabilize

the LPS (Schindler and Osborn, 1979; Hancock, 1984). The

toxicity of polymyxin is high, and to maintain viability

while still altering permeability, it should be used at

about 1 mg/L (Vaara and Vaara, 1983).

Categories of chemicals that increase outer membrane

permeability have been reviewed by Hancock (1984), Hancock

and Wong (1984), and Walker and Beveridge (1987). These

chemicals can be classified into three groups: (1) divalent

cation chelators such as EDTA and nitrilotriacetate, (2)

polycations such as aminoglycosides (e.g., polymyxin), and

(3) large monovalent cations such as cetrimide and Tris.

The permeabilizers with positive charge compete with and

displace membrane-stabilizing Mg2+ and Ca2+ ions in the

LPS.



Physical Treatments

The treatment of Gram-negative bacteria by physical

means such as heating, freeze-thawing, and freeze-drying

also causes perturbations in the outer membrane, thus

affecting cell permeability (Bennett et al., 1981; Mackey,

1983; Hitchener and Egan, 1977; Sinskey and Silverman,

1970; Ray and Speck, 1973).

Heating. Escherichia coli subjected to heating at

48C and treated with EDTA released up to 50% of the LPS.


1









The addition of Mg2+ (5mM) to the heating medium protected

the cells from injury (Hitchener and Egan, 1976).

Tsuchiado et al. (1985) reported that heat treatment of E.

coli at 550C in Tris buffer (pH 8) sensitized the cells to

crystal violet and the action of phospholipase.

Freeze-injury. Freeze-injury in bacteria was reviewed

by Ray and Speck (1973). Freeze-thawed microorganisms were

found to be sensitized to surfactants, lysozyme, UV

radiation, and selected metabolic inhibitors. Important

factors in cell death during freeze storage are cellular

crystal formation and prolonged exposure to concentrated

solutes (Ray and Speck, 1973).

Freeze-drying is similar to freeze-thawing although

freeze-dried cells stored under vacuum are viable for much

longer periods of time than frozen cells because

dehydration is beneficial for storage (Beuchat, 1978).

Sinskey and Silverman (1970) demonstrated that freeze-dried

E. coli were susceptible to antibiotics at concentrations

that were normally ineffective. Both freeze-thawed and

freeze-dried cells can repair permeability damage in

nutrient media (Sinskey and Silverman 1970; Ray and Speck,

1973).



Genetic Alterations of the Outer Membrane

Microorganisms with defined mutations which alter

cellular permeability lack or make reduced amounts of

structural cell envelope components. This subject has been










reviewed in regards to Gram-negative bacteria (Hancock,

1984). Information regarding Gram-positive mutants is

scarce. However, "rough" colonial variants of Lactobacillus

acidophilus were shown to have non-stainable blebs

protruding from the cell wall and were more sensitive to

freeze-damage and bile salts than "smooth" colonies

(Klaenhammer and Kleeman, 1981).

In Gram-negative bacteria, a large number of mutants

with altered outer membrane permeability have been isolated

(Hancock, 1984). Mutants have been isolated with

alterations of porins, LPS, and lipoprotein. Porin-

deficient mutants have been shown to have higher resistance

to hydrophilic antibiotics in some cases (Hancock, 1984).

Mutants lacking porin proteins apparently compensate by

creating phospholipid patches, creating a pathway for

hydrophobic substances (Nikaido and Vaara, 1985). Deep-

rough organisms with LPS mutations have been shown to have

hyper-susceptibility to hydrophobic antibiotics, deter-

gents, and EDTA (Hancock, 1984). Other outer membrane gene

mutations include: tolA,B (colicin tolerant), tolC (OmpF

protein), TolD,E (LPS), acrA (lipid A phosphate), and Ipo

(Braun lipoprotein).

In summary, enzyme biosynthesis in microorganisms has

been shown to increase enzyme activity by several

thousandfold in a matter of hours. The regulation of enzyme

synthesis determines whether biochemical pathways may

proceed and therefore is among the most important levels of










metabolic control. Microbial enzyme synthesis as a tool in

assessing chemical toxicity has not received widespread

attention.

Cell permeability to toxicants is a critical factor

determining the sensitivity of microbial toxicity

bioassays. In Gram-negative bacteria, the cell envelope

limits the diffusion of hydrophilic and hydrophobic

chemicals. Physical, chemical, or genetic manipulations

that alter cell permeability may increase the sensitivity

of microbial toxicity tests.













CHAPTER 3
ENZYME BIOSYNTHESIS VERSUS ENZYME ACTIVITY
AS A BASIS FOR MICROBIAL TOXICITY TESTING



Introduction

A common endpoint measurement employed in toxicity

assessment is the inhibition of enzyme activity (see

Bitton, 1983; Bitton and Dutka, 1986; Dutka and Bitton,

1986, for reviews). For example, Christensen et al. (1982)

determined the effects of 141 water pollutants and other

chemicals on the activity of eight enzymes in vitro. At an

upper concentration limit of 10-2M, 115 (81.6%) of the

chemicals were inhibitory to one or more of -the enzymes

tested. In addition to screening for toxicity, enzyme

activity assays may give insight into the mechanisms of

action of toxic chemicals.

An alternative approach in toxicity testing is to

measure inhibition of enzyme biosynthesis. In bacteria,

certain adaptive or inducible enzymes are kept at low

levels and energy for producing substantial amounts of

these catalysts is conserved until such time as the

required substrate becomes available or a repressor is

removed. This phenomenon is known as enzyme induction or

de novo enzyme biosynthesis. The classic model of an

inducible enzyme is 3-galactosidase (Jacob and Monod,

1961).









In Escherichia coli, P-galactosidase is produced by a

cluster of genes known as the lactose operon.

The production of this enzyme in most wild-type E. coli

strains is induced by the presence of lactose or synthetic

lactose analogs such as isopropyl-P-D-thiogalactoside

(IPTG). The inducers modify a repressor protein allowing

RNA polymerase transcription of the lac genes. In

addition, the presence of optimal levels of cAMP are needed

to function with the operon promoter site (Dobrogosz,

1981).

Several studies have dealt with the inhibition of 3-

galactosidase activity (Lederberg, 1950; Katayama, 1984;

Katayama, 1986) as well as inhibition of P-galactosidase

biosynthesis (Koch, 1963, Nakada and Magasanik, 1964). The

scope of these studies was limited to assessing the effects

of heavy metals and certain well known metabolic poisons.

Reinhartz and coworkers first suggested the use of 3-

galactosidase biosynthesis to assess the toxicity of

environmental pollutants (Reinhartz et al., 1987).

The purpose of the present research was to compare the

response of E. coli to selected chemicals, using microbial

toxicity assays based on P-galactosidase activity and 3-

galactosidase biosynthesis, respectively. Emphasis was

placed on differentiating between inhibition of existing

enzyme activity and inhibition of potential enzyme

biosynthesis. Relative sensitivity was judged on the basis

of responses to the heavy metals Hg2+, Cu2+ and Cd2+ and









the organic 3,4-dicchlorophenol (3,4-DCP), formaldehyde,

Hydrothol, phenol, sodium dodecyl sulfate (SDS), and

toluene. These results were compared to IC50s achieved

with other microbial systems, Daphnia bioassay and fish

bioassay.



Materials and Methods



Test Bacteria

The culture maintenance and enzyme assay procedures

followed closely those of Miller (1972). Escherichia coli,

strain C3000 (ATCC# 15597), was maintained in 40% glycerol

at -150C. Cells were grown by inoculating 50 mL minimal

media in a 125 mL Erlenmeyer flask with 50 uL of glycerol

culture. The bacteria were incubated at 350C in a shaking

water bath with an oscillation rate of 100 rev/min. They

were harvested in the log growth phase, having reached an

optical density of 0.2-0.4 (420 nm, light path = 1.0 cm).

The minimal media contained the following

constituents: K2HPO4, 10.5 g/L; KH2PO4, 4.5 g/L; (NH4)2SO4,

1.0 g/L; sodium citrate 2H20, 0.5 g/L; yeast extract, 0.5

g/L; MgSO4, 1 mL from a 20% stock solution; and glycerol,

10 mL from a 20% stock solution. The magnesium and glycerol

solutions were autoclaved separately from the salt

solution.










Test Chemicals and Reagents

Distilled water used in preparing chemical and reagent

solutions was sterilized by autoclaving. The chemicals

assessed for toxicity were: Hg2+ (HgCl2), Cu2+

(CuSO4*5H20), Cd2+ (CdC12), phenol, 3,4-dichlorophenol

(3,4-DCP), sodium dodecyl sulfate (SDS), formaldehyde,

toluene, and the aquatic herbicide Hydrothol (Pennwalt

Corporation, Philadelphia, PA). Hydrothol is the trade

name for the alkylamine salt of 7-oxabicyclo[2.2.l]heptane-

2,3-dicarboxylic acid. Stock solutions of heavy metals,

phenol, 3,4-DCP, SDS, formaldehyde, and hydrothol were

prepared in distilled water. Toluene was dissolved in

dimethylsulfoxide (DMSO).

Unless otherwise indicated, all chemicals and reagents

were obtained from Sigma Corporation (St. Louis, MO). IPTG

was prepared by dissolving 50 mg reagent in 50 mL distilled

water. Ortho-nitrophenyl-B-D-galactopyranoside (ONPG) was

prepared by dissolving 400 mg reagent in 100 mL distilled

water.

The buffer solution for the assay of enzyme activity

(Z-buffer) contained the following components:

Na2PO4-7H20, 16.1 g/L; NaH2PO4"H20, 5.5 g/L; KC1 0.75 g/L;

and MgSO4-7H20, 0.25 g/L. Other reagents employed in the

assays were: SDS, 0.1% (w/v); chloroform; and Na2CO3, 1M.











Test Procedures

Protocols for the enzyme activity and enzyme

biosynthesis assays are shown in Figures 3-1 and 3-2,

respectively. Each of the assays had the same basic steps:

grow cells in minimal medium, induce cells to produce 3-

galactosidase, and measure P-galactosidase. The protocols

differed with regard to the point in the sequence at which

cells were exposed to the toxicants. In the enzyme

activity assay, cells were induced before the exposure

step. Enzyme activity was therefore high at the beginning

of the toxicant contact period. A decrease of enzyme

activity relative to the controls during this period could

be attributed mainly to toxicant action on the enzyme. In

the enzyme biosynthesis test, induction followed the

exposure of cells to the toxicants. Enzyme activity at the

beginning of the toxicant contact period was therefore

negligible. At the end of the contact period, the inducer

was added and additional time was allowed for biosynthesis

of 3-galactosidase. Differences between final enzyme

levels (as measured by activity) were attributed mainly to

toxicant effect on the lac operon, rather than on the

enzyme itself.

All incubations were carried out at 350C in a shaking

water bath. Controls received distilled water in lieu of

toxicant. When toluene was the toxicant under

investigation, the control consisted of 5% DMSO. In the














ENT ZYME
ACTIVITY ASSAY



STEP 1. CELL GROWTH:
grow E. coli in minimal medium
overnight at 350C



STEP 2. ENZYME INDUCTION:
add 0.1 mL IPTG to 1.0 mL cells
incubate for 30 min.



STEP 3. EXPOSURE TO TOXICANT:
wash cells twice in distilled water
add 0.1 mL toxicant to 0.9 mL induced and washed cells
incubate for 30 min.



STEP 4. P-GALACTOSIDASE MEASUREMENT:
add 0.8 mL Z-buffer, 50 uL SDS,
50 4L chloroform and 0.2 mL ONPG
incubate until color develops
stop reaction with 1 mL cold Na2CO3
measure absorbance at 420nm


Figure 3-1. Protocol for P-galactosidase activity.














ENZYME
BIOSYNTHESIS S SAY



STEP 1. CELL GROWTH:
grow E. coli in minimal medium
overnight at 350C



STEP 2. EXPOSURE TO TOXICANT:
add 0.1 mL toxicant to 0.9 mL cells
incubate for 30 min.



STEP 3. ENZYME INDUCTION:
add 0.1 mL IPTG to 1.0 mL cells
incubate for 30 min.



STEP 4. P-GALACTOSIDASE MEASUREMENT:
add 0.8 mL Z-buffer, 50 UL SDS,
50 uL chloroform and 0.2 mL ONPG
incubate until color develops
stop reaction with 1 mL cold Na2CO3
measure absorbance at 420nm









Figure 3-2. Protocol for 5-galactosidase biosynthesis
activity.








enzyme activity test, blanks had distilled water

substituted for the ONPG, whereas in the enzyme

biosynthesis test, blanks had distilled water substituted

for the IPTG.

Beta-galactosidase was measured using ONPG. This

compound is colorless, but in the presence of 3-

galactosidase it is converted to galactose and o-

nitrophenol. The o-nitrophenol is yellow and can be

quantified by measuring its absorption at 420 nm. Cells

were treated with SDS and chloroform to release 3-

galactosidase immediately prior to adding ONPG. Color

development was allowed to continue for approximately 15

minutes, after which the reaction was stopped by adding

Na2CO3 solution.



Data Analysis

Preliminary range finding was carried out to determine

toxicant dilutions causing between 10% and 90% inhibition.

The degree of inhibition was determined on the basis of

measured absorbance values, considering the control to

represent 0% inhibition. Data were plotted in terms of

percent inhibition versus log final toxicant concentration.

The concentration giving 50% inhibition (IC50) was derived

from linear regression analysis of the data. At least three

replicate tests were carried out on each toxicant. These

definitive runs were conducted using five toxicant

dilutions for each test.






35




Results and Discussion

Comparison of Enzyme Activity and Enzyme
Biosynthesis Assays

The enzyme activity test was carried out with cells

centrifuged (5000 x g; 10 min.), washed twice, and

resuspended in distilled water. This treatment removed the

inducer and, in addition, avoided precipitation of heavy

metals by phosphate salts present in the growth medium.

Preliminary experiments confirmed the need for washing as

the toxicity of Cu2+ and Cd2+ was greatly diminished in the

presence of the growth medium. This effect was not observed

with Hg2+, however. Unwashed cells were used in the enzyme

biosynthesis assay because preliminary experiments

indicated that washed cells could not be induced.

Development of an enzyme biosynthesis test protocol

utilizing washed cells, however, could improve the assay's

sensitivity to heavy metals.

Responses of the two assays to heavy metals and toxic

organic are compared in Table 3-1. The two assays had

equivalent sensitivities for Cd2+ only. IC50s of Cu2+

Hg2+ and formaldehyde determined via enzyme biosynthesis

were significantly lower (p < 0.01) than those determined

via enzyme activity. The sensitivity of the respective

tests varied by a factor of 3 for copper, 39 for mercury,

and 90 for formaldehyde. The enzyme biosynthesis test was













TABLE 3-1. Relative sensitivity of P-galactosidase
activity and biosynthesis assays to selected toxicants.


IC50 (mg/L)a


Chemical


Cu2+

Hg2+


Formaldehyde


Phenol

SDS

Toluene

3,4-DCP


Enzyme
Activity Test


9.2 + 13.6

0.82 + 0.15

1.26 + 0.22

541 + 29.1


> 10,000

> 10,000

> 30,000


Enzyme
Biosynthesis Test


8.2 + 2.4

0.24 + 0.021

0.032 + 0.001

6.0 + 0.1

851 + 139

350 + 77

369* + 25

11.9 + 0.67


Hydrothol


4.6 + 0.1


a Mean + one std. dev. IC50s for biosynthesis assay
followed by an asterisk (*) are significantly different
(p < 0.01) from the enzyme activity test IC50s.








also more sensitive to phenol, SDS, and toluene.

Inhibition of 3-galactosidase activity was negligible to

these chemicals at concentrations as great as 10,000 mg/L.

It is interesting to note that the variability of the

enzyme biosynthesis test results was consistently less than

that of the enzyme activity test results. For example,

respective coefficients of variation for Cd2+ were 148%

versus 29%; those for formaldehyde were 5.4% versus 1.7%.

Based on these results it would appear that the assay

based on inhibition of 3-galactosidase activity is

moderately sensitive to heavy metals and insensitive to

toxic organic. These conclusions are consistent with

previous research on 3-galactosidase and other enzyme

systems. Lederberg (1950), in his work on the

characterization of 3-galactosidase, showed that Cu2+ and

Hg2+ salts inactivated enzyme activity at concentrations of

10-3 M (64 mg/L Cu2+, 200 mg/L Hg2+). Christensen et al.

(1982) noted that heavy metals caused a high degree of

inhibition to a wide spectrum of enzyme classes, whereas

the effect of organic chemicals appeared to be more

specific to the nature of the various enzyme classes. The

broad toxicity of heavy metals to enzymes suggests a

common, non-specific interference with enzyme function.

Metalloids such as arsenic may also be relatively toxic to

enzymes. For example, Reinhartz et al. (1987) determined

that the concentration of arsenite required to effect a

certain degree of inhibition of P-galactosidase activity









was eight times greater than the amount required to inhibit

to the same degree the biosynthesis of this enzyme. This

factor compares favorably to the differential sensitivity

found in this study between the respective tests. The

insensitivity of 5-galactosidase activity to organic

should not be surprising in view of the fact that benzene,

butyl alcohol, chloroform, isoamylalcohol, phenol, SDS and

toluene have been used in various studies to extract 3-

galactosidase from cells prior to measurement of enzyme

activity (Lederberg, 1950; Rotman, 1958; Miller, 1972).



Comparison of Enzyme Biosynthesis to other Toxicity Tests

Sensitivities of the (-galactosidase biosynthesis test

and other aquatic toxicity tests are compared in Table 3-2.

The alternative assays considered are based on INT-

dehydrogenase activity (wastewater dehydrogenase),

bioluminescence (Microtox) and mortality of aquatic

organisms (daphnids and fish), respectively. The enzyme

biosynthesis test was generally more sensitive than the

wastewater dehydrogenase test and compared favorably with

the others. It was the least sensitive of all the

alternatives only for SDS. This result may be due to the

limited permeability of wild-type E. coli strains (Sampson

and Benson, 1987).

In conclusion, the results clearly show de novo 3-

galactosidase biosynthesis to be promising as the basis for











TABLE 3-2. Sensitivity of the 5-galactosidase biosynthesis
test relative to other microbial systems,
Daphnia bioassay and fish bioassay.



Toxicity test endpoints (mg/L)


B-Gal. Wastewater Microtox Daphnia Fish
biosyn. dehydrog.
5-15 min 48 h 96h
Chemical IC50a IC50b EC50C LC50d LC50d



Cd2+ 8.2 -- 25-690 0.065 1-100

Cu2+ 0.24 0.59 0.28-19.5 0.02-.06 0.10-10

Hg2+ 0.032 0.07 0.02-0.06 0.03 0.01-0.9

Formaldehyde 6.0 11.6 8.7-904 -- 10-100

Phenol 851 1530 26-41 12-32 5-100

SDS 350 106 1.1-3.2 7-13 5-46

3,4-DCP 11.9 74 -- -

Hydrothol 4.6 -- 2.4e 0.36 0.3-1.6

Toluene 369 -- 50 310 20-36



a Based on inhibition of B-galactosidase biosynthesis (this
study).

b Based on inhibition of INT-dehydrogenase activity
(Dutton et al., 1986).

c Based on inhibition of bioluminescence (Chang et al.,
1981; Qureshi et al., 1982; McFeters et al., 1983;
Plotkin and Ram, 1984; Greene et al., 1985).

d Based on lethality (Finlayson, 1980; LeBlanc, 1980;
Pennwalt Corp., 1980; Qureshi et al., 1982;
McFeters et al., 1983).

e Bitton et al., unpublished.







40


testing the impact of environmental toxicants. On the

other hand, the direct measurement of P-galactosidase

activity to assess toxicity is not warranted, with the

possible exception of toxicity due to heavy metals or

metalloids.













CHAPTER 4
INHIBITION OF P-GALACTOSIDASE BIOSYNTHESIS IN
ESCHERICHIA COLI: A FUNCTION OF OUTER MEMBRANE
PERMEABILITY TO TOXICANTS


Introduction

Inhibition of enzyme biosynthesis by environmental

toxicants has received relatively little attention. Early

in the elucidation of the mechanism of enzyme synthesis

investigators demonstrated inhibition of enzyme synthesis

using metabolic poisons, such as azide, chloramphenicol,

formaldehyde, and 2,4-dinitrophenol (Monod, 1947; Hahn and

Wisseman, 1951; Koch, 1964; Nakada and Magasanik, 1964).

More recently, Naveh et al.(1984) developed a bioassay

for antibiotics that inhibit protein synthesis using a

mutant luminescent bacteria (Photobacterium leiognathi).

This dark mutant reverts to luminescence emission in the

presence of DNA-intercalating agents. The bacteria are

exposed to potential enzyme synthesis inhibitors, then

luminescence is induced with proflavin, a known mutagen.

Cenci et al. (1985) demonstrated that heavy metals

inhibit P-galactosidase biosynthesis in Escherichia coli.

The use of a toxicity screening assay based on the

inhibition of P-galactosidase biosynthesis in E. coli was

more fully developed by Reinhartz et al. (1987). These

investigators examined the inhibitory effect of several

pesticides and other toxicants on the synthesis of









P-galactosidase. The test was developed as a commercial

kit using freeze-dried E. coli (Orgenics Ltd.).

The complex envelope structure of gram-negative

bacteria is known to consist of cytoplasmic membrane, a

rigid peptidoglycan cell wall, and an outer membrane

(Nikaido and Vaara, 1985). The outer membrane is a matrix

composed of phospholipid (30%) and protein (50%),

surrounded by an external fringe of lipopolysaccharide

(LPS). The outer membrane structure is an effective

diffusion barrier to hydrophobic substances. The diffusion

of hydrophilic compounds is restricted by specific membrane

proteins that form water filled channels, called porins

(Nikaido and Vaara, 1985; Nakae, 1986). Enteric bacteria,

such as E. coli, are particularly resistant to hydrophobic

insult due to an additional hydrophilic antigenic component

(Koch and Schaechter, 1985).

The resistance of gram-negative microorganisms to

antibiotics due to the outer cell permeabiltiy barrier is

of serious clinical concern and has been addressed in

several comprehensive reviews (Costerton and Cheng, 1975;

Lugtenberg and Van Alphen, 1983; Godfrey and Bryan, 1984;

Hancock, 1984; Nikaido and Vaara, 1985; Nakae, 1986).

The objective of this research was to examine the

inhibition of (-galactosidase biosynthesis in E. coli by

selected environmental toxicants. The function of cell

permeability in determining overall assay sensitivity was

also an integral goal of these studies. Physical and








chemical treatments known to alter outer membrane

permeability were examined for their ability to increase

E. coli susceptibility to toxicants, as measured by

inhibition of 5-galactosidase biosynthesis. In addition,

E. coli mutants with altered membrane permeability were

screened for increased sensitivity to selected

environmental toxicants.



Materials and Methods

Test Bacteria

Assays were conducted using the following bacterial

strains: (1) a derivative of E. coli K12 (strain C3000;

ATCC# 15597), (2) various strains obtained from Dr. Barbara

Bachmann, Coli Genetic Stock Culture, Yale University, New

Haven, CT, and designated CGSC# 3004, 4698, 4699, 4923,

4924, 5163, 5634, 6098 and 6683, respectively, and (3)

strains DC2 and UB1005 obtained courtesy of Dr. David

Clark, Department of Microbiology, Southern Illinois

University, Carbondale, Illinois. To insure genetic

stability all strains were maintained in 40% glycerol at

-150C (Miller, 1972).



Test Chemicals and Reagents

Test chemicals and reagents used were: Cd2+ (CdC12),

phenol, pentachlorophenol (PCP), hexachlorocyclohexane

(lindane), sodium dodecyl sulphate (SDS), EDTA

(Na2EDTA'2H20), octylphenoxy polyethoxyethanol (Triton X-









100), polyoxythylene sorbitan monooleate (Tween 80),

benzydimethylhexadecylammonium chloride, benzethonium chlo-

ride, polyethylenimine (PEI), polymyxin B sulfate, and 7-

oxabicyclo-[2.2.1.]heptane-2,3-dicarboxylic acid (Hydro-

thol; Pennwalt Corporation, Philadelphia, PA). All test

chemicals with their exception of Hydrothol were purchased

from Sigma Corporation, St. Louis, MO. All stock solutions

were prepared in distilled water with two exceptions. PCP

was prepared by dissolution in dilute NaOH (0.01N; pH

adjusted to 7.0), and lindane was dissolved in methanol.

In the lindane tests, the final concentration of methanol

in controls and toxicant reaction mixtures was 2.5% (w/v).

At this concentration, methanol was not inhibitory to

enzyme synthesis.

Assay reagents were: isopropyl-f-D-thiogalactoside

(IPTG) and o-nitrophenyl-P-D-galactoside (ONPG) both of

which were obtained from Sigma Corp. (St. Louis, MO). IPTG

was prepared at 0.1% (w/v) and ONPG at 0.4% (w/v). The

solutions were stored for up to for one month in the dark

at 40C. Enzyme assay buffer (Z-buffer) contained:

Na2HPO4-7H20, 16.1 g/L; NaH2PO4"H20, 5.5 g/L; KC1 0.75 g/L;

and MgSO4-7H20, 0.25 g/L, and was adjusted to pH 7.0.

Other reagents employed were: SDS (0.1% and 10% w/v),

Na2CO3 (1M), chloroform; 3-[N-Morpholino]propane sulfonic

acid (MOPS).





45



P-Galactosidase Biosynthesis Assay Procedure

Bacteria for the 3-galactosidase biosynthesis assay

were grown by inoculating LB growth medium (1% tryptone,

0.5% yeast extract, 1% sodium chloride) with 50 uL of stock

glycerol culture. Cells were incubated at 350C overnight.

The culture was then diluted with fresh LB medium to

optical density A550 = 0.2 and allowed to grow to A550 =

0.6-0.65 (= 5x108 cells/mL).

Protocol for the 3-galactosidase biosynthesis toxicity

test is given in Figure 4-1. The assay consists of the

following basic steps: (1) grow cells, (2) wash cells, (3)

expose cells to toxicant, (4) cell induction for

3-galactosidase, and (5) measurement of (-galactosidase

activity.

Extraction of 3-galactosidase was normally

accomplished with SDS and chloroform as depicted in Figure

4-1 (see Step 4). It was also noted that 100 IL of SDS

(10%, w/v) could replace 50 4L chloroform plus 50 4L SDS

(0.1%, w/v) as an extractant of intracellular 3-

galactosidase. Increasing the SDS concentration does not

have an inhibitory effect on 3-galactosidase activity.

This observation is particularly useful when the assay is

adapted to a microplate reader where solvent and light may

interact causing serious interference in absorbance

readings (Appendix A).















P GALACTOS IDASE
BIOSYNTHESIS ASSAY



STEP 1. CELL GROWTH:
grow E. coli in LB medium
overnight at 350C



STEP 2. CELL PREPARATION:
dilute cells with fresh media to A550 = 0.2
allow to grow to A550 = 0.6
wash cells in NaC1 (0.85%)



STEP 2. EXPOSURE TO TOXICANT:
add 0.9 mL toxicant to 0.1 mL cells
incubate reaction mixture for 30 min.



STEP 3. ENZYME INDUCTION:
to each 1 mL reaction mixture add
0.1 mL IPTG, 0.4 mL BGAL buffer, and 0.5 mL growth media
incubate for 30 min.



STEP 4. P-GALACTOSIDASE MEASUREMENT:
add 0.8 mL Z-buffer, 50 uL SDS,
50 4L chloroform and 0.2 mL ONPG
incubate until color develops
stop reaction with 1 mL cold Na2CO3
measure absorbance at 420nm








Figure 4-1. Protocol for P-galactosidase
biosynthesis activity.


1









Blanks consisted of all assay components except the

inducer (IPTG) and accounted for any background enzyme

activity or non-enzymatic degradation of the substrate

(ONPG).



Data Analysis

The degree of sample inhibition was determined by

measuring A420 values of test samples with respect to the

control, which did not have any toxicant present (and

assigned 0% inhibition). Extrapolation of the sample

concentration giving 50% inhibition (IC50) was derived from

linear regression analysis in terms of percent inhibition

versus sample concentration (mg/L). Four or more

triplicate dilutions were run on each sample. Statistical

analysis consisted of significance tests using the t test,

one-way analysis of variance (ANOVA), and Dunnett's

multiple comparison test.



Physical Treatment Procedures

E. coli strain C3000 were used throughout the

following experiments unless otherwise indicated.

Heating. Cells were harvested by centrifugation,

washed, split into two fractions and resuspended in Z-

buffer or in 0.85% (w/v) NaCl. Cells were subjected to

heating for 15, 30 and 60 minutes at 500C in a hot air









incubator following the protocol of Mackey (1983). The (-

galactosidase biosynthesis toxicity assay was run on the

heat treated samples.

Freeze-thaw. The protocol of freeze-thawing of saline

washed cells (Mackey, 1983) completely inhibited 3-

galactosidase biosynthesis. Therefore, cells were concen-

trated (2X) by centrifugation, resuspended in MOPS buffer

(0.02M, pH 7) and frozen at -150C for 1, 7, 14, and 21

days. Thawing was at 350C on a rotary shaker. The B-

galactosidase biosynthesis protocol (Figure 4-1) was

modified as follows: (1) frozen cells were thawed in a 350C

water bath for 5 minutes, (2) then 0.1 mL thawed cells were

added to 0.1 mL concentrated LB growth medium (5X), 0.1 mL

IPTG, and 0.7 mL Z-buffer, (3) then 1.0 mL of toxicant

dilution or control water was added, (4) this 2.0 mL

reaction mixture was incubated at 350C for 3 hours and

5-galactosidase production was measured (as in Figure 4-1).

Freeze-drying. Cells were grown to A550 = 0.65,

centrifuged, concentrated two times by resuspension in half

the original volume in sterilized 2% gelatin (after Sinskey

and Silverman, 1970), or in MOPS (0.02M, pH 7) containing

a-D-trehalose (22.5 g/L), NaC1 (0.07%, w/v), and MgC12

(0.0012%, w/v) after Reinhartz et al. (1987). Three

milliliter aliquots were frozen for 4-6 hours at -600C in

serum bottles, then freeze-dried for 20-24 hours, and








sealed while still under vacuum. A manifold-type freeze

dryer was employed (model FDX-3-55A, FTS Systems, Inc.,

Stoneridge, NY).

Osmotic shock. The methods of Forsberg et al. (1972)

and Falla et al. (1988) with minor modification were used.

Bacterial cells (A550 = 0.6) were washed twice in NaC1

(0.85%) at 10,000 x g at 40C for 10 minutes, and the

supernatants were decanted. Then the cells were suspended

in half the original volume with sucrose (0.5M) and washed

three times at 10,000 x g at 4C for 15 minutes. Between

sucrose washings cells were incubated with shaking for 15

minutes at 250C. The 0-galactosidase biosynthesis assay

followed the normal protocol (Figure 4-1), except cells

were not washed further with saline and induction was

extended to 90 minutes.



Chemical Treatment Procedures

Penicillin. Penicillin G (Sigma Corp., St. Louis, MO)

1662 units/mg was prepared fresh and filter sterilized (0.2

4M). Cells were diluted in fresh LB to A550 = 0.3 and

amended with penicillin (0.05 mg/mL; final concentration).

After 60 minutes of exponential growth, cells were washed

in NaC1 (0.85%). The 3-galactosidase biosynthesis toxicity

assay was performed at this point.

Tris-EDTA. The procedure of Leive (1967) was

followed. Cells were washed in Tris buffer (0.1 M, pH 8),

concentrated 10X, treated with EDTA (0.5mM) for 30 seconds








and diluted 10-fold to original volume with saline. All

steps were conducted at room temperature, as cold

centrifugation (4C) with Tris impairs metabolic processes

(Leive, 1967 and confirmed in this study).

Polyethylenimine (PEI) and Polymyxin. The poly-

cationic polymers PEI and polymyxin were screened for

inhibition of (-galactosidase biosynthesis. Stock polymer

solutions were prepared by dissolution in distilled water.

Sub-lethal concentrations of 7.5 mg/L and 2 mg/L (final

concentration; w/v) were determined for PEI and polymyxin,

respectively. Polycation treatment consisted of a 30

minute incubation with cells at 350C (at 2 or 7.5 mg/L),

followed by washing the treated cells with NaCl (0.85%).

The (-galactosidase biosynthesis toxicity assay was

performed at this point.

Others. Other chemical treatments included: Tris

(Irvin et al., 1981; Elespuru and Moore, 1986), CaC12

treatment (Silhavy et al., 1984), novobiocin (Brock, 1960;

Ryan, 1979), EDTA at 1000 mg/L, the non-ionic detergents

Tween 80 at 1% (w/v) and Triton X-100 at 50 mg/L, the

anionic detergent SDS at 50 mg/L, and the cationic

detergents benzydimethylhexadecylammonium chloride and

benzethonium chloride, both at 0.5 mg/L. These treatments

were not effective at sensitizing 3-galactosidase

biosynthesis to SDS or PCP, and therefore no results are

reported here.











Results and Discussion



Preliminary Experiments

Concentration of growth media. In initial studies

with E. coli C3000, a wild type strain, the effect of

growth media on sensitivity of cells to toxicants was

examined (Table 4-1). Cells were grown overnight in full

strength LB, LB/4, or LB/10 and the culture was adjusted to

A550 = 0.2 with fresh LB, LB/4, or LB/10. Cells were not

washed as is depicted in the definitive protocol (Figure 4-

1). In addition, as noted in Table 4-1, the reaction

mixture (cells to toxicant ratio is 9 to 1) differs from

the definitive mixture (cells to toxicant ratio is 1 to 9).

The results show that cells grown in 1/10 strength

growth medium were significantly more susceptible

(p < 0.05) to cadmium and PCP than cells grown in LB or

LB/4 strength media. This effect was not demonstrated by

SDS.

The effect of washing cells. The effect of various

washing treatments on the inhibition of P-galactosidase

biosynthesis by selected chemicals is given in Table 4-2.

In this case the cells are grown in full strength LB, but

diluted 10-fold with test toxciant. Cadmium toxicity, as

demonstrated above, appears to be a function of media














TABLE 4-1. Effect of growth media on inhibition of
P-galactosidase biosynthesis in E. coli C3000

------------------------------------------------------

(IC50s; mg/L)b
Media----------------------------------
concentrationa Cadmium PCP SDS



LB (control) 8.2 + 0.40 35 + 1.5 >5000

LB/4 6.7 + 1.93 45 + 4.0 >5000

LB/10 2.2*+ 0.20 24*+ 0.9 >5000



a Cells grown in LB, LB/4, or LB/10 and adjusted to A550 =
0.2. Test reaction mixture = (0.9 mL cells + 0.1 mL
toxicant).

b Mean + one std. dev. Means followed by an asterisk (*)
indicates significant difference (p = 0.05) from controls.















TABLE 4-2. Effect of washing treatments on inhibition
of P-galactosidase biosynthesis in E. coli C3000



(IC50s; mg/L)b
Washing --------------------------------------
treatmenta Cadmium SDS PCP



Nonwashed 0.7 + 0.18 > 5000 21 + 4.7
(control)

LB media 1.0 + 0.19 > 5000 ---


NaCl 1.3 + 0.18 395 + 80 17 + 1.0
(0.85%)

MOPS buffer 0.13 + 0.006 272 + 62 19 + 2.2
(0.02M; pH 7)



a Cells grown in LB to A550 = 0.6. Test mixture = (0.1 mL
cells + 0.9 mL toxicant).
b Mean + one std. dev. Means followed by an asterisk (*)
are significantly different (p = 0.05) from controls.


I









strength. Cadmium may be completed with LB growth medium

components (e.g., 1% tryptone, 0.5% yeast extract, or 1%

NaCi).

E. coli C3000 washed with either NaCl or MOPS were

significantly more sensitive (p < 0.05) to SDS than

unwashed cells (Table 4-2). MOPS washed cells were also

significantly more sensitive to cadmium. The ability of

SDS to lyse cells is apparently related to the ionic

strength of the suspension medium. In a related study,

Corwin et al. (1971) found that washing E. coli K12 in

phosphate buffer (0.1M) significantly increased SDS

susceptibility, but lower ionic strength phosphate buffer

(0.01M) or Tris buffer did not.



Mutants with Possible Outer Membrane Alterations

Several E. coli strains that carry mutations that were

thought to affect permeability were screened for

sensitivity to SDS and PCP (Table 4-3). P-galactosidase

biosynthesis assay was carried out according to the

standard protocol (Figure 4-1). The most sensitive strain

examined was EWlb with a mutation resulting in reduced

synthesis of Omp F porin protein, a major outer membrane

porin protein (Whitney, 1971; Misra and Reeves, 1987).

In strain EWlb, the Tol C mutation results in a

deficient production of Omp F porin protein. Porins are

key hydrophilic pathways, thus a decreased sensitivity to

water soluble compounds might be expected (Hancock, 1984).












TABLE 4-3. Median inhibitory concentration data for
P-galactosidase biosynthesis in E. coli strains with
mutations that may be relevant to cell permeability



(IC50s; mg/L)b

Strain CGSC# Mutations PCP SDS
name



C3000 --- .(wild type; control) 25 + 0.9 395 + 80

EWlb 5634 tolC5 1.0"+ 0.15 26 + 1.5

RE103 4698 cmlA, rpsL101 6 + 1.0 335 + 40

D22 5163 envAl, rpsL173 9 + 1.0 185 + 5

A592 4923 tonA21, tonAl 9 + 1.0 270 + 23

RE107 4699 ompF625, rpsLl01 14*+ 1.1 255 + 10

C600 3004 tonA21 23 + 2.0 270 + 14

A593 4924 tonA21, tolB2 27 + 1.9 250 + 24

X2844 6683 tsx-462::TnlO 44 + 4.2 285*+ 31

BW322 6098 zia-207::Tnl0 46 + 2.9 204*+ 12

DC2 --- abs mutant of 14 + 4.2
UB1005

UB1005 --- nalA37 34 + 7.2

a Cells grown in LB to A550 = 0.6.
Test reaction mixture = (0.1 mL cells + 0.9 mL toxicant).

b Mean + one std. dev. Means followed by an asterisk (*)
are significantly different (p = 0.05) than controls.








However, outer membrane protein mutants apparently

compensate this structural void by filling in the outer

membrane with phospholipids, thus enhancing diffusion of

hydrophobic compounds (Nikaido and Vaara, 1985).

Wild type versus outer membrane mutant. The wild type

E. coli strain (C3000) and the outer membrane mutant strain

(EW1b) were compared with respect to chemical inhibition of

P-galactosidase biosynthesis (Tables 4-4 and 4-5). Cells

washed in saline and diluted 10-fold with toxicant (as in

Table 4-5) are generally more sensitive than in unwashed,

undiluted cells (as in Table 4-4). This is particularly

true for cadmium and SDS.

The median inhibitory concentrations (IC50s) for a

heavy metal (Cd2+), a hydrophilic herbicide (Hydrothol), a

surface active compound (SDS), a mildly hydrophobic

organic (phenol; log Kow = 1.48 Brooke et al., 1986), and a

very hydrophobic organic (PCP; log Kow = 5.01 Westall et

al. 1985) indicated that EWlb was significantly more sen-

sitive (p < 0.05) to PCP and SDS than the wild type strain.

The response of EWlb to hydrophilic compounds, however, is

slightly less sensitive compared to wild type cells.



Physical Permeabilizing Treatments

Heating. Heating experiments were initiated by

screening of unwashed cells (strain C3000) incubated at

580C for 0, 10, 20, 30 minutes, followed by exposure to SDS

(100 mg/L), and then P-galactosidase biosynthesis assay.















TABLE 4-4. Median inhibitory concentration data
for 3-galactosidase biosynthesis in E. coli strains C3000
and EWlb grown in LB/10 medium


(IC50s; mg/L)a

C3000b EWlbb


Toxicant


Cadmium

Hydrothol


Phenol


PCP

SDS


2.2 + 0.20

9.8 + 0.29

1330 + 300


24 + 0.9

>5000


6.8 + 0.12

11.3 + 0.70

1680 + 160

2.5 + 0.12

42*+ 1.5


a Mean + one std. dev. Means followed by an asterisk (*)
are significantly more sensitive (p < 0.05) to toxicant
than the adjacent strain (C3000 or Ewlb).

b Cells grown in LB/10 and adjusted to A550 = 0.2.
Test reaction mixture = (0.9 mL cells + 0.1 mL toxicant).












TABLE 4-5. Median inhibitory concentration data
for the 5-galactosidase assay using E. coli
C3000 and EW1b grown in LB medium



(IC50s; mg/L)a

Toxicant C3000b EWlbb



Cadmium 1.3 + 0.18 1.7 + 0.21

Hydrothol 9.1"+ 0.25 15.2 + 0.65

Phenol 950 + 38 1475 + 248

PCP 25 + 0.9 1.0 + 0.15

SDS 395 + 80 26 + 1.5

EDTA 2130 + 446 > 1000

Triton X-100 110 + 9 ---

BZ chloridec 2.5 + 0.08 ---

BMHA chloride 0.97 + 0.205 ---

PEIe 50 + 0.5 ---



a Mean + one std. dev. Means followed by an asterisk (*)
are significantly more sensitive (p < 0.05) to toxicant
than the adjacent strain (C3000 or EWlb).

b Cells grown in LB to A550 = 0.6.
Test reaction mixture = (0.1 mL cells + 0.9 mL toxicnat).

c BZ chloride = Benzothonium chloride.

d BMHA chloride = Benzydimethylhexadecylammonium chloride.

e Young et al. (unpublished data).









Twenty minute exposure was promising (SDS; 100 mg/L = 65%

inhibition). However, when the experiment was rigorously

controlled, by washing cells in saline and heating at 500C

(after Mackey, 1983), P-galactosidase biosynthesis was

completely inhibited. Heating experiments were abandoned

after attempts to replicate the preliminary findings

failed.

Freeze-thaw and freeze-drying. Preliminary freeze-

drying experiments employing gelatin (Sinskey and

Silverman, 1970) and a MOPS solution (Reinhartz et al.,

1987) both proved satisfactory. The Reinhartz preparation

with MOPS buffer gave slightly elevated production of 3-

galactosidase in controls and was used in definitive

freeze-drying experiments.

E. coli C3000, freeze-dried or frozen for 1, 7, or 14,

days were compared with regard to their sensitivity to SDS

and PCP (Table 4-6). Freeze-drying does not sensitize

cells as readily as freeze-thawing after 7 days. However,

freezing was more stressful to cells, decreasing enzyme

biosynthesis over time to such an extent that the

experiment could not be extended to 21 days. On the other

hand, no such decrease in overall 3-galactosidase synthesis

occurs in freeze-dried cells for up to two months.

Sinskey and Silverman (1970) showed that freeze-dried

cells were sensitized to actinomycin D as measured by

inhibition of 3-galactosidase biosynthesis. They noted

that synthesis of 3-galactosidase was delayed 300 minutes














TABLE 4-6. The effect of physical permeabilization
treatments and the inhibition of (-galactosidase
biosynthesis in E. coli (C3000)



(IC50s; mg/L)a

Treatment SDS PCP



Non-treated control 395A+ 80 25E+ 0.9

Freeze-thaw
day 1 107B+ 8 11F+ 0.7

day 7 60C+ 5 4G+ 0.2

day 14 52C+ 2 1.5H+ 0.10


Freeze-dry 115D+ 20 81+ 1.5

Osmotic shock > 450 71+ 1.2


a Mean + one std. dev. Means followed by the same letter
are not significantly different (p = 0.05).









in minimal medium and 150 minutes in 0.1% casamino acids

medium. In this study, (-galactosidase synthesis is delayed

approximately 30 minutes with wild type cells (C3000) and

60 minutes with an outer membrane mutant (EWlb). The

increase in recovery time may be partly attributed to the

richer recovery media used in this study (LB media versus

minimal media).

Osmotic shock. A procedure designed to isolate or

remove the outer bacterial membrane (Falla et al., 1988),

successfully sensitized cells to PCP, but not to SDS (Table

4-6). Washing in sucrose did not increase cell

susceptibility to SDS, the opposite response of earlier

washing experiments (Table 4-2).

In a related study, Maruo et al. (1969) reported

forming sphaeroplasts and protoplasts (membrane prepa-

rations) using lysozyme and Tris-EDTA in sucrose. 3-galac-

tosidase biosynthesis was demonstrated, but at 0.1-1.0

percent the enzyme activity of intact cells (and in our

hands sphaeroplast preparations did not produce 3-galac-

tosidase). Of particular interest was that addition of PCP

(5 X 10-6M; 0.05 mg/L) to membrane preparations resulted in

complete inhibition of (-galactosidase biosynthesis. Intact

cells were not tested with PCP in this study, but as E.

coli K12 was the test bacterium one would expect the level










of inhibition to occur at PCP concentrations 100-1000 times

higher (e.g., 5-50 mg/L). This observation reflects the

capacity of the E. coli outer membrane to resist the

permeation of hydrophobic chemicals, such as PCP.



Chemical Permeabilizing Treatments

Penicillin and Tris-EDTA. Tris-EDTA and penicillin

treatment both succeeded in sensitizing strain C3000 to PCP

(Table 4-7). Tris-EDTA treatment of E. coli Ewlb did not

sensitize cells to cadmium or SDS (Table 4-8). Tris-EDTA

treated EWlb cells appear to be sensitized to PCP.

Tris-EDTA treatment was difficult to replicate due to

high toxicity in some tests. Tris-EDTA treatment is toxic

after a few minutes (Leive and Kollin, 1967), and therefore

must be removed or diluted. In addition, Tris-EDTA

treatment must be conducted at room temperature as low

temperature (40C) causes Tris to be highly toxic releasing

cell nucleotides (Leive and Kollin, 1967).

Penicillin treatment of cells must be conducted in

actively growing cultures. Then penicillin can intercalate

with dividing cell walls and permeabilize the outer

membrane (Hamilton-Miller, 1966).

PEI and Polymyxin. E. coli C3000 treated with PEI

(7.5 mg/L) and polymyxin (2 mg/L) were exposed to selected

















TABLE 4-7. Inhibition of 0-galactosidase biosynthesis by
PCP in E. coli C3000 treated with Tris-EDTA and penicillin



% Inhibition


PCP (mg/L) Non-treated Tris-EDTA Penicillin



0 0 0 0

2.25 0 0 0

22.5 56 + 7.9 86 + 6.2 98 + 1.0

------------------------------------------------------
















TABLE 4-8. Inhibition of 3-galactosidase
biosynthesis by selected toxciants in E. coli EW1b
treated with Tris-EDTA



% Inhibitiona


Toxicant Non-treated Tris-EDTA
(mg/L)


Control 0 0 0


Cadmium 0.5 42 + 5.5 0

2.5 100 100


PCP 0.9 17 + 2.6 21 + 8.1

1.8 50 + 5.2 77 + 6.4


SDS 5 9 + 3.5 0

10 52 + 4.9 65 + 8.5



a Mean + one std. dev.








65


toxicants (Table 4-9). PEI treatment resulted in a

significant increase (p = 0.05) in the toxicity of

Hydrothol, PCP, and EDTA. This sensitization is even more

pronounced for cells treated with polymyxin.

E. coli EWlb treated with polymyxin (Table 4-10) were

rendered significantly more sensitive (p < 0.05) to SDS,

EDTA, and lindane, but not to PCP. It is not clear why

EW1b, an outer membrane mutant, was not sensitized to PCP

while it was dramatically sensitized to another hydrophobic

compound (e.g., lindane). It is noteworthy that PCP is

more hydrophobic than lindane. The octanol-water partition

coefficients (log Kow) for PCP and lindane, are 5.01 and

3.53, respectively (Westall et al., 1985; Hermens et al.,

1985).

It has been proposed that EDTA and polycationic

aminoglycosides, such as polymyxin B, destabilize the Gram-

negative outer membrane by displacing Mg2+ from

lipopolysaccahride molecules (Hancock and Wong, 1984).

EDTA removes LPS Mg2+ by chelation, while polymyxin

apparently binds to the membrane competing for Mg2+ binding

sites (Hancock and Wong, 1984; Vaara and Vaara, 1983).

In conclusion, the sensitivity of 3-galactosidase

biosynthesis, in E. coli, to toxic chemicals was enhanced

by physical and chemical permeabilizing treatments.

Overall, the most sensitive response to surface active

agents and to hydrophobic toxicants was achieved employing
















TABLE 4-9. Median inhibitory concentration data
for (-galactosidase biosynthesis in E. coli C3000 treated
with PEI and polymyxin


IC50s; (mg/L)a


Toxicant


Hydrothol


Phenol


PCP

SDS


Non-treated
control


9.1 + 0.25

950 + 38

25 + 0.9


PEI


7.2*+ 0.22

1173 + 180

17*+ 1.7


395 + 80


Polymyxin



6.1 + 0.31

1015 + 59

4.7 + 0.13

100 + 5


2130 + 446

1.3 + 0.18


1075 + 153

1.5 + 0.22


a Mean + one std. dev. Means followed by an asterisk (*)
indicates a significantly more sensitive response
(p < 0.05) to test toxicant than controls.


EDTA


Cadmium
















TABLE 4-10. Median inhibitory concentration data for
5-galactosidase biosynthesis in E. coli EWlb
treated with polymyxin



IC50s; (mg/L)a

Toxicant Non-treated Polymyxin
control


PCP 1.0 + 0.15 1.1 + 0.03

SDS 26 + 1.5 16 +1.1

EDTA > 1000 490 + 29

Lindane > 100 15 + 0.9

Cadmium 1.7 + 0.21 2.2 + 0.52



a Mean + one std. dev. Means followed by an asterisk (*)
indicates that a significantly more sensitive (p < 0.05)
response to test toxicant than controls.







68


an outer membrane mutant (E. coli EWlb) treated with

polymyxin.














CHAPTER 5
ASSESSMENT OF 5-GALACTOSIDASE BIOSYNTHESIS: TOXICITY
TESTING IN WATER AND WASTEWATER



Introduction

Microbial assays have been widely applied for

wastewater toxicity screening (see Bitton and Dutka, 1986;

Dutka and Bitton, 1986, for reviews). The purpose this

study was to evaluate induction of the lac operon in E.

coli for toxicity screening of wastewater. Comparisons were

made with Ceriodaphnia dubia and bacterial luminescence

(Microtox) bioassays.

The Microtox system (Microbics Corporation, Carlsbad,

CA) is based on inhibition of luminescence of the marine

bacterium, Photobacterium phosphoreum. The commercial

availability of Microtox is an integral part of its success

as well as the fact that it has been validated in

comparative studies with conventional toxicity bioassays

(Qureshi et al. 1982; Plotkin and Ram, 1985).

Disadvantages inherent in the assay include: (1) the assay

is osmotically balanced with 2% NaC1, possibly causing

metal-chloride complexation of heavy metals, particularly

cadmium, and (2) luminescence decays with time, requiring

precise timing and limiting the number of samples that can

be processed simultaneously.










Ceriodapnia dubia are among organisms recommended for

measuring the acute toxicity of effluents by the U.S. EPA

(Peltier and Weber, 1985). The toxicity endpoint in this

assay is lethality over 24-48 hours. The main difficulty in

conducting the assay is culture maintenance and the

duration of the test.

The sensitivity of E. coli 3-galactosidase

biosynthesis and the two bioassays described above was

judged on the basis of the inhibitor response to various

chemicals and wastewater effluents. Previously (see

Chapter 4), it was found that chemical treatment E. coli

with polymyxin (2 mg/L; final concentration) increased

significantly the sensitivity of the assay- to surface

active and hydrophobic chemicals. In addition, the use of a

mutant with an outer membrane protein alteration (Tol C

gene), sensitized further by polymyxin treatment,

resulted in a greatly improved toxicity bioassay. In the

present study, polymyxin-treated E. coli (strain EWlb) was

employed for all definitive toxicity testing.

The Buckman wastewater treatment plant, Jacksonville,

Florida, was used a a model wastewater system. This

facility had a mean flow of 1.85 m3/s in 1986. Primary

sludge and waste activated sludge are treated with a

polymer and thickened by centrifugation prior to

incineration, with centrate returned to the plant

headworks.









Materials and Methods



Sampling and Activated Sludge Treatment

Wastewater samples were collected from the collection

system, plant influent and secondary effluent before

chlorination. Activated sludge treatment was simulated by

allowing the collected samples to settle for one hour, then

two-thirds (2000 mL) of the liquid volume was decanted. A

1400 mL aliquot of settled wastewater was mixed with 600 mL

return activated sludge from the Buckman plant and aerated

for 4 hours. The aeration period was followed by one hour

of settling. Then 600 mL of treated effluent was decanted

from the batch reactor. Samples were stored at 40C for 24

hours before analysis.



Test Bacteria

Assays were conducted using a derivative of E. coli

K12 (strain C3000, a phage host) and E. coli EWlb, a strain

obtained from the Coli Genetic Stock Culture (CGSC #

5634)), Dr. Bachmann, Curator, Yale University, New Haven,

CT. To insure genetic stability all strains were maintained

in 40% glycerol at 150C.



Test Chemicals and Reagents

The test chemicals assessed for toxicity were: Cd2+

(CdC12), Cu2+ (CuSO4'5H20), CN- (KCN), phenol,

pentachlorophenol (PCP),- lindane, sodium dodecyl sulphate










(SDS), EDTA (Na2EDTA*2H20), methanol, and Hydrothol

(Pennwalt Corporation, Philadelphia, PA). All stock

solutions were prepared in distilled water with the

following two exceptions. PCP was prepared by dissolution

in dilute NaOH (0.01N) and pH was adjusted to 7.0. Lindane

was dissolved in methanol. In the case of lindane, the

final concentration of methanol in the assay was 2.5%

(w/v). This level of methanol was included in controls and

was not inhibitory to enzyme biosynthesis.

Dilutions of toxicant stock solutions were made in

reconstituted, moderately hard freshwater that contained

the following components: NaHCO3, 96 mg/L; CaSO42H20, 60

mg/L; MgSO4, 60 mg/L; and KC1, 4 mg/L (Peltier and Weber,

1985). The pH at equilibrium was 7.4-7.8.

(-galactosidase assay reagents were: isopropyl-p-D-

thiogalactoside (IPTG) and o-nitrophenyl-3-D-galactoside

(ONPG), both obtained from Sigma Corp. (St. Louis, MO).

IPTG was prepared at 0.1% (w/v) and ONPG at 0.4% (w/v).

Both solutions were filter sterilized and stored protected

from light at 40C for up to one month. Enzyme assay buffer

(pH 7.0) contained: Na2HPO4*7H20, 16.1 g/L; NaH2PO4*H20,

5.5 g/L; KC1 0.75 g/L; and MgSO4*7H20, 0.25 g/L. Other

reagents employed were: SDS, 0.1% (w/v); Na2CO3 (1M); and

chloroform.

P-Galactosidase Biosynthesis Assay Procedure

Protocol for the 3-galactosidase biosynthesis toxicity

test was given previously in given Figure 4-1. Briefly,










the assay consisted of the following steps: (1) grow cells,

(2) wash cells, (3) expose cells to test sample, (4) cell

induction for 3-galactosidase, and (5) measurement of

3-galactosidase. activity. Bacteria were grown by

inoculating LB growth medium (1% tryptone, 0.5% yeast

extract, 1% sodium chloride) with 50 4L of stock glycerol

culture. The culture was incubated at 350C overnight then

diluted with fresh LB to an absorbance of 0.2-0.3 at 550nm

and allowed to grow to A550 = 0.6 (= 5x108 cells/mL).

Controls contained only dilution water (0 mg/L; no

toxicant). Typically, the controls produced the highest

level of 3-galactosidase, unless the test chemical causes

stimulation of enzyme induction. Blanks consisted of all

assay components except the inducer (IPTG). The blanks

indicated any background enzyme activity or non-enzymatic

degradation of the substrate (ONPG).

The degree of sample inhibition was determined on the

basis of measuring A420 values with respect to the controls

(assigned 0% inhibition). The sample concentration giving

50% inhibition (IC50) was derived from linear regression

analysis in terms of percent inhibition versus sample

concentration. Four or more triplicate dilutions were run

on each sample.


1






74


Polymyxin Permeabilizing Treatment

Polymyxin B sulfate (Sigma Corp., St. Louis, MO) was

screened for inhibition of P-galactosidase biosynthesis in

saline-washed cells and a sublethal concentration of 2 mg/L

(in strains C3000 and EWlb) was determined. Polymyxin was

dissolved in distilled water and stock solutions were kept

at 40C for up to one month. Polymyxin treatment (2mg/L

w/v; final concentration) consisted of exposing saline-

washed cells at 350C for 30 minutes, and then the cells

were washed in saline again to remove excess antibiotic.



Microtox and Ceriodaphnia dubia Bioassays

Microtox and C. dubia bioassays were conducted

concurrently with (-galactosidase in an adjacent laboratory

by students under the direction of Dr. Koopman (Dept.

Environmental Engineering, University of Florida).

Lypholized Photobacterium phosphoreum (Microbics

Corporation, Carlsbad, CA) were reconstituted for use in

the Microtox test. Data were tabulated and reduced

according to the Microtox Operating Manual (Microbics,

1982). All assays were carried out at 15C with a 15

minute contact time with the toxicant. Samples causing 50%

reduction in bacterial luminescence compared to controls

are referred to as EC50s.

Ceriodaphnia dubia bioassays were conducted according

to U.S. EPA guidelines (Peltier and Weber, 1985). Results

were based on organism mortality after 48 hours (LC50s.).











Expression of Wastewater Toxicity

Toxic units (Brown, 1968) were used to express

wastewater toxicity as a proportion of its lethal threshold

concentration. The threshold value was taken as the

respective IC50, EC50, or LC50. Thus:


100 % Waste
Toxic Units (TU) = ---------------------
IC50, EC50, or LC50


where the IC50, EC50, or LC50 is expressed in % waste.



Results and Discussion



P-galactosidase versus Microtox and C. Dubia Assays: Effect

of Selected Chemicals

Inhibitory effects of selected chemicals toward

P-galactosidase biosynthesis, Microtox, and C. dubia assays

were compared (Table 5-1). The P-galactosidase assay was

conducted using E. coli EWlb, which was washed with saline

and subsequently treated with polymyxin. Ceriodapnia

dubia was the most sensitive assay tested for 7 of 9

compounds. P-galactosidase was 1 to 10 times less

sensitive than C. dubia bioassay, except for phenol where

it was 100 times less sensitive. Microtox compared

favorably with C. dubia for phenol and was the most

sensitive assay for SDS. Microtox, however, was greater

than 100 times less sensitive for cadmium than C. dubia.














TABLE 5-1. Median inhibitory concentrations (mg/L)
of toxic pollutants as determined by Ceriodapnia
dubia, Microtox, and 5-galactosdiase biosynthesis assaysa



Assay (LC50, EC50 & ICSO)b
Chemical ---------------------------------------------
C. dubiac Microtoxc 3-galactosidase



Cadmium 0.15 + 0.011 25 0.46 + 0.021

Copper 0.03 + 0.006 0.5 + 0.05 0.5 + 0.01

Cyanide 1.0 + 0.35 2.8 + 0.01 3.6 + 0.42

Phenol 14 + 7.1 11 + 1.9 1540 + 190

PCP 0.3 + 0.04 1.2 + 0.21 1.1 + 0.03

Lindane 1.5 + 0.23 --- 15 + 0.93

SDS 10 + 2.9 1.5 + 0.33 16 + 1.1

EDTA 98 + 9.3 --- 490 + 29

Methanol 11000 + 3000 42000 + 5700 43,800 + 5720



a 3-galactosidase bioassay with E. coli EWlb treated with
polymxin.

b Mean + one std. dev.

c Voiland et al. (unpublished data).









Microtox employs a marine test organism

(Photobacterium phosphoreum) that requires a relatively

high osmotic solution strength. This is provided in the

Microtox assay by adjusting the sample to 2.0% NaCl.

Hinwood and McCormick (1987) substituted sucrose (20 %) for

the NaCl in the Microtox assay and demonstrated

significantly higher toxicity with certain metals (e.g.,

cadmium, nickel, and zinc). This led to the suggestion

that chloro-complexes may be reducing metal toxicity

(Hinwood and McCormick, 1987).

Wastewater Toxicity. Two toxic wastewater samples

from the Buckman wastewater system in Jacksonville,

Florida, were more than five times more toxic to

P-galactosidase biosynthesis in polymyxin treated EWlb

cells than in untreated EWlb cells (Table 5-2). Wild type

cells (C3000) were not inhibited by the samples. The

results indicated that toxicity was caused by a hydrophobic

organic compound, as C3000 and EWlb cells respond equally

to heavy metals and hydrophilic compounds (Table 4-1).

Toxicity of wastewater samples was determined by the

three assays on two occasions (Figure 5-1). The inhibitory

effect to the 5-galactosidase assay and the Microtox system

was comparable in two of four toxic wastewater samples (5b

and 8d). Inhibition of P-galactosidase biosynthesis corre-

lated with C. dubia lethality in one of four toxic samples

(sample 2e). The P-galactosidase assay was not inhibited

by three samples (8d, 10a, and cent.) that demonstrated































I I
5a 5b


I 1 I
8d 10 cent.


WASTEWATER STATIONS


2e 8 8c 8d 10a cent.
WASTEWATER STATIONS.


Figure 5-2.


Wastewater toxicity to 3-galactosidase
biosynthesis and other assays. Comparison to
Microtox-top; C. dubia-bottom.


SBGAL December 1987

Microtox



---








-E; *y ^-,.-?-.









toxicity in the C. dubia bioassay. Sample 8c was toxic to

5-galactosidase at the highest effluent concentration

(90%), but was not inhibitory to C. dubia.

Conclusions regarding complex effluents are often

difficult to formulate, but the P-galactosidase response

was less sensitive overall than Microtox or C. dubia on

these two sampling dates. Further testing of wastewater

samples is required to determine if inhibition of 3-galac-

tosidase biosynthesis would be useful in screening

wastewater toxicity.







TABLE 5-2. Effect of polymyxin treatment on test
sensitivity to toxic wastewater samples



Test Wastewater Sample (Toxic units)
organism ---------------------------------
5b 8d


C3000 0 0


EWlb 1.1 1.9


EWlb + polymyxin > 9 > 9













CHAPTER 6
INHIBITION OF BIOSYNTHESIS OF ENZYMES CONTROLLED BY
DIFFERENT OPERONS: A COMPARISON OF
P-GALACTOSIDASE, a-GLUCOSIDASE AND TRYPTOPHANASE



Introduction

The main focus of the three previous chapters was to

determine the effect of toxic chemicals and wastewater on

5-galactosidase synthesis in Escherichia coli. The present

chapter addresses the effect of toxic chemicals on the

inducible biosynthesis of other enzymes, namely,

tryptophanase in E. coli and a-glucosidase in Bacillus

licheniformis.

The kinetics of enzyme induction and derepression of

3-galactosidase, a-glucosidase, and tryptophanase are under

similar overall genetic regulatory control (e.g., enzyme

formation is initiated when genetically regulated

repressors are inactivated). However, each of the operon

systems has unique regulatory features (Pardee and

Prestidge, 1961; Pollock, 1961; 1963; Bilezikian et

al.,1967; Botsford and DeMoss, 1971).

Comparisons of the inhibitory response of different

operons is limited to a few examples in the literature. The

preferential inhibition of enzyme synthesis by chloram-

phenicol was studied by Sypherd et al. (1962). Sublethal

amounts of chloramphenicol (0.8 ug/mL) preferentially












inhibited the synthesis of tryptophanase, 3-galactosidase,

and citritase by 70%, 68%, and 58%, respectively. D-serine

deaminase synthesis was not inhibited.

Alkaline phosphatase synthesis was about five times

more sensitive to procaine hydrochloride than was

(-galactosidase synthesis (Tribhuwan and Pradhan, 1977).

The investigators suggested that procaine, a membrane

active anesthetic, more easily inhibited periplasmic

alkaline phosphatase than intracellular 3-galactosidase

(Tribhuwan and Pradhan, 1977).

Finally, Pollock (1963) showed that actinomycin D

(0.05 4g/mL) halted a-glucosidase biosynthesis without

affecting penicillinase formation in B. licheniformis. It

was proposed that the differential effect of actinomycin

may be due to differences in its affinity for the DNA of

the different genes.

The objective of this study was to compare three

inducible enzyme systems; 3-galactosidase and

tryptophanase, in E. coli, and a-glucosidase in B.

licheniformis. Biosynthesis of 3-galactosidase is induced

by lactose or lactose-analogs. This enzyme catalyzes the

degradation of lactose to galactose and glucose.

Tryptophanase degrades tryptophan to indole, pyruvate and

ammonia. It is induced by tryptophan. Biosynthesis of a-

glucosidase, an extracellular enzyme, is induced by








maltose. The enzyme degrades maltose into glucose residues

acting at the 1,4-a-glycoside linkages.



Materials and Methods



Test Bacteria

Assays were conducted using a laboratory E. coli

strain (C3000) and E. coli (EWlb) obtained from the Coli

Genetic Stock Culture (CGSC# 5634) Yale University, New

Haven, CT. Bacillus licheniformis (strain 749) was

obtained from the Bacillus Genetic Stock Center (BGSC#

5A20), Ohio State University, Columbus, OH. To insure

genetic stability all strains were maintained in 40%

glycerol at -150C.



Test Chemicals

The test chemicals assessed for toxicity were: Cd2+

(CdCl2), phenol, pentachlorophenol (PCP), sodium dodecyl

sulphate (SDS), and Hydrothol (Pennwalt Corporation,

Philadelphia, PA). All stock solutions were prepared in

distilled water with one exception. PCP was prepared b'

dissolution in dilute NaOH (0.01N) and pH was adjusted to

7.0.



General Assay Protocols

The B-galactosidase, tryptophanase, and a-glucosidase

assays had the following- basic steps in common: (1) cell









growth, (2) washing of cells, (3) exposure to toxic test

sample dilution, (4) induction of enzyme biosynthesis, and

(5) measurement of enzyme activity. In all three cases

bacteria were grown overnight by inoculating growth media

with 50 LL of stock glycerol culture. The cultures were

incubated at 350C overnight and then the cultures were

diluted with fresh growth medium to A550 = 0.2 and allowed

to grow to A550 = 0.6-0.7 (= 5x108 cells/mL in the case of

E. coli).



Assay for (-Galactosidase Biosynthesis

Induction and assay for B-galactosidase biosynthesis

were undertaken according to the methodology described

earlier (Figure 4-1).



Assay for Tryptophanase Biosynthesis

Tryptophanase biosynthesis was determined by a

modification of the color test for indole with Ehrlich's

reagent (Pardee and Prestidge, 1961). The modified assay

is outlined in Figure 6-1. E. coli were grown overnight in

a medium consisting of the following ingredients: casein

hydrolysate without tryptophan (10g/L; ICN Nutritional

Biochem., Cleveland, OH), yeast extract (5g/L), and NaC1

(10 g/L).

Phosphate buffer for tryptophan induction contained

KH2PO4 (13.6 g/L) adjusted to pH = 7.8. Ehrlich's reagent
consists of 5 parts p-dimethyl-amino-benzaldehyde (5 %,















TRYPTOPHANASE BIOSYNTHESIS
ASSAY


STEP 1. CELL GROWTH:
grow Escherichia coli
overnight at 350C



STEP 2: CELL PREPARATION:
dilute cells with fresh medium to A550=0.2
allow to grow up to A550=0.6
wash cells once in distilled water



STEP 3: EXPOSURE TO TOXICANT:
add 0.9 mL toxicant to 0.1 mL cells
incubate 30 min.



STEP 4: ENZYME INDUCTION:
add 0.4 mL buffer, 0.1 mL L-tryptophan
and 0.5 mL fresh medium
incubate for 120 min.



STEP 5: TRYPTOPHANASE MEASUREMENT:
add 1.0 mL Ehrlich's reagent
incubate 15 min.
measure absorbance at 568nm


Figure 6-2.


Protocol for tryptophanase
biosynthesis bioassay.








w/v) in 95% ethanol and 12 parts acid-alcohol (16 mL conc.

H2S04 in 200 mL 95% ethanol). The incorporation of

pyridoxal phosphate cofactor (Pardee and Prestidge, 1961)

did not increase sensitivity of the assay, and was not

included in definitive experiments. Similar observations

were made by Botsford and DeMoss (1971).



Assay for a-Glucosidase Biosynthesis

The protocol for inhibition of a-glucosidase is

outlined in Figure 6-2. B. licheniformis (strain 749) was

grown overnight in trypticase soy broth-without dextrose

(27.5 g/L) plus yeast extract (5 g/L). In some studies the

growth medium was amended with polyoxyethylene sorbitan

monooleate (Tween 80, 10 g/L; w/v).

Optimal assay conditions were based on previous work

conducted on induction of a-glucosidase in this strain

(Pollock, 1963). The induction assay buffer was the Z-

buffer used previously in assay for 5-galactosidase

activity (Chapter 4). The inducer solution, maltose, was

dissolved in water (4 %; w/v) and then autoclaved. The p-

nitrophenyl-a-D-glucoside (PNaG) chromogen solution was

prepared by dissolving 0.4g/100 mL in distilled water. This

solution was filter sterilized and stored at 40C in an

amber bottle.

a-Glucosidase was assayed by measuring the absorption

at 420nm of the p-nitrophenol that is liberated by enzyme

hydrolysis of PNaG. No extractant was necessary to measure














.--GLUCOSI DASE
BIOSYNTHESIS ASSAY


STEP 1. CELL GROWTH:
grow Bacillus licheniformis
overnight at 30C



STEP 2: CELL PREPARATION:
dilute cells with fresh medium to A550=0.2
allow to grow up to A550=0.6
wash cells once in distilled water



STEP 3: EXPOSURE TO TOXICANT:
add 0.9 mL toxicant to 0.1 mL cells
incubate 30 min.



STEP 4: ENZYME INDUCTION:
add 0.4 mL buffer, 0.1 mL maltose
and 0.5 mL fresh medium
incubate for 60 min.



STEP 5: a-GLUCOSIDASE MEASUREMENT:
add 0.2 mL NPaG
incubate until color develops (= 30-60 min)
stop reaction with 1 mL Na2CO3
measure absorbance at 420nm


Figure 6-3. Protocol for a-glucosidase biosynthesis assay.









a-glucosidase production because it is an extracellular

enzyme. Full expression of the enzyme requires cell lysis

with lysozyme or sonication (Pollock, 1961).

Data Analysis

In all three assays, controls were incubated without

toxicant in dilution water (0 mg/L; no toxicant).

Typically, the controls produced the most enzyme, unless

stimulation had occurred. Blanks consisted of all assay

components except the inducer. Blanks indicated

constitutive background enzyme activity or non-enzymatic

degradation of the substrate (e.g., ONPG, NPaG or Ehrlich's

reagent).

The degree of sample inhibition was determined by

measuring absorbance with respect to the control (assigned

0% inhibition). The sample concentration giving 50%

inhibition (IC50) was derived from linear regression

analysis in terms of percent inhibition versus sample

concentration (mg/L). Four or more sample dilutions, in

triplicate, were run on each test chemical. Statistical

analysis consisted of significance tests using the t test,

one-way analysis of variance (ANOVA), and Dunnett's,

multiple comparison test.

Results and Discussion



Relative Sensitivity of Different Operons to Toxicants

The relative response of a-glucosidase, tryptophanase,

and P-galactosidase biosynthesis to selected toxicants are








given in Tables 6-1 and 6-2. The compounds tested included

a heavy metal (cadmium), a detergent (SDS), a herbicide

(hydrothol) and two phenols (phenol and PCP).

Inhibition of 5-galactosidase in wild type E. coli

(C3000) is resistant to hydrophobic chemicals (PCP) and

surface active chemicals (SDS), as was discussed in

previous chapters. This finding is corroborated by the

response with tryptophanase synthesis in E. coli (C3000).

A significantly more sensitive (p < 0.05) response to SDS

and PCP was obtained with E. coli (EWlb), which has a

deficient outer membrane due to a mutation in the tol C

gene (Whitney, 1971; Davies and Reeves, 1975).

In B. licheniformis a-glucosidase biosynthesis was

significantly more sensitive (p < 0.05) to SDS and PCP than

E. coli C3000. Gram-positive bacteria, such as B.

licheniformis, do not have an outer membrane and therefore

tend to be more sensitive than Gram-negative bacteria to

hydrophobic compounds (Koch and Schaechter, 1985). The

inhibitory effect to a-glucosidase biosynthesis in B.

licheniformis was amplified by incorporation of 1% Tween 80

in the growth medium (Table 6-2). Nonionic detergents,

such as Tween (polyoxyethylene esters of sorbitol), cause

bacilli to grow in more dispersed form (Davis et al.,

1980). Sensitization to toxicants by the addition of Tween

could be explained by the increased cell surface area

available for toxicant interaction.














TABLE 6-1. Inhibition of tryptophanase and
5-galactosidase in E. coli



(IC50; mg/L)a
Toxicants --------------------- ---
Tryptophan.b P-gal.c P-gal.d



Cadmium 0.5 + 0.05 1.3 + 0.18 0.5 + 0.02

SDS 180 + 5 395 + 80 16 + 1.1

Hydrothol 3.1 + 0.10 9.8 + 0.28 11.3 + 0.70

Phenol 1960 + 120 1330 + 300 1540 + 190

PCP 19 + 1.8 25 + 0.9 1.1 + 0.03



a Mean + one std. dev.

b Tryptophanase biosynthesis in E. coli (strain C3000).

c .-galactosidase biosynthesis in E. coli (strain C3000).

d 3-galactosidase biosynthesis in E. coli (strain EWlb).














TABLE 6-2. Inhibition of a-glucosidase biosynthesis
in B. licheniformis



(IC50; mg/L)a
Toxicants -----------------------------------------
Non-treated Tween 80 (1%)
control


Cadmium 1.4 + 0.05 ---

SDS 65 + 19 12 + 0.5

Hydrothol 7.2 + 0.26 4.4 + 0.30

Phenol 1405 + 150 760 + 55

PCP 1.8 + 0.04 1.0 + 0.01



a Mean + one std. dev. Means followed by an asterisk (*)
are significantly different (p < 0.05) from controls.









In conclusion, Bernhart and Vestal (1983) reported

that a-glucosidase activity (in vitro) was relatively

sensitive to heavy metals (except copper), but completely

insensitive to PCP, phenol, and SDS. The present study

clearly demonstrates that the inhibition of a-glucosidase

biosynthesis is sensitive to PCP, phenol and SDS. It is

reiterated here, as it was for 3-galactosidase (in Chapter

3), that enzyme biosynthesis is more sensitive to toxicants

than enzyme activity. Furthermore, biosynthesis of

a-glucosidase, in B. licheniformis, was more sensitive to

toxciants than either 3-galactosidase or tryptophanse

biosynthesis, in E. coli. Disparity in sensitivity between

the two biosynthetic systems may be a function of

differences in cell permeability.




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ENZYME BIOSYNTHESIS IN BACTERIA
AS A BASIS FOR TOXICITY TESTING
By
RONALD JOHN DUTTON
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
mi
fit
1988

Copyright
by
Ronald John
1988
Dutton

To my parents,
Mary and Adam Dutton,
and in memory of their daughter,
Anita Christine Dutton

ACKNOWLEDGEMENTS
The author would like to acknowledge and thank the
chairman of his doctoral committee, Dr. Gabriel Bitton, for
his insight, encouragement, and enthusiasm during the
course of this study, and for his assistance in developing
this dissertation. The author is also grateful to the other
members of his committee, Dr. Thomas L. Crisman, Dr. W.
Lamar Miller, Dr. Seymour S. Block, and Dr. Ben L. Koopman.
Special thanks are extended to Dr. Koopman for providing
considerable advice and editorial guidance.
The author is indebted to Ms. Orna Agami for her
excellent technical assistance.
The author also wishes to thank his fellow students,
in particular, Ms. Judy Awong, for their support during the
course of this study.
This work was supported in part by funds provided by
grant No. CES-8619073 from the National Science Foundation
and grant Nos. WM152 and WM222 from the Florida Department
of Environmental Regulation.
Finally, the author acknowledges with gratitude his
parents, Mary and Adam Dutton, for their encouragement and
assistance, and his wife, Nana, .for her love and
understanding.
IV

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS iv
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
2 LITERATURE REVIEW 5
Part I: Enzyme Biosynthesis 5
Introduction 5
Regulation of Enzyme Synthesis 5
Adaptive Enzymes 8
Part II: Enzyme Biosynthesis as a Basis
for Toxicity Testing 11
Inhibition of Enzyme Synthesis 11
Inhibition of the Synthesis of Enzymes
Controlled by Different Operons 13
Part III: The Role of the Bacterial
Cell Envelope 13
Introduction 13
Gram-positive Bacteria 14
Gram-negative Bacteria 15
Part IV: Cell Envelope Alterations Affecting
Permeability to Toxicants 18
Introduction 18
Growth Conditions 19
Chemical Treatments 21
Physical Treatments 2 3
Genetic Alterations of the Outer Membrane 24
Conclusions 25
3 ENZYME BIOSYNTHESIS VERSUS ENZYME ACTIVITY
AS A BASIS FOR MICROBIAL TOXICITY TESTING 27
Introduction 27
Materials and Methods 29
Test Bacteria 29
Test Chemicals and Reagents 30
Test Procedures 31
Data Analysis 34
v

Results and-Discussion 35
Comparison of Enzyme Activity and
Enzyme Biosynthesis Assays.. 35
Comparison Enzyme Biosynthesis to other
Toxicity Tests 38
4 INHIBITION OF (5-GALACTOSIDASE BIOSYNTHESIS IN
ESCHERICHIA COLI: A FUNCTION OF OUTER MEMBRANE
PERMEABILITY TO TOXICANTS 41
Introduction 41
Materials and Methods 43
Test Bacteria 43
Test Chemicals and Reagents 44
3-Galactosidase Biosynthesis Assay Procedures.. 45
Data Analysis 47
Physical Treatments 47
Chemical Treatments 49
Results and Discussion 51
Preliminary Experiments 51
Mutants with Possible Outer Membrane
Alterations 54
Physical Treatments 56
Chemical Treatments 6 2
5 ASSESSMENT OF f3-GALACT0SIDASE BIOSYNTHESIS:
TOXICITY TESTING IN WATER AND WASTEWATER 6 9
Introduction 69
Materials and Methods 71
Sampling and Activated Sludge Treatment 71
Test Bacteria 71
Test Chemicals and Reagents 71
p-Galactosidase Biosynthesis Assay Procedures.. 72
â–  Polymyxin Treatment 7 4
Microtox and Ceriodaphnia dubia Bioassays 74
Expression of Wastewater Toxicity 75
Results and Discussion ' 75
3-Galactosidase versus Microtox and
C.dubia Bioassays: Effect of Selected
Chemicals 7 5
6 INHIBITION BIOSYNTHESIS OF ENZYMES CONTROLLED BY
DIFFERENT OPERONS: A COMPARISON OF
(3-GALACTOSIDASE, a-GLUCOSIDASE AND
TRYPTOPHANASE 8 0
Introduction 80
Materials and Methods 82
Test Bacteria 82
Test Chemicals 82
General Assay Protocols 82
Assay for 0-Galactosidase Biosynthesis 83
Assay for' Tryptophanase Biosynthesis 83
vi

Assay for a-glucosidase Biosynthesis 85
Data Analysis 87
Results and Discussion 87
Relative Sensitivity of Different Operons to
Toxicants 87
7 CONCLUSIONS 9 2
APPENDICES
A MINIATURIZATION OF (3-GALACTOSIDASE
BIOSYNTHESIS ASSAY 95
B AN ALTERNATE SUBSTRATE FOR (3-GALACTOSIDASE
DETERMINATION 9 8
C DEVELOPMENT OF OTHER ENZYME BIOSYNTHESIS
TOXICITY TESTS 100
REFERENCES 104
BIOGRAPHICAL SKETCH 116
Vll

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
ENZYME BIOSYNTHESIS IN BACTERIA
AS A BASIS FOR TOXICITY TESTING
By
Ronald John Dutton
December 1988
Chairman: Gabriel Bitton
Major Department: Environmental Engineering Sciences
Toxicity assessment of hazardous materials and toxic
wastewaters is greatly facilitated by the availability of
inexpensive short-term toxicity tests. The purpose of
this research was to investigate rapid toxicity assays
based on the inhibition of inducible enzyme biosynthesis in
bacteria.
An assay based on the inhibition of (3-galactosidase
biosynthesis was compared to a similar assay based on the
inhibition of [3-galactosidase activity. In both tests,
Escherichia coli were induced to synthesize (3-galactosidase
by exposure to isopropyl-(3-thiogalactoside (IPTG). The
induction step preceded contact of the cells with toxic
chemicals in the enzyme activity assay, whereas in the
enzyme biosynthesis test, IPTG was added following
viii

contact of cells with the toxicant. Relative sensitivity
was judged on the basis of responses to heavy metals and
organic toxicants of environmental importance. Comparison
of these results to median inhibitory concentration data
(IC50s) achieved with other microbial systems, Daphnia
bioassay, and fish bioassay indicate that the enzyme
activity test was sensitive to heavy metals, but was
insensitive to organic toxicants. The test based on
inhibition of (3-galactosidase biosynthesis was sensitive to
both heavy metals and organics.
It was found that if wild type E. coli were subjected
to physical and chemical treatments known to alter the
outer membrane, the sensitivity of the (3-galactosidase bio¬
synthesis test to surface active and hydrophobic chemicals
could be significantly increased. The use of E. coli
(EWlb) with an outer membrane mutation (TolC gene),
sensitized further by chemical treatment with polymyxin (2
mg/L), resulted in an improved bioassay for chemical and
wastewater toxicity.
Toxic inhibition of (3-galactosidase and tryptophanase
biosynthesis, both in E. coli, and of a-giucosidase
biosynthesis in Bacillus licheniformis was also
investigated. The response of a-glucosidase biosynthesis
to selected toxicants was the most sensitive. Disparity in
sensitivity between the biosynthetic systems in E. coli and
B. lichenif ormis may be a function of differences in cell
permeability to the test chemicals.
IX

CHAPTER 1
INTRODUCTION
Production of synthetic chemicals has greatly
escalated since World War II. It was estimated that there
are approximately 100,000 chemicals in commerce with
about 1000 new ones added annually (McCutcheon, 1980).
It is important to assess the risks posed by these
potentially toxic substances to the environment. Current
concerns include the effects of acid precipitation,
widespread leaching of waste disposal sites, and point
and non-point source discharges to natural waters.
Protection of the environment is mandated by various
federal laws (e.g., Clean Air Act, Clean Water Act,
Resource Conservation and Recovery Act). Assessment of
the risks to the environment involves obtaining
information on (1) the concentration of specific
toxicants, (2) the source and fate of the toxicants, (3)
the potential target species which may be harmed by the
toxic effect, and (4) toxicity data for the pertinent
species (Camougis, 1985).
The "workhorse" in monitoring pollution effects has
been the acute toxicity test (Buikema et al. 1982).
Information generated from toxicity tests can be used to
predict . environmental effects of toxicants; compare
1

toxicants, test conditions, or test species; and regulate
the environmental discharge of the toxicants.
Toxicity tests employing a variety of organisms are
presently applied to detect toxic chemicals (McFeters et
al. 1983). Toxicity tests using bacteria began to
receive serious consideration after the realization that
a microbial bioassay, for the detection of mutagens (the
Ames Test), proved to be useful in screening for
potential animal carcinogens (Ames et al., 1975; Devoret,
1979 ) .
The basis for the use of microorganisms in toxicity
testing includes the following: (1) they possess the
majority of the same biochemical pathways present in
higher organisms, (2) they exhibit a significantly
organized membrane structure, (3) they play an
instrumental role in nutrient cycling, and (4) they
represent the first level at the base of the food chain.
Endpoints of microbial toxicity tests are based on the
measurement of growth inhibition, oxygen uptake, heat
production, substrate uptake, ATP content,
bioluminescence, and enzyme activity (Bitton, 1983; Liu
and Dutka, 1984; Bitton and Dutka, 1986; Dutka and
Bitton, 1986) .
In classical enzyme studies it is conventional to
study the kinetics of enzyme inhibition with known
metabolic inhibitors. It logically followed that
environmental scientists would apply this principle to

assess the toxicity of environmental toxicants (e.g.,
insecticides versus acetylcholinesterase). Consequently,
short-term enzyme assays (in vitro and in vivo) for
toxicity screening were developed using dehydrogenase,
lipase, luciferase, esterases, carbonic anhydrase,
ribonuclease, urease, acetylcholinesterase, and ATPase,
(Christensen et al., 1982; Bitton, 1983 ; Obst et al.
1988) .
Little attention has been given to the effect of
toxic chemicals on enzyme biosynthesis. This approach per
se should be more sensitive than enzyme inhibition,
because both inhibition of enzyme formation (protein
synthesis) and inhibition of enzyme activity are
measured concurrently. Short-term toxicity screening
tests based on inhibition of bacterial enzyme synthesis
should be feasible because of the rapid kinetics of
enzyme biosynthesis in bacteria.
This dissertation assesses the effectiveness of
enzyme biosynthesis in bacteria as a basis for toxicity
testing. This task was approached in the following
steps: (1) the response of (3-galactosidase activity
versus (3-galactosidase biosynthesis to selected toxic
chemicals was compared, (2) the inhibition of
(3-galactosidase biosynthesis in Escherichia coli was
addressed more fully, with a detailed consideration of
cell permeability to toxicants, (3) the response of
enzyme biosynthesis to toxic wastewaters was examined,

4
and (4) the inhibition of (3-galactosidase, tryptophanase,
and a-glucosidase biosynthesis to selected toxic
chemicals was compared.

CHAPTER 2
LITERATURE REVIEW
Part I: Enzyme Biosynthesis
Introduction
Prokaryotic and eukaryotic cells contain complex
regulatory mechanisms that control the concentration of
their enzymes. Enzymes may be synthesized continuously
("constitutive enzymes") or only in response to a specific
stimuli ("adaptive enzymes"). The regulation of enzyme
synthesis is perhaps the most important level of metabolic
control because it determines whether or not a biosynthetic
or catabolic reaction pathway may in fact operate (Walker,
1983 ) .
Regulation of Enzyme Synthesis
Bacteria have adapted to survive and proliferate in
environments with widely fluctuating carbon and nitrogen
sources. The bacterial cells must be able to derive as
much energy as possible from sporadically available
nutrients (Walker, 1983). Catabolic and biosynthetic
pathways mediate these adaptive processes. Bacterial cells
switch all the enzymes in a pathway on or off, and the
genes coding for these enzymes are usually coordinated in a
sequence referred to as an operon.
5

6
The functioning of an operon involved in enzyme
synthesis is summarized in the following steps: (1)
inactivation of repressor (e.g., induction), (2) RNA
transcription from DNA template (operon region) forming
precursor mRNA's, (3) maturing/processing of mRNA, (4)
translation of mature mRNA into enzyme molecules at the
ribosomes, and (5) formation of active sites and other
steps necessary for full enzyme activity (activation).
The first definitive model of the genetic regulatory
control of enzyme synthesis was the lactose operon (Jacob
and Monod, 1961). The research culminated in a nobel prize
for the French investigators Jacques Monod, Francois Jacob,
and Andres Lwoff, in 1965. The genetic regulatory control
of the lactose operon is depicted in Figure 2-1.
In response to addition of lactose (or synthetic
lactose analogs), the lactose operon in Escherichia coli
increases the number of (3-galactosidase molecules by a
factor of 1000 within 1-2 hours. This factor is a function
of enzyme synthesis within the cells and the increase in
cells due to growth. In contrast, the induction of
tryptophan oxygenase in rat liver (by tryptophan) leads to
an eightfold increase in enzyme over a period of 6 hours.
These differences in rate and amount are typical of the
responses in bacterial and animal cells (Walker, 1983).

*
i
galacto
Figure
v
V
polycistronic
m RNA
V
-galactosida galactoside
/3 -galacto_s¡dase permease transacetylase
' v \ \
\ s \ .
\ \ s'. \
\
\
'V
-e + glucose <-
hA.
allolactose <
lactose •<:
acetyl CoA
A
LACTOS
JJ
cell membrane
2-1. The pathway of lactose catabolism and
regulation by the the lactose operon.

8
Adaptive Enzymes
Adaptive enzymes are classified as "inducible" or
"repressible" (Braun, 1965; Brock, 1979). Enzyme
induction was defined as "a relative increase in the rate
of synthesis of a specific enzyme resulting from exposure
to a chemical substance" (Cohn et al., 1953). Each
substrate induces the synthesis of the enzymes necessary
for its own catabolism. For example, the presence of the
substrate lactose induces production of (3-galactosidase,
which degrades lactose to galactose and glucose.
Enzyme repression is defined as "a relative decrease,
resulting from the exposure of cells to a given substance,
in the rate of synthesis of an specific enzyme" (Vogel,
1957). When a particular substance is absent, the specific
enzyme is derepressed and synthesized. For example, the
absence of phosphate activates (derepresses) synthesis of
alkaline phosphatase. In both enzyme induction and enzyme
derepression, the net result is that a repressor in the
operon region of the geonome is diverted from its
"clogging" function, and enzyme biosynthesis is initiated
(Braun, 1965). Examples of bacterial operons and various
adaptive enzymes are given in Tables 2-1 and 2-2.
Many microbial carbon sources (e.g., lactose,
galactose, arabinose, maltose, and a number of trioses and
pentoses) are catabolized by inducible enzymes.

9
TABLE 2-1. Some operons in bacteria
Operons
Number
genes
of
Function
lac
3
transport
and hydrolysis of (3-galactoside
gal
4
conversion
phosphate
. of galactose to glucose-1-
hut
4
histidine
utilization
leu
4
conversion
leucine
. of a-ketoisovalerate to
ara
5
transport
and utilization of arabinose
bio
5
synthesis
of biotin
trp
5
synthesis
of tryptophan
arg
8
synthesis
of arginine
his
9
synthesis
of histidine
Source: adapted after Lehninger (1982) and Walker (1983).

10
TABLE 2-2. Examples of some adaptive enzymes
Enzyme
Microorganism
Inducer/repressor
(3-galactosidase
Escherichia coli
lactose, IPTG
galactokinase
E. coli
fucose
tryptophanase
E. coli
tryptophan
alk. phosphotase
E. coli
phosphate
a-amylase
Bacillus spp.
starch
a-glucosidase
Bacillus spp.
maltose
p-lactamase
Bacillus spp.
penicillin
glucose isomerase
B. coagulans
D-xylose
histidase
Klebsiella aeroqenes
uronic acid
pullanase
Aerobacter spp.
maltose
urease
Proteus rettgeri
urea
tryptophan
Pseudomonas spp.
kynurenine
oxygenase
Source: adapted after Demain (1971), Wang et al. (1979),
and Reed (1982).

Inducible catabolic enzymes are expressed only after carbon
sources of a higher energy value have first been depleted,
thus economizing cell energy expenditure. This phenomenon
(e.g., repression of [3-galactosidase biosynthesis in the
presence of glucose) is known as "catabolite repression"
(Brock, 1979).
Enzyme regulation in biosynthetic pathways (for amino
acids, biotin, thiamine, other vitamins) is controlled
primarily by repressible enzymes in analogous systems to
those
regulating
catabolite operons (Wang
et al.,
1979;
Walker
, 1983).
Upon
induction
catabolic
enzymes
have
been
observed
to
increase
several thousandfold in
activity, while hundredfold changes have been observed in
the specific activity of enzymes in biosynthetic pathways
(Demain, 1971).
Part II; Enzyme Biosynthesis as a Basis
for Toxicity Testing
Inhibition of Enzyme Biosynthesis
In the first studies to examine the effect of
antimicrobial agents on enzyme biosynthesis, Monod (1944;
1947) showed that 2,4-dinitrophenol and chloramphenicol
inhibited (3-galactosidase biosynthesis. Hahn and Wisseman
(1951) later demonstrated that chloramphenicol, aureomycin,
terramycin, and acridine inhibited metabolism of lactose,
arabinose, maltose, and acetate. They attributed this to
inhibition of adaptive enzyme formation. Inhibition of

S-galactosidase formation by selected antimicrobial agents
was also demonstrated by Koch (1964) and Nakada and
Magasanik (1964). Kaminski (1963) showed that
staphylococcal penicillinase formation was completely
inhibited by anionic detergents at concentrations (1.0
mg/L) that did not inhibit growth. Kucera et al. (1965)
observed inhibition of staphylococcoal penicillinase
formation by 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T)
at 100 mg/L.
More recently, Naveh et al. (1984) and Ulitzur (1986)
have developed a bioassay for antibiotics that inhibit
protein synthesis, employing a dark mutant luminescent
bacterium (Photobacterium leiognathi). The bacterial
mutant reverts back to luminescence in the presence of DNA-
intercalating agents. The bacteria are exposed to test
samples (potential protein synthesis inhibitors) and then
the luminescence system is induced with a mutagen (e.g.,
proflavin). The relative change in light production is
measured with a photometer.
Cenci et al. (1985) demonstrated that heavy metals
inhibit (3-galactosidase synthesis in E. coli. And
Reinhartz et al. (1987) examined the effect of several
common pollutants on the synthesis of (3-galactosidase. The
assay was developed as a commercial toxicity test kit using
freeze-dried E. coli (Toxi-Chromotest, Orgenics Ltd. ,
Yavne, Israel).

Inhibition of the Synthesis of Enzymes Controlled by
Different Operons
The preferential inhibition of enzyme synthesis by
chloramphenicol was examined by Sypherd et al. (1962).
Sublethal amounts of chloramphenicol (0.8ug/mL) inhibited
the synthesis of (3-galactosidase, galactoside permease,
tryptophanase, and citritase by 68%, 64%, 70% and 58%,
respectively. However, chloramphenicol at this
concentration failed to inhibit D-serine deaminase and any
constitutive enzyme activity (e.g., hexokinase, glucose
dehydrogenase, acid phosphatase, and pyrophosphatase).
Pollock (1963) showed that actinomycin D (0.05y.g/mL)
will halt a-glucosidase induction without affecting
penicillinase induction in B.subtilis. Furthermore,
inhibition of alkaline phosphatase, a repressible
periplasmic enzyme, was about five times more sensitive
than p-galactosidase induction in E. coli exposed to
procaine hydrochloride (Tribhuwan and Pradhan, 1977).
Part III; The Role of the Bacterial Cell Envelope
- in Resistance to Toxicants
Introduction
Gram-positive and Gram-negative bacteria all produce
cell walls (with the exception of mycoplasmas) that
surround an inner cytoplasmic membrane. Peptidoglycan or
murein is a common component of these walls, conferring

mechanical rigidity to the cells (Nikaido and Vaara, 1985).
All Gram-negative bacteria contain an additional layer, the
outer membrane, composed of phospholipids,
lipopolysaccharide (LPS), and protein (Nakae, 1986). Both
Gram-positive and Gram-negative bacteria may also be
surrounded by globular protein coats or extensive fibrillar
carbohydrate capsules (Costerton and Cheng, 1975). These
capsules may play important roles in pathogenesis or
adhesion and are a potential diffusion barrier to chemicals
(Godfrey and Bryan, 1984).
Gram-positive Bacteria
Peptidoglycan is the major component of the Gram¬
positive cell wall, forming a thick fibrous structure with
a mainly negative charge (Costerton and Cheng, 1975). The
cell wall is usually interspersed with covalently linked
teichoic and teichuronic acid polymers that do not form
coherent or continuous structures. The Gram-positive cell
wall with its net negative charge is analogous to an ion
exchange bed that excludes very large molecules and may
adsorb positively charged particles onto structural
polymers (Costerton and Cheng, 1975). The main bulk of the
Gram-positive cell wall probably does not act as barrier to
toxicants since its molecular seiving function is
restricted to molecules larger than 100,000 daltons
(Godfrey and Bryan, 1984). In addition, as Gram-positive
bacteria do not have an outer membrane, they tend to be

i 3
more sensitive than Gram-negative bacteria to hydrophobic
compounds such as detergents, dyes, and certain antibiotics
(Koch and Schaechter, 1985).
Gram-negative Bacteria
The outer membrane structure of Gram-negative bacteria
is very complex (Figure 2-2). The cell wall consists of a
thin peptidoglycan layer (0.8nm to 30nm thick) that does
not contain teichoic acids, but is covalently linked to
lipoprotein molecules (Costerton and Cheng, 1975). External
to the cell wall, Gram-negative bacteria have a complex
outer membrane composed of phospholipids, lipopoly-
saccharides (LPS) and proteins.
Most studies conducted on the molecular structure and
permeability of the Gram-negative outer membrane have
focused primarily on enteric bacteria. The resistance of
these organisms to antibiotics has been addressed in
several excellent reviews (Costerton and Cheng, 1975;
Godfrey and Bryan, 1984; Hancock, 1984; Nikaido and Vaara,
1985; Nakae, 1986).
Phospholipids. A phospholipid bilayer is the first
«»
layer external to the peptidoglycan cell wall, and is very
similar structurally to the inner cytoplasmic membrane.
(Nikaido and Vaara, 1985). The phospholipid content of the
outer membrane is much less than in the

o CO
Llpopolyaacctiaríde
• Inner leaflet
íy' Psptldoplycan
Outer
membrane
Inner
membrane
legend
Protein
i> Lip o p o ly sa ccha ride
O Uplda
Lipoprotein
Cytoplasm
The structure of the Gram-negative cell wall
and membrane layers. (From Godfrey and Bryan,
1984; reprinted by permission; copyright
Academic Press).
Figure 2-2.

cytoplasmic membrane. But like its inner counterpart, it
is an effective barrier to hydrophilic compounds (Koch and
Scnaechter, 1985).
Proteins. About half the mass of the outer membrane
is made of proteins (Nikaido and Vaara, 1985; Nakae, 1986).
The proteins of the outer membrane are embedded in the
phospholipid bilayer. In E. coli K12 the major proteins
are OmpA, OmpF, and OmpC, and murein lipoprotein. In E.
coli B OmpC is missing (Mizushima, 1987).
The murein lipoprotein covalently anchors the outer
membrane to the underlying cell wall. The other major
membrane proteins (Omp proteins) form water filled channels
called porins. The porins allow small hydrophilic
substances to penetrate through the outer membrane. Certain
minor membrane porin proteins may be important in enhancing
specific solute uptake: LamB (maltose), PhoE (phosphate,
other anions), Tsx (nucleotide), TonA (Fe3+), BtuB (vitamin
B12) (Nakae, 1986). The size of the major porins in
enteric bacteria are about 1.1-1.2 run, allowing substances
smaller than 600-800 daltons to diffuse through. In
Pseudomonas aeroqinosa the porin diameter is about 2 nm
o
(Nikaido and Vaara, 1985).
Lipolysaccharide (LPS). The LPS forms the external
fringe of the outer membrane and is composed of hydrophilic
oligosaccharide segments and a covalently bonded
hydrophobic segment, called lipid A, that anchors the LPS
to the underlying outermembrne phospholipid (Koch and

Schaechter, 1985; Naikaido and Vaara, 1985; Raetz, 1987).
The LPS constitutes about 20% of the outer membrane by
weight (Nakae, 1986). Adjacent LPS molecules are apparently
stabilized covalently by Mg2+ cross-bridging (Leive, 1974;
Nikaido and Vaara, 1985). It is the LPS,' along with the
O-antigen component in enterics, that give Gram-negative
bacteria a hydrophilic coat that effectively excludes
hydrophobic substances (Koch and Schaechter, 1985).
In summary, Gram-negative bacteria have external to
their cell wall a complex outer membrane. The outer
membrane has an external fringe (LPS) that is hydrophilic,
and excludes hydrophobic substances. Inside the LPS, the
outer membrane is composed of a lipoprotein and protein
matrix that create water filled channels called porins. In
enteric bacteria, hydrophilic substances are excluded above
600-700 daltons due to the channel size of the porins. In
addition, Gram-negative bacteria, particularly enteric
bacteria, have evolved an outer membrane defense to
hydrophobic chemicals. The implications of this
permeability barrier in Gram-negative bacteria must be
considered when conducting toxicity tests.
Part IV: Cell Envelope Alterations Affecting
Permeability to Toxicants
Introduction
The composition of the bacterial cell envelope is
affected directly by the conditions of the surrrounding

19
environment. The integrity of the cell envelope depends on
nutritional, chemical, physical, and genetic factors.
Manipulation or changes in these parameters can result in
altered cell permeability to antibiotics, dyes, detergents,
substrates, and other chemicals (Leive, 1968; Sinskey and
Silverman, 1970; Unemoto and Macleod, 1975; Nikaido, 1976;
Hitchener and Egan, 1977; Bennett et al., 1981; Huoang et
al. 1983; Mackey, 1983; Hancock and Wong, 1984; Hancock,
1984; Brown and Williams, 1985).
Growth Conditions
Growth conditions can significantly alter the outer
membrane of Gram-negative bacteria (Brown and Williams,
1975; Beuchat, 1978). Ionic strength and osmotic balance
due to magnesium, sodium, and potassium affect the
stability of the outer membrane. Alterations in the outer
membrane resulting from growth in phosphate and magnesium-
limited cultures are associated with increased resistance
to EDTA and polymyxin (Finch and Brown, 1975; Brown and
Melling, 1969), cationic proteins (Finch and Brown, 1978),
and cold shock (Kenward and Brown, 1978). Gilbert and
Brown (1980) found that E. coli cultures became sensitized
to chlorophenol and phenoxyethanol when carbon was
limiting. This was not the case with magnesium or
phosphate-limited cultures.
Pseudomonas fluorescens grown on glucose show higher
resistance to actinomycin D and EDTA than cells grown on

succinate (Walker and Durham, 1975). Oxygen consumption in
E. coli was inhibited in the presence of phenol and
phenoxyethanol when succinate, pyruvate or acetate were
used as substrates, but was stimulated if the substrate was
glucose, mannitol, or lactose (Hugo and Street, 1952).
This was explained partly in terms of intracellular or
membrane associated enzymes for the different substrates
(Hugo, 1967).
Analogous responses to media composition may also
occur in Gram-positive bacteria. Bacillus megaterium
grown in magnesium and carbon-limited media became
sensitized to chlorhexidine and phenoxyethanol with
increasing growth rate, while phosphate-limited cultures
were not (Gilbert and Brown, 1980). Sensitivity was
increased two-fold for phenoxyethanol and up to ten-fold
for chlorhexidine. Protoplasts of these cultures were
sensitized to a similar extent indicating that alterations
in cell wall permeability were involved.
Gram-positive bacteria grown in glycerol to enhance
cell lipid content, or in biotin-deficient media to deplete
lipids, showed altered resistance to antimicrobial agents
(Hugo and Stretton, 1966 ; Hugo and Franklin, 1968 ; Hugo and
Davidson, 1973). Lipid "fattened" staphylococci (18% cell
lipid increase) were resistant to phenols with side chains
longer than 4 carbon groups. Uptake studies suggested that
the larger phenols were bound in the cell lipid fraction.

Chemical Treatments
Introduction. Various chemical treatments have been
shown to alter cell permeability (e.g.., detergents,
Miozzari et al., 1972, Arnold and Johnson, 1982; solvents,
Beuchat, 1978, Dobrogosz, 1981, Chaudary, 1984;
antibiotics, Nakao et al., 1973; calcium chloride,
Bezinger, 1978; EDTA, Leive, 1968; tris(hydroxymethyl)-
aminomethane (Tris), Irvin et al., 1981; and polycations,
Vaara and Vaara, 1983, Hancock, 1984). An important aspect
of these treatments is the change in permeability and
whether or not this change impairs metabolism. It is
critical to this study that enzyme biosynthesis proceeds
following a particular sensitizing treatment. Several
treatments are discussed in more detail below.
EDTA treatment. Ethylenediaminetetracetate (EDTA)
treatment (10_4M) in Tris buffer results in up to 50%
release in the LPS of E. coli, an increase in permeability
to antibiotics (Leive, 1968; Russel et al., 1973), and an
increase in cell hydophobicity (Mackey, 1983). It was
postulated that EDTA disorganizes the outer membrane by
chelating Ca^+ and Mg^+ in the LPS (Leive, 1974). However,
cells treated with EDTA and subsequently grown in media
were able to repair outer membrane damage in less than one
hour (Scudamore et al., 1979).
Tris treatment. Tris buffer appears to permeabilize
the cell envelope (Irvin et al., 1981). A mutant E. coli

strain treated with Tris buffer for 1.5-2.5 hours released
the peripiasmic enzyme alkaline phosphatase and was
sensitized to lysozyme. Tris treatment at 4°C, but not at
37°C, has been shown to release the cell nucleotide pool
as indicated by increased absorbance of cell supernatant at
260 nm (Leive and Kollin, 1967). The release of the
nucleotide pool induces RNA breakdown and impairs metabolic
processes. Therefore in permeability studies where
metabolic studies are important, washing with Tris should
be at room temperature.
Polycation treatment. Growth in subinhibitory
concentrations of a number of polycationic agents (e.g.,
protamine, lysine, spermine, streptomycin, lysozyme,
amikacin, gentamicin, and polymyxin (Vaara and Vaara, 1983;
Walker and Beveridge, 1987) sensitizes Gram-negative
bacteria to hydrophobic antibiotics (Vaara and Vaara,
1983). Polymyxin B appears to be the most effective
polycationic agent, sensitizing Salmonella typhimurium and
E. coli to detergents and hydrophobic antibiotics (Vaara
and Vaara, 1983 ) .
Polymyxins are antibiotics produced by Bacillus
polymyxa. They are characterized by a high molecular weight
(1000-1200), a heptapeptide ring, a high content of
diaminobutyric acid, and a side chain ending in a
methyloctanoic acid residue (Godfrey and Bryan, 1984).
Unlike many polycations, polymyxin does not release LPS. It
appears that polymyxin causes rodlike projections or blebs

23
in the outer membrane (Lounatmaa et al., 1976). Polymyxin
interacts with the LPS and its action may be due to
competitive displacement of divalent cations that stabilize
the LPS (Schindler and Osborn, 1979; Hancock, 1984). The
toxicity of polymyxin is high, and to maintain viability
while still altering permeability, it should be used at
about 1 mg/L (Vaara and Vaara, 1983).
Categories of chemicals that increase outer membrane
permeability have been reviewed by Hancock (1984), Hancock
and Wong (1984), and Walker and Beveridge (1987 ). These
chemicals can be classified into three groups: (1) divalent
cation chelators such as EDTA and nitrilotriacetate, (2)
polycations such as aminoglycosides (e.g., polymyxin), and
(3) large monovalent cations such as cetrimide and Tris.
The permeabilizers with positive charge compete with and
displace membrane-stabilizing Mg2+ and Ca2+ ions in the
LPS.
Physical Treatments
The treatment of Gram-negative bacteria by physical
means such as heating, freeze-thawing, and freeze-drying
also causes perturbations in the outer membrane, thus
affecting cell permeability (Bennett et al., 1981; Mackey,
1983; Hitchener and Egan, 1977; Sinskey and Silverman,
1970; Ray and Speck, .1973).
Heating. Escherichia coli subjected to heating at
48 °C and treated with EDTA released up to 50% of the LPS.

The addition of Mg2 + (5mM) to the heating medium protected
the cells from injury (Hitchener and Egan, 1976).
Tsuchiado et al. (1985) reported that heat treatment of E.
coli at 55°C in Tris buffer (pH 8) sensitized the cells to
crystal violet and the action of phospholipase.
Freeze-injury. Freeze-injury in bacteria was reviewed
by Ray and Speck (1973). Freeze-thawed microorganisms were
found to be sensitized to surfactants, lysozyme, UV
radiation, and selected metabolic inhibitors. Important
factors in cell death during freeze storage are cellular
crystal formation and prolonged exposure to concentrated
solutes (Ray and Speck, 1973).
Freeze-drying is similar to freeze-thawing although
freeze-dried cells stored under vacuum are viable for much
longer periods of time than frozen cells because
dehydration is beneficial for storage (Beuchat, 1978).
Sinskey and Silverman (1970) demonstrated that freeze-dried
E. coli were susceptible to antibiotics at concentrations
that were normally ineffective. Both freeze-thawed and
freeze-dried cells can repair permeability damage in
nutrient media (Sinskey and Silverman 1970; Ray and Speck,
1973).
Genetic Alterations of the Outer Membrane
Microorganisms with defined mutations which alter
cellular permeability lack or make reduced amounts of
structural cell envelope components. This subject has been

reviewed in regards to Gram-negative bacteria (Hancock,
1984). Information regarding Gram-positive mutants is
scarce. However, "rough" colonial variants of Lactobacillus
acidophilus were shown to have non-stainable blebs
protruding from the cell wall and were more sensitive to
freeze-damage and bile salts than "smooth" colonies
(Klaenhammer and Kleeman, 1981).
In Gram-negative bacteria, a large number of mutants
with altered outer membrane permeability have been isolated
(Hancock, 1984). Mutants have been isolated with
alterations of porins, LPS, and lipoprotein. Porin-
deficient mutants have been shown to have higher resistance
to hydrophilic antibiotics in some cases (Hancock, 1984).
Mutants lacking porin proteins apparently compensate by
creating phospholipid patches, creating a pathway for
hydrophobic substances (Nikaido and Vaara, 1985). Deep-
rough organisms with LPS mutations have been shown to have
hyper-susceptibility to hydrophobic antibiotics, deter¬
gents, and EDTA (Hancock, 1984). Other outer membrane gene
mutations include: tolA,B (colicin tolerant), tolC (OmpF
protein), TolD,E (LPS), acrA (lipid A phosphate), and lpo
(Braun lipoprotein).
In summary, enzyme biosynthesis in microorganisms has
been shown to increase enzyme activity by several
thousandfold in a matter of hours. The regulation of enzyme
synthesis determines whether biochemical pathways may
proceed and therefore is among the most important levels of

26
metabolic control. Microbial enzyme synthesis as a tool in
assessing chemical toxicity has not received widespread
attention.
Cell permeability to toxicants is a critical factor
determining the sensitivity of microbial toxicity
bioassays. In Gram-negative bacteria, the cell envelope
limits the diffusion of hydrophilic and hydrophobic
chemicals. Physical, chemical, or genetic manipulations
that alter cell permeability may increase the sensitivity
of microbial toxicity tests.

CHAPTER 3
ENZYME BIOSYNTHESIS VERSUS ENZYME ACTIVITY
AS A BASIS FOR MICROBIAL TOXICITY TESTING
Introduction
A common endpoint measurement employed in toxicity
assessment is the inhibition of enzyme activity (see
Bitton, 1983; Bitton and Dutka, 1986; Dutka and Bitton,
1986, for reviews). For example, Christensen et al. (1982)
determined the effects of 141 water pollutants and other
chemicals on the activity of eight enzymes in vitro. At an
upper concentration limit of 10“^M, 115 (81.6%) of the
chemicals were inhibitory to one or more of -the enzymes
tested. In addition to screening for toxicity, enzyme
activity assays may give insight into the mechanisms of
action of toxic chemicals.
An alternative approach in toxicity testing is to
measure inhibition of enzyme biosynthesis. In bacteria,
certain adaptive or inducible enzymes are kept at low
levels and energy for producing substantial amounts of
these catalysts is conserved until such time as the
required substrate becomes available or a repressor is
removed. This phenomenon is known as enzyme induction or
de novo enzyme biosynthesis. The classic model of an
inducible enzyme is p-galactosidase (Jacob and Monod,
1961).
27

28
In Escherichia coli, (3-galactosidase is produced by a
cluster of genes known as the lactose operon.
The production of this enzyme in most wild-type E. coli
strains is induced by the presence of lactose or synthetic
lactose analogs such as isopropyl-3-D-thiogalactoside
(IPTG). The inducers modify a repressor protein allowing
RNA polymerase transcription of the lac genes. In
addition, the presence of optimal levels of cAMP are needed
to function with the operon promoter site (Dobrogosz,
1981) .
Several studies have dealt with the inhibition of (3-
galactosidase activity (Lederberg, 1950; Katayama, 1984;
Katayama, 1986) as well as inhibition of p-galactosidase
biosynthesis (Koch, 1963, Nakada and Magasanik, 1964). The
scope of these studies was limited to assessing the effects
of heavy metals and certain well known metabolic poisons.
Reinhartz and coworkers first suggested the use of (3-
galactosidase biosynthesis to assess the toxicity of
environmental pollutants (Reinhartz et al., 1987).
The purpose of the present research was to compare the
response of E. coli to selected chemicals, using microbial
toxicity assays based on 3- galactosidase biosynthesis, respectively. Emphasis was
placed on differentiating between inhibition of existing
enzyme activity and inhibition of potential enzyme
biosynthesis. Relative sensitivity was judged on the basis
of responses to the heavy metals Hg^+, Cu^+ and Cd^+ and

29
the organics 3,4-dicchlorophenol (3,4-DCP), formaldehyde,
Hydrothol, phenol, sodium dodecyl sulfate (SDS), and
toluene. These results were compared to IC50s achieved
with other microbial systems, Daphnia bioassay and fish
bioassay.
Materials and Methods
Test Bacteria
The culture maintenance and enzyme assay procedures
followed closely those of Miller (1972). Escherichia coli,
strain C3000 (ATCC# 15597), was maintained in 40% glycerol
at -15°C. Cells were grown by inoculating 50 mL minimal
media in a 125 mL Erlenmeyer flask with 50 uL of glycerol
culture. The bacteria were incubated at 35°C in a shaking
water bath with an oscillation rate of 100 rev/min. They
were harvested in the log growth phase, having reached an
optical density of 0.2-0.4 (420 nm, light path = 1.0 cm).
The minimal media contained the following
constituents: K2HP04, 10.5 g/L; KH2P04, 4.5 g/L; (NH4)2S04,
1.0 g/L; sodium citrate 2H20, 0.5 g/L; yeast extract, 0.5
g/L; MgS04, 1 mL from a 20% stock solution; and glycerol,
10 mL from a 20% stock solution. The magnesium and glycerol
solutions were autoclaved separately from the salt
solution.

30
Test Chemicals and Reagents
Distilled water used in preparing chemical and reagent
solutions was sterilized by autoclaving. The chemicals
assessed for toxicity were: Hg2+ (HgCl2), Cu2+
(CuS04•5H20), Cd2+ (CdCl2), phenol, 3,4-dichlorophenol
(3,4-DCP), sodium dodecyl sulfate (SDS), formaldehyde,
toluene, and the aquatic herbicide Hydrothol (Pennwalt
Corporation, Philadelphia, PA). Hydrothol is the trade
name for the alkylamine salt of 7-oxabicyclo[2.2.1]heptane-
2, 3-dicarboxylic acid. Stock solutions of heavy metals,
phenol, 3,4-DCP, SDS, formaldehyde, and hydrothol were
prepared in distilled water. Toluene was dissolved in
dimethylsulfoxide (DMSO).
Unless otherwise indicated, all chemicals and reagents
were obtained from Sigma Corporation (St. Louis, MO). IPTG
was prepared by dissolving 50 mg reagent in 50 mL distilled
water. Ortho-nitrophenyl-B-D-galactopyranoside (ONPG) was
prepared by dissolving 400 mg reagent in 100 mL distilled
water.
The buffer solution for the assay of enzyme activity
(Z-buffer) contained the following components:
Na2P04 * 7H20, 16.1 g/L; NaH2PO4‘H20, 5.5 g/L; KC1 0.75 g/L;
and MgS04*7H20, 0.25 g/L. Other reagents employed in the
SDS, 0.1% (w/v); chloroform; and Na2C02, 1M.
assays were:

-i ±
Test Procedures
Protocols for the enzyme activity and enzyme
biosynthesis assays are shown in Figures 3-1 and 3-2,
respectively. Each of the assays had the same basic steps:
grow cells in minimal medium, induce cells to produce (3-
galactosidase, and measure (3-galactosidase. The protocols
differed with regard to the point in the sequence at which
cells were exposed to the toxicants. In the enzyme
activity assay, cells were induced before the exposure
step. Enzyme activity was therefore high at the beginning
of the toxicant contact period. A decrease of enzyme
activity relative to the controls during this period could
be attributed mainly to toxicant action on the enzyme. In
the enzyme biosynthesis test, induction followed the
exposure of cells to the toxicants. Enzyme activity at the
beginning of the toxicant contact period was therefore
negligible. At the end of the contact period, the inducer
was added and additional time was allowed for biosynthesis
of (3-galactosidase. Differences between final enzyme
levels (as measured by activity) were attributed mainly to
toxicant effect on the lac operon, rather than on the
enzyme itself.
All incubations were carried out at 35°C in a shaking
water bath. Controls received distilled water in lieu of
toxicant. When toluene was the toxicant under
investigation, the control consisted of 5% DMSO. In the

32
ENZYME
ACTIVITY ASSAY
STEP 1. CELL GROWTH:
grow E. coli in minimal medium
overnight at 35°C
STEP 2. ENZYME INDUCTION:
add 0.1 mL IPTG to 1.0 mL cells
incubate for 30 min.
STEP 3. EXPOSURE TO TOXICANT:
wash cells twice in distilled water
add 0.1 mL toxicant to 0.9 mL induced and washed cells
incubate for 30 min.
STEP 4. Í3-GALACTOSIDASE MEASUREMENT:
add 0.8 mL Z-buffer, 50 uL SDS,
50 uL chloroform and 0.2 mL ONPG
incubate until color develops
stop reaction with 1 mL cold Na2C03
measure absorbance at 420nm
Figure 3-1. Protocol for [3-galactosidase activity.

33
ENZYME
BIOSYNTHESIS ASSAY
STEP 1. CELL GROWTH:
grow E. coli in minimal medium
overnight at 35°C
STEP 2. EXPOSURE TO TOXICANT:
add 0.1 mL toxicant to 0.9 mL cells
incubate for 30 min.
STEP 3. ENZYME INDUCTION:
add 0.1 mL IPTG to 1.0 mL cells
incubate for 30 min.
STEP 4. (3-GALACTOSIDASE MEASUREMENT:
add 0.8 mL Z-buffer, 50 ]±L SDS,
50 uL chloroform and 0.2 mL ONPG
incubate until color develops
stop reaction with 1 mL cold Na2C03
measure absorbance at 420nm
Figure 3-2. Protocol for f3-galactosidase biosynthesis
activity.

34
enzyme activity test, blanks had distilled water
substituted for the ONPG, whereas in the enzyme
biosynthesis test, blanks had distilled water substituted
for the IPTG.
Beta-galactosidase was measured using ONPG. This
compound is
colorless, but in
the presence
of
(3-
galactosidase
it
is converted
to galactose
and
o-
nitrophenol.
The
o-nitrophenol
is yellow and
can be
quantified by measuring its absorption at 420 nm. Cells
were treated with SDS and chloroform to release (3-
galactosidase immediately prior to adding ONPG. Color
development was allowed to continue for approximately 15
minutes, after which the reaction was stopped by adding
Na2C03 solution.
Data Analysis
Preliminary range finding was carried out to determine
toxicant dilutions causing between 10% and 90% inhibition.
The degree of inhibition was determined on the basis of
measured absorbance values, considering the control to
represent 0% inhibition. Data were plotted in terms of
percent inhibition versus log final toxicant concentration.
The concentration giving 50% inhibition (IC50) was derived
from linear regression analysis of the data. At least three
replicate tests were carried out on each toxicant. These
definitive runs were conducted using five toxicant
dilutions for each test.

35
Results and Discussion
Comparison of Enzyme Activity and Enzyme
Biosynthesis Assays
The enzyme activity test was carried out with cells
centrifuged (5000 x g? 10 min.), washed twice, and
resuspended in distilled water. This treatment removed the
inducer and, in addition, avoided precipitation of heavy
metals by phosphate salts present in the growth medium.
Preliminary experiments confirmed the need for washing as
the toxicity of Cu2+ and Cd2+ was greatly diminished in the
presence of the growth medium. This effect was not observed
with Hg2+, however. Unwashed cells were used in the enzyme
biosynthesis assay because preliminary experiments
indicated that washed cells could not be induced.
Development of an enzyme biosynthesis test protocol
utilizing washed cells, however, could improve the assay's
sensitivity to heavy metals.
Responses of the two assays to heavy metals and toxic
organics are compared in Table 3-1. The two assays had
equivalent sensitivities for Cd2+ only. IC50s of Cu2+,
Hg2+ and formaldehyde determined via enzyme biosynthesis
were significantly lower (p < 0.01) than those determined
via enzyme activity. The sensitivity of the respective
tests varied by a factor of 3 for copper, 39 for mercury,
and 90 for formaldehyde. The enzyme biosynthesis test was

36
TABLE 3-1. Relative sensitivity of [3-galactosidase
activity and biosynthesis assays to selected toxicants.
Chemical
IC50
(mg/L)a
Enzyme
Activity Test
Enzyme
Biosynthesis Test
Cd2+
9.2 + 13.6
8.2 + 2.4
Cu2+
0.82 + 0.15
0.24*+ 0.021
Hg2 +
1.26 + 0.22
0.032*+ 0.001
Formaldehyde
541 + 29.1
6.0*+ 0.1
Phenol
> 10,000
851*+ 139
SDS
> 10,000
350*+ 77
Toluene
> 30,000
369*+ 25
3,4-DCP
--
11.9 + 0.67
Hydrothol
--
4.6 + 0.1
a Mean + one std. dev. IC50s for biosynthesis assay
followed by an asterisk (*) are significantly different
(p < 0.01) from the enzyme activity test IC50s.

37
also more sensitive to phenol, SDS, and toluene.
Inhibition of (3-galactosidase activity was negligible to
these chemicals at concentrations as great as 10,000 mg/L.
It is interesting to note that the variability of the
enzyme biosynthesis test results was consistently less than
that of the enzyme activity test results. For example,
respective coefficients of variation for Cd^+ were 148%
versus 29%; those for formaldehyde were 5.4% versus 1.7%.
Based on these results it would appear that the assay
based on inhibition of (3-galactosidase activity is
moderately sensitive to heavy metals and insensitive to
toxic organics. These conclusions are consistent with
previous research on [3-galactosidase and other enzyme
systems. Lederberg (1950 ), in his work on the
characterization of (3-galactosidase, showed that Cu^+ and
Hg2+ salts inactivated enzyme activity at concentrations of
10-3 M (64 mg/L Cu2+, 200 mg/L Hg^+). Christensen et al.
(1982) noted that heavy metals caused a high degree of
inhibition to a wide spectrum of enzyme classes, whereas
the effect of organic chemicals appeared to be more
specific to the nature of the various enzyme classes. The
broad toxicity of heavy metals to enzymes suggests a
common, non-specific interference with enzyme function.
Metalloids such as arsenic may also be relatively toxic to
enzymes. For example, Reinhartz et al. (1987) determined
that the concentration of arsenite required to effect a
certain degree of inhibition of (3-galactosidase activity

38
was eight times greater than the amount required to inhibit
to the same degree the biosynthesis of this enzyme. This
factor compares favorably to the differential sensitivity
found in this study between the respective tests. The
insensitivity of (3-galactosidase activity to organics
should not be surprising in view of the fact that benzene,
butyl alcohol, chloroform, isoamylalcohol, phenol, SDS and
toluene have been used in various studies to extract (3-
galactosidase from cells prior to measurement of enzyme
activity (Lederberg, 1950; Rotman, 1958; Miller, 1972).
Comparison of Enzyme Biosynthesis to other Toxicity Tests
Sensitivities of the |3- and other aquatic toxicity tests are compared in Table 3-2.
The alternative assays considered are based on INT-
dehydrogenase activity (wastewater dehydrogenase),
bioluminescence (Microtox) and mortality of aquatic
organisms (daphnids and fish), respectively. The enzyme
biosynthesis test was generally more sensitive than the
wastewater dehydrogenase test and compared favorably with
the others. It was the least sensitive of all the
alternatives only for SDS. This result may be due to the
limited permeability of wild-type E. coli strains (Sampson
and Benson, 1987).
In conclusion, the results clearly show de novo (3-
galactosidase biosynthesis to be promising as the basis for

39
TABLE 3-2. Sensitivity of the (3-galactosidase biosynthesis
test relative to other microbial systems,
Daphnia bioassay and fish bioassay.
Chemical
Toxicity test endpoints
(mg/L)
B-Gal.
biosyn.
IC50a
Wastewater
dehydrog.
IC50b
Microtox
5-15 min
EC50c
Daphnia
48 h
LC50d
Fish
96h
LC50d
Cd2+
8.2
—
25-690
0.065
1-100
Cu2 +
0.24
0.59
0.28-19.5 0
.02-.06
0.
10-10
Hg2 +
0.032
0.07
0.02-0.06
0.03
0.
01-0.9
Formaldehyde
6.0
11.6
8.7-904
--
10-100
Phenol
851
1530
26-41
12-32
5-100
SDS
350
106
1.1-3.2
7-13
5-46
3,4-DCP
11.9
74
--
--
--
Hydrothol
4.6
--
2.4e
0.36
0
.3-1.6
Toluene
369
—
50
310
20-36
a Based on inhibition of p-galactosidase biosynthesis (this
study) .
b Based on inhibition of INT-dehydrogenase activity
(Dutton et al., 1986).
c Based on inhibition of bioluminescence (Chang et al.,
1981; Qureshi et al., 1982; McFeters et al., 1983;
Plotkin and Ram, 1984; Greene et al., 1985).
d Based on lethality (Finlayson, 1980; LeBlanc, 1980;
Pennwalt Corp., 1980; Qureshi et al., 1982;
McFeters et al., 1983).
e
Bitton et al., unpublished.

40
testing the impact of environmental toxicants. On the
other hand, the direct measurement of (3-galactosidase
activity to assess toxicity is not warranted, with the
possible exception of toxicity due to heavy metals or
metalloids.

CHAPTER 4
INHIBITION OF g-GALACTOSIDASE BIOSYNTHESIS IN
ESCHERICHIA COLI: A FUNCTION OF OUTER MEMBRANE
PERMEABILITY TO TOXICANTS
Introduction
Inhibition of enzyme biosynthesis by environmental
toxicants has received relatively little attention. Early
in the elucidation of the mechanism of enzyme synthesis
investigators demonstrated inhibition of enzyme synthesis
using metabolic poisons, such as azide, chloramphenicol,
formaldehyde, and 2,4-dinitrophenol (Monod, 1947; Hahn and
Wisseman, 1951; Koch, 1964; Nakada and Magasanik, 1964).
More recently, Naveh et al.(1984) developed a bioassay
for antibiotics that inhibit protein synthesis using a
mutant luminescent bacteria (Photobacterium leiognathi).
This dark mutant reverts to luminescence emission in the
presence of DNA-intercalating agents. The bacteria are
exposed to potential enzyme synthesis inhibitors, then
luminescence is induced with proflavin, a known mutagen.
Cenci et al. (1985) demonstrated that heavy metals
inhibit [3-galactosidase biosynthesis in Escherichia coli.
The use of a toxicity screening assay based on the
inhibition of p-galactosidase biosynthesis in E. coli was
more fully developed by Reinhartz et al. (1987). These
investigators examined the inhibitory effect of several
pesticides and other toxicants on the synthesis of
41

42
p-galactosidase. The test was developed as a commercial
kit using freeze-dried E. coli (Orgenics Ltd.).
The complex envelope structure of gram-negative
bacteria is known to consist of cytoplasmic membrane, a
rigid peptidoglycan cell wall, and an .outer membrane
(Nikaido and Vaara, 1985). The outer membrane is a matrix
composed of phospholipid (30%) and protein (50%),
surrounded by an external fringe of lipopolysaccharide
(LPS). The outer membrane structure is an effective
diffusion barrier to hydrophobic substances. The diffusion
of hydrophilic compounds is restricted by specific membrane
proteins that form water filled channels, called porins
(Nikaido and Vaara, 1985; Nakae, 1986). Enteric bacteria,
such as E. coli, are particularly resistant to hydrophobic
insult due to an additional hydrophilic antigenic component
(Koch and Schaechter, 1985).
The resistance of gram-negative microorganisms to
antibiotics due to the outer cell permeabiltiy barrier is
of serious clinical concern and has been addressed in
several comprehensive reviews (Costerton and Cheng, 1975;
Lugtenberg and Van Alphen, 1983; Godfrey and Bryan, 1984;
Hancock, 1984; Nikaido and Vaara, 1985; Nakae, 1986).
The objective of this research was to examine the
inhibition of (3-galactosidase biosynthesis in E. coli by
selected environmental toxicants. The function of cell
permeability in determining overall assay sensitivity was
also an integral goal of these studies. Physical and

43
chemical treatments known to alter â–  outer membrane
permeability were examined for their ability to increase
E. coli susceptibility to toxicants, as measured by
inhibition of (3-galactosidase biosynthesis. In addition,
E. coli mutants with altered membrane permeability were
screened for increased sensitivity to selected
environmental toxicants.
Materials and Methods
Test Bacteria
Assays were conducted using the following bacterial
strains: (1) a derivative of E. coli K12 (strain C3000 ;
ATCC# 15597), (2) various strains obtained from Dr. Barbara
Bachmann, Coli Genetic Stock Culture, Yale University, New
Haven, CT, and designated CGSC# 3004, 4698, 4699, 4923,
4924 , 5163 , 5634 , 6098 and 6683 , respectively, and (3)
strains DC2 and UB1005 obtained courtesy of Dr. David
Clark, Department of Microbiology, Southern Illinois
University, Carbondale, Illinois. To insure genetic
stability all strains were maintained in 40% glycerol at
-150C (Miller, 1972).
Test Chemicals and Reagents
Test chemicals and reagents used were: Cd^+ (CdCl2),
phenol, pentachlorophenol (PCP), hexachlorocyclohexane
(lindane), sodium dodecyl sulphate (SDS), EDTA
(Na2EDTA-2H20), octylphenoxy polyethoxyethanol (Triton X-

44
100), polyoxythylene sorbitan monooleate (Tween 80),
benzydimethylhexadecylammonium chloride, benzethonium chlo¬
ride, polyethylenimine (PEI), polymyxin B sulfate, and 7-
oxabicyclo-[2.2.1.]heptane-2,3-dicarboxylic acid (Hydro-
thol; Per.nwalt Corporation, Philadelphia, PA). All test
chemicals with ther exception of Hydrothol were purchased
from Sigma Corporation, St. Louis, MO. All stock solutions
were prepared in distilled water with two exceptions. PCP
was prepared by dissolution in dilute NaOH (0.01N; pH
adjusted to 7.0), and lindane was dissolved in methanol.
In the lindane tests, the final concentration of methanol
in controls and toxicant reaction mixtures was 2.5% (w/v).
At this concentration, methanol was not inhibitory to
enzyme synthesis.
Assay reagents were: isopropyl-f3-D-thiogalactoside
(IPTG) and o-nitrophenyl-(3-D-galactoside (ONPG) both of
which were obtained from Sigma Corp. (St. Louis, MO). IPTG
was prepared at 0.1% (w/v) and ONPG at 0.4% (w/v). The
solutions were stored for up to for one month in the dark
at 4°C. Enzyme assay buffer (Z-buffer) contained:
Na2HP04•7H20, 16.1 g/L; NaH2PO4'H20, 5.5 g/L; KCl 0.75 g/L;
and MgSO4-7H20, 0.25 g/L, and was adjusted to pH 7.0.
Other reagents employed were: SDS (0.1% and 10% w/v),
Na2C02 (1M), chloroform; 3-[N-Morpholino]propane sulfonic
acid (MOPS).

3-Galactosidase Biosynthesis Assay Procedure
Bacteria for the (3-galactosidase biosynthesis assay
were grown by inoculating LB growth medium (1% tryptone,
0.5% yeast extract, 1% sodium chloride) with 50 uL of stock
glycerol culture. Cells were incubated at 35°C overnight.
The culture was then diluted with fresh LB medium to
optical density A55Q = 0.2 and allowed to grow to A55Q =
0.6-0.65 (= 5x10^ cells/mL).
Protocol for the (3-galactosidase biosynthesis toxicity
test is given in Figure 4-1. The assay consists of the
following basic steps: (1) grow cells, (2) wash cells, (3)
expose cells to toxicant, (4) cell induction for
(3-galactosidase, and (5) measurement of (3-galactosidase
activity.
Extraction of (3-galactosidase was normally
accomplished with SDS and chloroform as depicted in Figure
4-1 (see Step 4). It was also noted that 100 uL of SDS
(10%, w/v) could replace 50 uL chloroform plus 50 y.L SDS
(0.1%, w/v) as an extractant of intracellular (3-
galactosidase. Increasing the SDS concentration does not
have an inhibitory effect on (3-galactosidase activity.
This observation is particularly useful when the assay is
adapted to a microplate reader where solvent and light may
interact causing serious interference in absorbance
readings (Appendix A).

(3 — GALACTOSIDASE
BIOSYNTHESIS ASSAY
STEP 1. CELL GROWTH:
grow E. coli in LB medium
overnight at 35°C
STEP 2. CELL PREPARATION:
dilute cells with fresh media to A55Q = 0.2
allow to grow to A550 = 0.6
wash cells in NaOl (0.85%)
STEP 2. EXPOSURE TO TOXICANT:
add 0.9 mL toxicant to 0.1 mL cells
incubate reaction mixture for 30 min.
STEP 3. ENZYME INDUCTION:
to each 1 mL reaction mixture add
0.1 mL IPTG, 0.4 mL BGAL buffer, and 0.5 mL growth media
incubate for 30 min.
STEP 4. (3-GALACTOSIDASE MEASUREMENT:
add 0.8 mL Z-buffer, 50 y.L SDS,
50 uL chloroform and 0.2 mL ONPG
incubate until color develops
stop reaction with 1 mL cold Na2C03
measure absorbance at 420nm
Figure 4-1. Protocol for |3-galactosidase
biosynthesis activity.

Blanks consisted of all assay components except the
inducer (IPTG) and accounted for any background enzyme
activity or non-enzymatic degradation of the substrate
(ONPG).
Data Analysis
The degree of sample inhibition was determined by
measuring A420 values of test samples with respect to the
control, which did not have any toxicant present (and
assigned 0% inhibition). Extrapolation of the sample
concentration giving 50% inhibition (IC50) was derived from
linear regression analysis in terms of percent inhibition
versus sample concentration (mg/L). Four or more
triplicate dilutions were run on each sample. Statistical
analysis consisted of significance tests using the t test,
one-way analysis of variance (ANOVA), and Dunnett's
multiple comparison test.
Physical Treatment Procedures
E. coli strain C3000 were used throughout the
following experiments unless otherwise indicated.
Heating. Cells were harvested by centrifugation,
washed, split into two fractions and resuspended in 2-
buffer or in 0.85% (w/v) NaCl. Cells were subjected to
heating for 15, 30 and 60 minutes at 50°C in a hot air

48
incubator following the protocol of Mackey (1983). The (3-
galactosidase bicsynthesis toxicity assay was run on the
heat treated samples.
Freeze-thaw. The protocol of freeze-thawing of saline
washed cells (Mackey, 1983) completely inhibited (3-
galactosidase biosynthesis. Therefore, cells were concen¬
trated (2X) by centrifugation, resuspended in MOPS buffer
(0.02M, pH 7) and frozen at -15°C for 1, 7, 14, and 21
days. Thawing was at 3 5°C on a rotary shaker. The (3-
galactosidase biosynthesis protocol (Figure 4-1) was
modified as follows: (1) frozen cells were thawed in a 35°C
water bath for 5 minutes, (2) then 0.1 mL thawed cells were
added to 0.1 mL concentrated LB growth medium (5X), 0.1 mL
IPTG, and 0.7 mL Z-buffer, (3) then 1.0 mL of toxicant
dilution or control water was added, (4) this 2.0 mL
reaction mixture was incubated at 35 °C for 3 hours and
(3-galactosidase production was measured (as in Figure 4-1).
Freeze-drying. Cells were grown to A55Q = 0.65,
centrifuged, concentrated two times by resuspension in half
the original volume in sterilized 2% gelatin (after Sinskey
and Silverman, 1970), or in MOPS (0.02PI, pH 7) containing
a-D-trehalose (22.5 g/L), NaCl (0.07%, w/v), and MgCl2
(0.0012%, w/v) after Reinhartz et al. (1987). Three
milliliter aliquots were frozen for 4-6 hours at -60°C in
serum bottles, then freeze-dried for 20-24 hours, and

sealed while still under vacuum. A manifold-type freeze
dryer was employed (model FDX-3-55A, FTS Systems, Inc.,
Stoneridge, NY).
Osmotic shock. The methods of Forsberg et al. (1972)
and Falla et al. (1988) with minor modification were used.
Bacterial cells (A550 = 0.6) were washed twice in NaCl
(0.85%) at 10,000 x g at 4°C for 10 minutes, and the
supernatants were decanted. Then the cells were suspended
in half the original volume with sucrose (0.5M) and washed
three times at 10,000 x g at 4°C for 15 minutes. Between
sucrose washings cells were incubated with shaking for 15
minutes at 25 °C. The (3-galactosidase biosynthesis assay
followed the normal protocol (Figure 4-1), except cells
were not washed further with saline and induction was
extended to 90 minutes.
Chemical Treatment Procedures
Penicillin. Penicillin G (Sigma Corp., St. Louis, MO)
1662 units/mg was prepared fresh and filter sterilized (0.2
UM) . Cells were diluted in fresh LB to A55Q = 0.3 and
amended with penicillin (0.05 mg/mL; final concentration).
After 60 minutes of exponential growth, cells were washed
in NaCl (0.85%). The [3-galactosidase biosynthesis toxicity
assay was performed at this point.
Tris-EDTA. The procedure of Leive (1967) was
followed. Cells were washed in Tris buffer (0.1 M, pH 8),
concentrated 10X, treated with EDTA (0.5mM) for 30 seconds

5C
and diluted 10-fold to original volume with saline. All
steps were conducted at room temperature, as cold
centrifugation (4°C) with Tris impairs metabolic processes
(Leive, 1967 and confirmed in this study).
Polyethylenimine (PEI) and Polymyxin. The poly-
cationic polymers PEI and polymyxin were screened for
inhibition of p-galactosidase biosynthesis. Stock polymer
solutions were prepared by dissolution in distilled water.
Sub-lethal concentrations of 7.5 mg/L and 2 mg/L (final
concentration; w/v) were determined for PEI and polymyxin,
respectively. Polycation treatment consisted of a 30
minute incubation with cells at 35°C (at 2 or 7.5 mg/L),
followed by washing the treated cells with NaCl (0.85%).
The 0-galactosidase biosynthesis toxicity assay was
performed at this point.
Others. Other chemical treatments included: Tris
(Irvin et al., 1981; Elespuru and Moore, 1986), CaCl2
treatment (Silhavy et al., 1984), novobiocin (Brock, I960;
Ryan, 1979), EDTA at 1000 mg/L, the non-ionic detergents
Tween 80 at 1% (w/v) and Triton X-100 at 50 mg/L, the
anionic detergent SDS at 50 mg/L, and the cationic
detergents benzydimethylhexadecylammonium chloride and
benzethonium chloride, both at 0.5 mg/L. These treatments
were not effective at sensitizing f3-galactosidase
biosynthesis to SDS or PCP, and therefore no results are
reported here.

Results and Discussion
Preliminary Experiments
Concentration of growth media. In initial studies
with E. coli C3000 , a wild type strain, the effect of
growth media on sensitivity of cells to toxicants was
examined (Table 4-1). Cells were grown overnight in full
strength LB, LB/4, or LB/10 and the culture was adjusted to
A550 = 0.2 with fresh LB, LB/4, or LB/10. Cells were not
washed as is depicted in the definitive protocol (Figure 4-
1). In addition, as noted in Table 4-1, the reaction
mixture (cells to toxicant ratio is 9 to 1) differs from
the definitive mixture (cells to toxicant ratio is 1 to 9).
The results show that cells grown in 1/10 strength
growth medium were significantly more susceptible
(p < 0.05) to cadmium and PCP than cells grown in LB or
LB/4 strength media. This effect was not demonstrated by
SDS.
The effect of washing cells. The effect of various
washing treatments on the inhibition of (3-galactosidase
biosynthesis by selected chemicals is given in Table 4-2.
In this case the cells are grown in full strength LB, but
diluted 10-fold with test toxciant. Cadmium toxicity, as
demonstrated above, appears to be a function of media

o
TABLE 4-1. Effect of growth media on inhibition of
(3-galactosidase biosynthesis in E. coli C3000
Media
concentration3
(IC50s; mg/L)k
Cadmium
PCP
SDS
LB (control)
8.2 + 0.40
35 + 1.5
>5000
LB/4
6.7 + 1.93
45 + 4.0
>5000
LB/10
2.2*+ 0.20
24*+ 0.9
>5000
a Cells grown in LB, LB/4, or LB/10 and adjusted to A55Q =
0.2. Test reaction mixture = (0.9 mL cells + 0.1 mL
toxicant).
b Mean + one std. dev. Means followed by an asterisk (*)
indicates significant difference (p = 0.05) from controls.

TABLE 4-2. Effect of washing treatments on inhibition
of pi-galactosidase biosynthesis in E. coli C3000
Washing
treatment3-
(IC50s; mg/L)k
Cadmium
SDS
PCP
Nonwashed
0.7 + 0.18
> 5000
21
+
4.7
(control)
LB media
1.0 + 0.19
> 5000
—
NaCl
1.3 + 0.18
395*+ 80
17
+
1.0
(0.85%)
MOPS buffer
0.13*+ 0.006
272*+ 62
19
+
2.2
(0.02M; pH 7)
a Cells grown in LB to A55Q = 0.6. Test mixture = (0.1 mL
cells + 0.9 mL toxicant).
k Mean + one std. dev. Means followed by an asterisk (*)
are significantly different (p = 0.05) from controls.

o
TABLE 4-3. Median inhibitory concentration data for
(3-galactosidase biosynthesis in E. coli strains with
mutations that may be relevant to cell permeability
Strain
namea
CGSC#
Mutations
(IC50s;
PCP
mg/L)k
SDS
C3000
—
(wild type; control)
25 +
0.9
395 +
80
EWib
5634
tolC5
1.0*+
0.15
26* +
1.5
RE10 3
4698
cmlA, rpsLIOl
6* +
1.0
335 +
40
D22
5163
envAl, rpsL173
9* +
1.0
•Jr
185 +
5
A592
4923
tonA21, tonAl
9* +
1.0
270* +
23
RE107
4699
ompF625, rpsLIOl
14* +
1.1
255* +
10
C600
3004
tonA21
23 +
2.0
270* +
14
A593
4924
tonA21, tolB2
27 +
1.9
250*+
24
X2844
6683
tsx-462::TnlO
44 +
4.2
285* +
31
BW3 22
6098
zia-207;:TnlO
46 +
2.9
204* +
12
DC 2
—
abs mutant of
UB1005
14*±
4.2
—
UB1005
—
nalA37
34 +
7.2
—
a Cells grown in LB to A55Q = 0.6.
Test reaction mixture = (0.1 mL cells + 0.9 mL toxicant).
k Mean + one std. dev. Means followed by an asterisk (*)
are significantly different (p = 0.05) than controls.

3 O
However, outer membrane protein mutants apparently
compensate this structural void by filling in the outer
membrane with phospholipids, thus enhancing diffusion of
hydrophobic compounds (Nikaido and Vaara, 1985).
Wild type versus outer membrane mutant. The wild type
E. coli strain (C3000) and the outer membrane mutant strain
(EWlb) were compared with respect to chemical inhibition of
[3-galactosidase biosynthesis (Tables 4-4 and 4-5). Cells
washed in saline and diluted 10-fold with toxicant (as in
Table 4-5) are generally more sensitive than in unwashed,
undiluted cells (as in Table 4-4). This is particularly
true for cadmium and SDS.
The median inhibitory concentrations (IC50s) for a
heavy metal (Cd^+), a hydrophilic herbicide (Hydrothol), a
surface active compound (SDS), a mildly hydrophobic
organic (phenol; log Kow = 1.48 Brooke et al., 1986), and a
very hydrophobic organic (PCP; log Kow = 5.01 Westall et
al. 1985) indicated that EWlb was significantly more sen¬
sitive (p < 0.05) to PCP and SDS than the wild type strain.
The response of EWlb to hydrophilic compounds, however, is
slightly less sensitive compared to wild type cells.
Physical Permeabilizing Treatments
Heating. Heating experiments were initiated by
screening of unwashed cells (strain C3000) incubated at
58°C for 0, 10, 20, 30 minutes, followed by exposure to SDS
(100 mg/L) , and then f3-galactosidase biosynthesis assay.

57
TABLE 4-4. Median inhibitory concentration data
for (3-galactosidase biosynthesis in E. coli strains C3000
and EWlb grown in LB/10 medium
Toxicant
(IC50s;
mg/L)a
C3000b
EWlbb
Cadmium
2.2* +
0.20
6.8 +
0.12
Hydrothol
9.8 +
0.29
11.3 +
0.70
Phenol
1330 +
300
1680 +
160
PCP
24 +
0.9
2.5* +
0.12
SDS
>5000
42* +
1.5
a Mean + one std. dev. Means followed by an asterisk (*)
are significantly more sensitive (p < 0.05) to toxicant
than the adjacent strain (C3000 or Ewlb).
b Cells grown in LB/10 and adjusted to A550 = 0.2.
Test reaction mixture = (0.9 mL cells + 0.1 mL toxicant).

TABLE 4-5. Median inhibitory concentration data
for the [3-galactosidase assay using E. coli
C3000 and EWlb grown in LB medium
(IC50s; mg/L)a
Toxicant
C3000b
EWlbb
Cadmium
1.3
+
0.18
1.7 + 0.21
Hydrothol
9.1
*±
0.25
.15.2 + 0.65
Phenol
950
+
38
1475 + 248
PCP
25
+
0.9
1.0*+ 0.15
SDS
395
+
80
26*+ 1.5
EDTA
2130
+
446
> 1000
Triton X-100
110
+
9
—
BZ chloride0
2.5
+
0.08
—
BMHA chloride^
0.97
+
0.205
—
PEIe
50
+
0.5
a Mean + one std. dev. Means followed by an asterisk (*)
are significantly more sensitive (p < 0.05) to toxicant
than the adjacent strain (C3000 or EWlb).
b Cells grown in LB to A550 = 0.6.
Test reaction mixture = (0.1 mL cells + 0.9 mL toxicnat).
c BZ chloride = Benzothonium chloride.
b BMHA chloride = Benzydimethylhexadecylammonium chloride.
e Young et al. (unpublished data).

59
Twenty minute exposure was promising (SDS; 100 mg/L = 65%
inhibition),. However, when the experiment was rigorously
controlled, by washing cells in saline and heating at 50°C
(after Mackey, 1983), (3-galactosidase biosynthesis was
completely inhibited. Heating experiments were abandoned
after attempts to replicate the preliminary findings
failed.
Freeze-thaw and freeze-drying. Preliminary freeze¬
drying experiments employing gelatin (Sinskey and
Silverman, 1970) and a MOPS solution (Reinhartz et al.,
1987) both proved satisfactory. The Reinhartz preparation
with MOPS buffer gave slightly elevated production of (3-
galactosidase in controls and was used in definitive
freeze-drying experiments.
E. coli C3000, freeze-dried or frozen for 1, 7, or 14,
days were compared with regard to their sensitivity to SDS
and POP (Table 4-6). Freeze-drying does not sensitize
cells as readily as freeze-thawing after 7 days. However,
freezing was more stressful to cells, decreasing enzyme
biosynthesis over time to such an extent that the
experiment could not be extended to 21 days. On the other
hand, no such decrease in overall (3-galactosidase synthesis
occurs in freeze-dried cells for up to two months.
Sinskey and Silverman (1970) showed that freeze-dried
cells were sensitized to actinomycin D as measured by
inhibition of (3-galactosidase biosynthesis. They noted
that synthesis of (3-galactosidase was delayed 300 minutes

TABLE 4-6. The effect of physical perraeabilization
treatments and the inhibition of (3-galactosidase
biosynthesis in E. coli (C3000)
(IC50s;
mg/L)a
Treatment
SDS
POP
Non-treated control
3 95a+
80
25e+
0.9
Freeze-thaw
day 1
107b+
8
llF+
0.7
day 7
60c+
5
4g+
0.2
day 14
5 2c+
2
1.5H+
0.10
Freeze-dry
115d+
20
00
H
1 +
1.5
Osmotic shock
> 450
71±
1.2
a Mean + one std. dev. Means followed by the same letter
are not significantly different (p = 0.05).

in minimal medium and 150 minutes in 0.1% casamino acids
medium. In this study, p-galactosidase synthesis is delayed
approximately 30 minutes with wild type cells (C3000) and
60 minutes with an outer membrane mutant (EWlb). The
increase in recovery time may be partly attributed to the
richer recovery media used in this study (LB media versus
minimal media).
Osmotic shock. A procedure designed to isolate or
remove the outer bacterial membrane (Falla et al., 1988),
successfully sensitized cells to POP, but not to SDS (Table
4-6). Washing in sucrose did not increase cell
susceptibility to SDS, the opposite response of earlier
washing experiments (Table 4-2).
In a related study, Maruo et al. (1969) reported
forming sphaeroplasts and protoplasts (membrane prepa¬
rations) using lysozyme and Tris-EDTA in sucrose. (3-galac-
tosidase biosynthesis was demonstrated, but at 0.1-1.0
percent the enzyme activity of intact cells (and in our
hands sphaeroplast preparations did not produce (3-galac-
tosidase). Of particular interest was that addition of POP
(5 X 10"^M; 0.05 mg/L) to membrane preparations resulted in
complete inhibition of (3-galactosidase biosynthesis. Intact
cells were not tested with POP in this study, but as E.
coli K12 was the test bacterium one would expect the level

62
of inhibition to occur at PCP concentrations 100-1000 times
higher (e.g.
, 5-50 mg/L) .
This
observation reflects
the
capacity of
the E. coli
outer
membrane to resist
the
permeation of hydrophobic chemicals, such as PCP.
Chemical Permeabilizing Treatments
Penicillin and Tris-EDTA. Tris-EDTA and penicillin
treatment both succeeded in sensitizing strain C3000 to PCP
(Table 4-7). Tris-EDTA treatment of E. coli Ewlb did not
sensitize cells to cadmium or SDS (Table 4-8). Tris-EDTA
treated EWlb cells appear to be sensitized to PCP.
Tris-EDTA treatment was difficult to replicate due to
high toxicity in some tests. Tris-EDTA treatment is toxic
after a few minutes (Leive and Kollin, 1967), and therefore
must be removed or diluted. In addition, Tris-EDTA
treatment must be conducted at room temperature as low
temperature (4°C) causes Tris to be highly toxic releasing
cell nucleotides (Leive and Kollin, 1967).
Penicillin treatment of cells must be conducted in
actively growing cultures. Then penicillin can intercalate
with dividing cell walls and permeabilize the outer
membrane (Hamilton-Miller, 1966).
PEI and Polymyxin. E. coli C3000 treated with PEI
(7.5 mg/L) and polymyxin (2 mg/L) were exposed to selected

63
TABLE 4-7. Inhibition of [3-galactosidase biosynthesis by
PCP in E. coli C3000 treated with Tris-EDTA and penicillin
% Inhibition
PCP (mg/L)
Non-treated
Tris-EDTA
Penicillin
0
0
0
0
2.25
0
0
0
22.5
56 + 7.9
86 + 6.2
98 + 1.0

TABLE 4-8. Inhibition of (3-galactosidase
biosynthesis by selected toxciants in E. ooli EWlb
treated with Tris-EDTA
% Inhibition3
Toxicant
(mg/L)
Non
-treated
Tris-EDTA
Control
0
0
0
Cadmium
in
o
42
+
5.5
0
2.5
100
100
PCP
0.9
17
+
2.6
21
+ 8.1
h-1
00
50
+
nj
m
77
+ 6.4
SDS
5
9
+
3.5
0
10
52
+
4.9
65
+ 8.5
a
Mean + one std. dev.

toxicants (Table 4-9). PEI treatment resulted in a
significant increase (p = 0.05) in the toxicity of
Hydrothol, PCP, and EDTA. This sensitization is even more
pronounced for cells treated with polymyxin.
E. coli EWlb treated with polymyxin (Table 4-10) were
rendered significantly more sensitive (p < 0.05) to SDS,
EDTA, and lindane, but not to PCP. It is not clear why
EWlb, an outer membrane mutant, was not sensitized to PCP
while it was dramatically sensitized to another hydrophobic
compound (e.g., lindane). It is noteworthy that PCP is
more hydrophobic than lindane. The octanol-water partition
coefficients (log Kow) for PCP and lindane, are 5.01 and
3.53, respectively (Westall et al., 1985; Hermens et al.,
1985).
It has been proposed that EDTA and polycationic
aminoglycosides, such as polymyxin B, destabilize the Gram¬
negative outer membrane by displacing Mg^+ from
lipopolysaccahride molecules (Hancock and Wong, 1984).
EDTA removes LPS Mg^+ by chelation, while polymyxin
apparently binds to the membrane competing for Mg^+ binding
sites (Hancock and Wong, 1984; Vaara and Vaara, 1983).
In conclusion, the sensitivity of (3-galactosidase
biosynthesis, in E. coli, to toxic chemicals was enhanced
by physical and chemical permeabilizing treatments.
Overall, the most sensitive response to surface active
agents and to hydrophobic toxicants was achieved employing

66
TABLE 4-9. Median inhibitory concentration data
for (3-galactosidase biosynthesis in E. coli C3000 treated
with PEI and polymyxin
IC50s; (mg/L)a
Toxicant Non-treated PEI Polymyxin
control
Hydrothol
9.1
+
0.25
7.2* +
0.22
6.1* +
0.31
Phenol
950
+
38
1173 +
180
1015 +
59
PCP
25
+
0.9
17* +
1.7
4.7* +
0.13
SDS
395
+
80
—
100*+
5
EDTA
2130
+
446
1075 +
153
—
Cadmium
1.3
+
0.18
1.5 +
0.22
—
a Mean + one std. dev. Means followed by an asterisk (*)
indicates a significantly more sensitive response
(p < 0.05) to test toxicant than controls.'

67
TABLE 4-10. Median inhibitory concentration data for
(3-galactosidase biosynthesis in E. coli EWlb
treated with polymyxin
Toxicant
IC 5 0 s ;
(mg/L)a
Non-treated
control
Polymyxin
PCP
1.0 + 0.15
1.1 + 0.03
SDS
26 + 1.5
16* + 1.1
EDTA
> 1000
490*+ 29
Lindane
> 100
15*+ 0.9
Cadmium
1.7 + 0.21
2.2 + 0.52
a Mean + one std. dev. Means followed by an asterisk (*)
indicates that a significantly more sensitive (p < 0.05)
response to test toxicant than controls.

an outer membrane mutant (E. coli EWlb) treated
polymyxin.
68
with

CHAPTER 5
ASSESSMENT OF (3-GALACTOSIDASE BIOSYNTHESIS: TOXICITY
TESTING IN WATER AND WASTEWATER
Introduction
Microbial assays have been widely applied for
wastewater toxicity screening (see Bitton and Dutka, 1986;
Dutka and Bitton, 1986, for reviews). The purpose this
study was to evaluate induction of the lac operon in E.
coli for toxicity screening of wastewater. Comparisons were
made with Ceriodaphnia dubia and bacterial luminescence
(Microtox) bioassays.
The Microtox system (Microbios Corporation, Carlsbad,
CA) is based on inhibition of luminescence of the marine
bacterium, Photobacterium phosphoreum. The commercial
availability of Microtox is an integral part of its success
as well as the fact that it has been validated in
comparative studies with conventional toxicity bioassays
(Qureshi et al. 1982; Plotkin and Ram, 1985).
Disadvantages inherent in the assay include: (1) the assay
is osmotically balanced with 2% NaCl, possibly causing
metal-chloride complexation of heavy metals, particularly
cadmium, and (2) luminescence decays with time, requiring
precise timing and limiting the number of samples that can
be processed simultaneously.
69

70
Ceriodapnia dubia are among organisms recommended for
measuring the acute toxicity of effluents by the U.S. EPA
(Peltier and Weber, 1985). The toxicity endpoint in this
assay is lethality over 24-48 hours. The main difficulty in
conducting the assay is culture maintenance and the
duration of the test.
The sensitivity of E. ooli (3-galactosidase
biosynthesis and the two bioassays described above was
judged on the basis of the inhibitor response to various
chemicals and wastewater effluents. Previously (see
Chapter 4), it was found that chemical treatment E. coli
with polymyxin (2 mg/L; final concentration) increased
significantly the sensitivity of the assay- to surface
active and hydrophobic chemicals. In addition, the use of a
mutant with an outer membrane protein alteration (Tol C
gene), sensitized further by polymyxin treatment,
resulted in a greatly improved toxicity bioassay. In the
present study, polymyxin-treated E. coli (strain EWlb) was
employed for all definitive toxicity testing.
The Buckman wastewater treatment plant, Jacksonville,
Florida, was used a a model wastewater system. This
facility had a mean flow of 1.85 m-^/s in 1986. Primary
sludge and waste activated sludge are treated with a
polymer and thickened by centrifugation prior to
incineration, with céntrate returned to the plant
headworks.

Materials and Methods
Sampling and Activated Sludge Treatment
Wastewater samples were collected from the collection
system, plant influent and secondary effluent before
chlorination. Activated sludge treatment was simulated by
allowing the collected samples to settle for one hour, then
two-thirds (2000 mL) of the liquid volume was decanted. A
1400 mL aliquot of settled wastewater was mixed with 600 mL
return activated sludge from the Buckman plant and aerated
for 4 hours. The aeration period was followed by one hour
of settling. Then 600 mL of treated effluent was decanted
from the batch reactor. Samples were stored at 4°C for 24
hours before analysis.
Test Bacteria
Assays were conducted using a derivative of E. coli
K12 (strain C3000, a phage host) and E. coli EWlb, a strain
obtained from the Coli Genetic Stock Culture (CGSC #
5634)), Dr. Bachmann, Curator, Yale University, New Haven,
CT. To insure genetic stability all strains were maintained
in 40% glycerol at 15°C.
Test Chemicals and Reagents
The test chemicals assessed for toxicity were: Cd2+
(CdCl2), ' cu2+ (CuSO4‘5H20) , CN" (KCN), phenol,
pentachlorophenol (PCP),- lindane, sodium dodecyl sulphate

72
(SDS) , EDTA (Na2EDTA•2H2O ) , methanol, and Hydrothol
(Pennwalt Corporation, Philadelphia, PA). All stock
solutions were prepared in distilled water with the
following two exceptions. PCP was prepared by dissolution
in dilute NaOH (0.0IN) and pH was adjusted to 7.0. Lindane
was dissolved in methanol. In the case of lindane, the
final concentration of methanol in the assay was 2.5%
(w/v). This level of methanol was included in controls and
was not inhibitory to enzyme biosynthesis.
Dilutions of toxicant stock solutions were made in
reconstituted, moderately hard freshwater that contained
the following components: NaHCC>3, 96 mg/L; CaSO42H20, 60
mg/L; MgS04, 60 mg/L; and KCl, 4 mg/L (Peltier and Weber,
1985). The pH at equilibrium was 7.4-7.8.
(5-galactosidase assay reagents were: isopropyl-(3-D-
thiogalactoside (IPTG) and o-nitrophenyl-f3-D-galactoside
(ONPG), both obtained from Sigma Corp. (St. Louis, MO).
IPTG was prepared at 0.1% (w/v) and ONPG at 0.4% (w/v).
Both solutions were filter sterilized and stored protected
from light at 4°C for up to one month. Enzyme assay buffer
(pH 7.0) contained: Na2HP04•7H20, 16.1 g/L; NaH2PO4'H20,
5.5 g/L; KCl 0.75 g/L; and MgSO4’7H20 , 0.25 g/L. Other
reagents employed were: SDS, 0.1% (w/v); Na2CÜ3 (1M); and
chloroform.
3-Galactosidase Biosynthesis Assay Procedure
Protocol for the (3-galactosidase biosynthesis toxicity
test was given previously in given Figure 4-1. Briefly,

73
the assay consisted of the following steps: (1) grow cells,
(2) wash cells, (3) expose cells to test sample, (4) cell
induction for (3-galactosidase, and (5) measurement of
(3-galactosidase . activity. Bacteria were grown by
inoculating LB growth medium (1% tryptone, 0.5% yeast
extract, 1% sodium chloride) with 50 y.L of stock glycerol
culture. The culture was incubated at 35°C overnight then
diluted with fresh LB to an absorbance of 0.2-0.3 at 550nm
and allowed to grow to A550 = 0.6 ( = 5x10® cells/mL).
Controls contained only dilution water (0 mg/L; no
toxicant). Typically, the controls produced the highest
level of |3-galactosidase, unless the test chemical causes
stimulation of enzyme induction. Blanks consisted of all
assay components except the inducer (IPTG). The blanks
indicated any background enzyme activity or non-enzymatic
degradation of the substrate (ONPG).
The degree of sample inhibition was determined on the
basis of measuring A420 values with respect to the controls
(assigned 0% inhibition). The sample concentration giving
50% inhibition (IC50) was derived from linear regression
analysis in terms of percent inhibition versus sample
concentration. Four or more triplicate dilutions were run
on each sample.

Polymyxin Permeabiiizinq Treatment
Polymyxin B sulfate (Sigma Corp., St. Louis, MO) was
screened for inhibition of f3-galactosidase biosynthesis in
saline-washed cells and a sublethal concentration of 2 mg/L
(in strains C3000 and EWlb) was determined. Polymyxin was
dissolved in distilled water and stock solutions were kept
at 4°C for up to one month. Polymyxin treatment (2mg/L
w/v; final concentration) consisted of exposing saline-
washed ceils at 35 °C for 30 minutes, and then the cells
were washed in saline again to remove excess antibiotic.
Microtox and Ceriodaphnia dubia Bioassays
Microtox and C. dubia bioassays were conducted
concurrently with ¡3-galactosidase in an adjacent laboratory
by students under the direction of Dr. Koopman (Dept.
Environmental Engineering, University of Florida).
Lypholized Photobacterium phosphoreum (Microbics
Corporation, Carlsbad, CA) were reconstituted for use in
the Microtox test. Data were tabulated and reduced
according to the Microtox Operating Manual (Microbics,
1982). All assays were carried out at 15°C with a 15
minute contact time with the toxicant. Samples causing 50%
reduction in bacterial luminescence compared to controls
are referred to as EC50s.
Ceriodaphnia dubia bioassays were conducted according
to U.S. EPA guidelines (Peltier and Weber, 1985). Results
were based on organism mortality after 48 hours (LC50s.).

Expression of Wastewater Toxicity
Toxic units (Brown, 1968) were used to express
wastewater toxicity as a proportion of its lethal threshold
concentration. The threshold value was taken as the
respective IC50, EC50, or LC50. Thus:
100 % Waste
Toxic Units (TU) =
IC50, EC50, or LC50
where the IC50, EC50, or LC50 is expressed in % waste.
Results and Discussion
3-galactosidase versus Microtox and C. Dubia Assays: Effect
of Selected Chemicals
Inhibitory effects of selected chemicals toward
(3-galactosidase biosynthesis, Microtox, and C. dubia assays
were compared (Table 5-1). The 3-galactosidase assay was
conducted using E. coli EWlb, which was washed with saline
and subsequently treated with polymyxin. Ceriodapnia
dubia was the most sensitive assay tested for 7 of 9
compounds. 3-galactosidase was 1 to 10 times less
sensitive than C. dubia bioassay, except for phenol where
it was
100 times
less
sensitive.
Microtox
compared
favorably
with C.
dubia
for phenol
and was
the most
sensitive
assay for
SDS.
Microtox,
however, was greater
than 100 times less sensitive for cadmium than C. dubia.

TABLE 5-1. Median inhibitory concentrations (mg/L)
of toxic pollutants as determined by Ceriodapnia
dubia, Microtox, and 0-galactosdiase biosynthesis assaysa
Chemical
Assay
(LC50, ]
EC50 &
IC50)b
C.
dubiac
Microtoxc
(3-galactosidase
Cadmium
0.15
+
0.011
25
0.46
+
0.021
Copper
0.03
+
0.006
0.5 +
0.05
0.5
+
0.01
Cyanide
1.0
+
0.35
2.8 +
0.01
3.6
+
0.42
Phenol
14
+
7.1
11 +
1.9
1540
+
190
PCP
0.3
+
0.04
1.2 +
0.21
1.1
+
0.03
Lindane
1.5
+
0.23
—
15
+
0.93
SDS
10
+
2.9
1.5 +
0.33
16
+
1.1
EDTA
98
+
9.3
—
490
+
29
Methanol
11000
+
3000
42000 +
5700
43,800
+
5720
a [3-galactosidase bioassay with E. coli EWlb treated with
polymxin.
b Mean + one std. dev.
c Voiland et al. (unpublished data).

Microtox employs a marine test organism
(Photobacterium phosphoreum) that requires a relatively
high osmotic solution strength. This is provided in the
Microtox assay by adjusting the sample to 2.0% NaCl.
Hinwood and McCormick (1987) substituted sucrose (20 %) for
the NaCl in the Microtox assay and demonstrated
significantly higher toxicity with certain metals (e.g.,
cadmium, nickel, and zinc). This led to the suggestion
that chloro-complexes may be reducing metal toxicity
(Hinwood and McCormick, 1987).
Wastewater Toxicity. Two toxic wastewater samples
from the Buckman wastewater system in Jacksonville,
Florida, were more than five times more toxic to
(3-galactosidase biosynthesis in polymyxin treated EWlb
cells than in untreated EWlb cells (Table 5-2). Wild type
cells (C3000) were not inhibited by the samples. The
results indicated that toxicity was caused by a hydrophobic
organic compound, as C3000 and EWlb cells respond equally
to heavy metals and hydrophilic compounds (Table 4-1).
Toxicity of wastewater samples was determined by the
three assays on two occasions (Figure 5-1). The inhibitory
effect to the (3-galactosidase assay and the Microtox system
was comparable in two of four toxic wastewater samples (5b
and 8d) . Inhibition of |3-galactosidase biosynthesis corre¬
lated with C. dubia lethality in one of four toxic samples
(sample 2e). The (5-galactosidase assay was not inhibited
by three samples (8d, 10a, and cent.) that demonstrated

TOXIC UNITS TOXIC UNITS
WASTEWATER STATIONS
Figure 5-2. Wastewater toxicity to (3-galactosidase
biosynthesis and other assays. Comparison to
Microtox-top; C. dubia-bottom.

79
toxicity in the C. dubia bioassay. Sample 8c was toxic to
(3-galactosidase at the highest effluent concentration
(90%), but was not inhibitory to C. dubia.
Conclusions regarding complex effluents are often
difficult to formulate, but the (3-galactosidase response
was less sensitive overall than Microtox or C. dubia on
these two sampling dates. Further testing of wastewater
samples is required to determine if inhibition of (3-galac¬
tosidase biosynthesis would be useful in screening
wastewater toxicity.
TABLE 5-2. Effect of polymyxin treatment on test
sensitivity to toxic wastewater samples
Test
organism
Wastewater Sample
(Toxic units)
5b
8d
C3000
0
0
EWlb
1.1
1.9
EWlb + polymyxin
> 9
> 9

CHAPTER 6
INHIBITION OF BIOSYNTHESIS OF ENZYMES CONTROLLED BY
DIFFERENT OPERONS: A COMPARISON OF
(3-GALACTOSIDASE, a-GLUCOSIDASE AND TRYPTOPHANASE
Introduction
The main focus of the three previous chapters was to
determine the effect of toxic chemicals and wastewater on
(3-galactosidase synthesis in Escherichia coli. The present
chapter addresses the effect of toxic chemicals on the
inducible biosynthesis of other enzymes, namely,
tryptophanase in E^_ coli and a-glucosidase in Bacillus
licheniformis.
The kinetics of enzyme induction and derepression of
(3-galactosidase, a-glucosidase, and tryptophanase are under
similar overall genetic regulatory control (e.g., enzyme
formation is initiated when genetically regulated
repressors are inactivated). However, each of the operon
systems has unique regulatory features (Pardee and
Prestidge, 1961; Pollock, 1961; 1963; Bilezikian et
al.,1967; Botsford and DeMoss, 1971).
Comparisons of the inhibitory response of different
operons is limited to a few examples in the literature. The
preferential inhibition of enzyme synthesis by chloram¬
phenicol was studied by Sypherd et al. (1962). Sublethal
amounts of chloramphenicol (0.8 ug/mL) preferentially
80

81
inhibited the synthesis of tryptophanase, (3-galactosidase,
and citritase by 70%, 68%, and 58%, respectively. D-serine
deaminase synthesis was not inhibited.
Alkaline phosphatase synthesis was about five times
more sensitive to procaine hydrochloride than was
(3-galactosidase synthesis (Tribhuwan and Pradhan, 1977).
The inves-tigators suggested that procaine, a membrane
active anesthetic, more easily inhibited periplasmic
alkaline phosphatase than intracellular (3-galactosidase
(Tribhuwan and Pradhan, 1977).
Finally, Pollock (1963) showed that actinomycin D
(0.05 ug/mL) halted a-glucosidase biosynthesis without
affecting penicillinase formation in B. licheniformis. It
was proposed that the differential effect of actinomycin
may be due to differences in its affinity for the DNA of
the different genes.
The objective
of
this study
was to compare
three
inducible enzyme
systems;
(3-galactosidase
and
tryptophanase, in
E.
coli, and
a-glucosidase
in B.
lichenif ormis. Biosynthesis of (3-galactosidase is induced
by lactose or lactose-analogs. This enzyme catalyzes the
degradation of' lactose to galactose and glucose.
Tryptophanase degrades tryptophan to indole, pyruvate and
ammonia. It is induced by tryptophan. Biosynthesis of a-
glucosidase, an extracellular enzyme, is induced by

maleóse. The enzyme degrades maltose into glucose residues
acting at the 1,4-a-glycoside linkages.
Materials and Methods
Test Bacteria
Assays were conducted using a laboratory E. coli
strain (C3000) and E. coli (EWlb) obtained from the Coli
Genetic Stock Culture (CGSC# 5634) , Yale University, New
Haven, CT. Bacillus licheniformis (strain 749) was
obtained from the Bacillus Genetic Stock Center (BGSC#
5A20), Ohio State University, Columbus, OH. To insure
genetic stability all strains were maintained in 40%
glycerol at -15°C.
Test Chemicals
The test chemicals assessed for toxicity were: Cd^+
(CdCl2), phenol, pentachlorophenol (PCP), sodium dodecyl
sulphate (SDS), and Hydrothol (Pennwalt Corporation,
Philadelphia, PA) . All stock solutions were prepared in
distilled water with one exception. PCP was prepared by
dissolution in dilute NaOH (0.01N) and pH was adjusted to
7.0.
General Assay Protocols
The 3-galactosidase, tryptophanase, and a-glucosidase
assays had the following- basic steps in common: (1) cell

33
growth, (2) washing of cells, (3) exposure to toxic test
sample dilution, (4) induction of enzyme biosynthesis, and
(5) measurement of enzyme activity. In all three cases
bacteria were grown overnight by inoculating growth media
with 50 y.L of stock glycerol culture. The cultures were
incubated at 35 °C overnight and then the cultures were
diluted with fresh growth medium to A550 = 0.2 and allowed
to grow to A550 = 0.6-0.7 (= 5x10^ cells/mL in the case of
E. coli).
Assay for (3-Galactosidase Biosynthesis
Induction and assay for (3-galactosidase biosynthesis
were undertaken according to the methodology described
earlier (Figure 4-1).
Assay for Tryptophanase Biosynthesis
Tryptophanase biosynthesis was determined by a
modification of the color test for indole with Ehrlich's
reagent (Pardee and Prestidge, 1961). The modified assay
is outlined in Figure 6-1. E. coli were grown overnight in
a medium consisting of the following ingredients: casein
hydrolysate without tryptophan (lOg/L; ICN Nutritional
Biochem., Cleveland, OH), yeast extract (5g/L), and NaCl
(10 g/L).
Phosphate buffer for tryptophan induction contained
KH2PO4 (13.6 g/L) adjusted to pH = 7.8. Ehrlich's reagent
consists of 5 parts p-dimethyl-amino-benzaldehyde (5 %,

TRYPTOPHANASE BIOSYNTHESIS
ASSAY
STEP 1. CELL GROWTH:
grow Escherichia coli
overnight at 35°C
STEP 2: CELL PREPARATION:
dilute cells with fresh medium to A55q=0.2
allow to grow up to A55q=0.6
wash cells once in distilled water
STEP 3: EXPOSURE TO TOXICANT:
add 0.9 mL toxicant to 0.1 mL cells
incubate 30 min.
STEP 4: ENZYME INDUCTION:
add 0.4 mL buffer, 0.1 mL L-tryptophan
and 0.5 mL fresh medium
incubate for 120 min.
STEP 5: TRYPTOPHANASE MEASUREMENT:
add 1.0 mL Ehrlich's reagent
incubate 15 min.
measure absorbance at 568nm
Figure 6-2.
Protocol for tryptophanase
biosynthesis bioassay.

85
w/v) in 95% ethanol and 12 parts acid-alcohol (16 mL cone.
H2SO4 in 200 mL 95% ethanol). The incorporation of
pvridoxal phosphate cofactor (Pardee and Prestidge, 1961)
did not increase sensitivity of the assay, and was not
included in definitive experiments. Similar observations
were made by Botsford and DeMoss (1971).
Assay for a-Glucosidase Biosynthesis
The protocol for inhibition of a-glucosidase is
outlined in Figure 6-2. B. licheniformis (strain 749) was
grown overnight in trypticase soy broth-without dextrose
(27.5 g/L) plus yeast extract (5 g/L). In some studies the
growth medium was amended with polyoxyethylene sorbitan
monooleate (Tween 80, 10 g/L; w/v).
Optimal assay conditions were based on previous work
conducted on induction of a-glucosidase in this strain
(Pollock, 1963). The induction assay buffer was the Z-
buffer used previously in assay for |3-galactosidase
activity (Chapter 4). The inducer solution, maltose, was
dissolved in water (4 %; w/v) and then autoclaved. The p-
nitrophenyl-a-D-glucoside (PNaG) chromogen solution was
prepared by dissolving 0.4g/100 mL in distilled water. This
solution was filter sterilized and stored at 4°C in an
amber bottle.
a-Glucosidase was assayed by measuring the absorption
at 420nm of the p-nitrophenol that is liberated by enzyme
hydrolysis of PNaG. No extractant was necessary to measure

86
a-GLUCOSIDASE
BIOSYNTHESIS ASSAY
STEP 1. CELL GROWTH:
grow Bacillus licheniformis
overnight at 30°C
STEP 2: CELL PREPARATION:
dilute cells with fresh medium to A55q=0.2
allow to grow up to A55q=0.6
wash cells once in distilled water
STEP 3: EXPOSURE TO TOXICANT:
add 0.9 mL toxicant to 0.1 mL cells
incubate 30 min.
STEP 4: ENZYME INDUCTION:
add 0.4 mL buffer, 0.1 mL maltose
and 0.5 mL fresh medium
incubate for 60 min.
STEP 5: a-GLUCOSIDASE MEASUREMENT:
add 0.2 mL NPaG
incubate until color develops (= 30-60 min)
stop reaction with 1 mL Na2C03
measure absorbance at 420nm
Figure 6-3.
Protocol for a-glucosidase biosynthesis assay.

37
o-glucosidase production because it is an extracellular
enzyme. Full expression of the enzyme requires cell lysis
with lysozyme or sonication (Pollock, 1961).
Data Analysis
In all three assays, controls were incubated without
toxicant in dilution water (0 mg/L; no toxicant).
Typically, the controls produced the most enzyme, unless
stimulation had occurred. Blanks consisted of all assay
components except the inducer. Blanks indicated
constitutive background enzyme activity or non-enzymatic
degradation of the substrate (e.g., ONPG, NPaG or Ehrlich's
reagent).
The degree of sample inhibition was determined by
measuring absorbance with respect to the control (assigned
0% inhibition). The sample concentration giving 50%
inhibition (IC50) was derived from linear regression
analysis in terms of percent inhibition versus sample
concentration (mg/L). Four or more sample dilutions, in
triplicate, were run on each test chemical. Statistical
analysis consisted of significance tests using the t test,
one-way analysis of variance (ANOVA), and Dunnett's,
multiple comparison test.
Results and Discussion
Relative Sensitivity of Different Operons to Toxicants
The relative response of a-glucosidase, tryptophanase,
and p-galactosidase biosynthesis to selected toxicants are

given in Tables 6-1 and 6-2. The compounds tested included
a heavy metal (cadmium), a detergent (SDS), a herbicide
(hvdrothol) and two phenols (phenol and PCP).
Inhibition of (3-galactosidase in wild type E. ooli
(C3000) is resistant to hydrophobic chemicals (PCP) and
surface active chemicals (SDS), as was discussed in
previous chapters. This finding is corroborated by the
response with tryptophanase synthesis in E. coli (C3000).
A significantly more sensitive (p < 0.05) response to SDS
and PCP was obtained with E. coli (EWlb), which has a
deficient outer membrane due to a mutation in the tol C
gene (Whitney, 1971; Davies and Reeves, 1975).
In B. licheniformis a-glucosidase biosynthesis was
significantly more sensitive (p < 0.05) to SDS and PCP than
E. coli C3000. Gram-positive bacteria, such as B.
licheniformis, do not have an outer membrane and therefore
tend to be more sensitive than Gram-negative bacteria to
hydrophobic compounds (Koch and Schaechter, 1985). The
inhibitory effect to a-glucosidase biosynthesis in B.
licheniformis was amplified by incorporation of 1% Tween 80
in the growth medium (Table 6-2). Nonionic detergents,
such as Tween (polyoxyethylene esters of sorbitol), cause
bacilli to grow in more dispersed form (Davis et al.,
1980). Sensitization to toxicants by the addition of Tween
could be explained by the increased cell surface area
available for toxicant interaction.

89
TABLE 6-1. Inhibition of tryptophanase and
(3-galactosidase in E. coli
Toxicants
(IC 5 0 ;
mg/L)a
Tryptophan.13
¡3-gal.c
(3-
gal
#d
Cadmium
0.5
+
0.05
1.3 +
0.18
0.5
+
0.02
SDS
180
+
5
395 +
80
16
+
1.1
Hydrothol
3.1
+
0.10
9.8 +
0.28
11.3
+
0.70
Phenol
1960
+
120
1330 +
300
1540
+
190
PCP
19
+
1.8
25 +
0.9
1.1
+
0.03
a Mean + one std. dev.
k Tryptophanase biosynthesis in E. coli (strain C3000).
c (3-galactosidase biosynthesis in E. coli (strain C3000).
d
|3-galactosidase biosynthesis in E. coli (strain EWlb) .

90
TABLE 6-2. Inhibition of a-glucosidase biosynthesis
in B. licheniformis
Toxicants
(IC 5 0; mg/L)a
Non-treated Tween 80 (1%)
control
Cadmium
1.4
+
0.05
—
SDS
65
+
19
12*+
0.
5
Hydrothol
7.2
+
0.26
4.4* +
0.
30
Phenol
1405
+
150
760* +
55
PCP
1.8
+
0.04
+ 1
*
O
T 1
0.
01
a Mean + one std. dev. Means followed by an asterisk (*)
are significantly different (p < 0.05) from controls.

91
In conclusion, Bernhart and Vestal (1983) reported
that a-glucosidase activity (in vitro) was relatively
sensitive to heavy metals (except copper), but completely
insensitive to PCP, phenol, and SDS. The present study
clearly demonstrates that the inhibition of a-glucosidase
biosynthesis is sensitive to PCP, phenol and SDS. It is
reiterated here, as it was for 3-galactosidase (in Chapter
3), that enzyme biosynthesis is more sensitive to toxicants
than enzyme activity. Furthermore, biosynthesis of
a-glucosidase, in B. licheniformis, was more sensitive to
toxciants than either (3-galactosidase or tryptophanse
biosynthesis, in E. coli. Disparity in sensitivity between
the two biosynthetic systems may be a function of
differences in cell permeability.

CHAPTER 7
CONCLUSIONS
The purpose of this study was to evaluate the
effectiveness of enzyme biosynthesis in bacteria for
toxicity testing. The criteria for evaluation included com¬
parisons between enzyme biosynthesis assays in different
operons. Also, direct comparisons were made to standard
acute toxicity tests (e.g., Microtox and Ceriodaphnia) .
The conclusions regarding bacterial enzyme bio¬
synthesis derived in the course of this study were as
follows:
1. Assay for f3-galactosidase activity in Escherichia coli
was sensitive to heavy metals (e.g., mercury, cadmium and
copper), but was not sensitive to organic toxicants (e.g.,
SDS, phenol, formaldehyde and toluene).
2. Assay for de novo biosynthesis of p-galactosidase was
sensitive to both organics and heavy metals.
3. The (3-galactosidase biosynthesis test employing wild
type E. coli (C3000) was generally more sensitive than a
wastewater dehydrogenase test and compared reasonably well
to values reported in the literature for inhibition of
bacterial luminescence (Microtox) and aquatic toxicity
tests (fish and Daphnia). However, the enzyme biosynthesis
test was notably less sensitive for sodium dodecyl sulfate
(SDS) and phenol.
92

9 3
4. A genetically altered outer membrane mutant (E. coll
EWlb) was mere than 10 times more sensitive to SDS and
pentachlorophenol (PCP) than a wild type strain (C3000).
5. In wild type E. coli (strain C3000), freeze-drying,
freeze-thawing, osmotic shock, and various chemical
treatments all successfully increased (3-galactosidase
biosynthesis sensitivity to SDS or PCP.
6. The most effective and reproducible treatment was with
polymyxin, a polycationic antibiotic. Polymyxin is known
to interface in the bacterial outermembrane lipopoly-
saccharide structure, compromising its integrity by
displacing magnesium and calcium.
7. The use of polymyxin treated E. coli (EWlb) dramatically
improved the (3-galactosidase biosynthesis assay response to
toxic wastewater samples. However, the response to toxic
wastewater samples was not as sensitive compared to
Microtox and Ceriodaphnia dubia bioassays.
8. In E. coli (C3000), biosynthesis of tryptophanase was
slightly more sensitive to SDS and Hydrothol than in
biosynthesis of (3-galactosidase. The opposite was true for
phenol.
9. In Bacillus licheniformis, a-glucosidase biosynthesis
was significantly more sensitive to SDS and PCP than in
assays with E. coli (C3000). Gram-positive bacteria, such
as B. lichenif ormis, do not have an outer membrane and
therefore are more susceptible to hydrophobic toxicants.

94
1C. 5. licheniformis a-gluccsidase biosynthesis assay,
amended with Tween 80, resulted in the most sensitive test
overall. This assay should be evaluated for toxicity
screening of wastewater.
Identifying each of the of the compounds in a complex
waste that cause toxicity would be time-consuming and
costly. The impact of of effluent toxicity depends on many
factors (e.g., pH, hardness, suspended solids, toxicant
interactions). Biological testing of effluents has become
an important part of the water quality-based approach for
controlling toxic pollutants (Peltier and Weber, 1985).
The role of microbial toxicity tests is presently
unclear. The greater sensitivity of traditional aquatic
toxicity tests (e.g., fish and daphnids) for certain
compounds must be weighed against the cost and ease of
conducting bacterial assays.
Improved sensitivity of microbial toxicity tests might
be achieved by concentration of toxic substances in water
as the quantity needed for microbial bioassay is small
(McFeters et al. 1983). In addition, as concluded from
this study, the permeability of cells to toxicants is a
critical parameter to be considered in toxicity testing
with microorganisms. Physical, chemical or genetic
manipulations of cell permeability may lead to more useful
short-term toxicity screening tests using bacteria.

APPENDIX A
MINIATURIZATION OF (3-GALACTOSIDASE
BIOSYNTHESIS ASSAY
This appendix describes a method for miniaturization
of the p-galactosidase enzyme biosynthesis protocol. The
procedure uses a 96-well microtiter plate reader called
Titertek Multiskan (Flow Laboratories Inc., McLean, VA) .
The use of microplate plate readers for routine enzyme
activity measurements is not a novel approach (Genta et
al., 1982; Ashour et al., 1987). In fact, a procedure for
(3-galactosidase was discussed by Reinhartz et al. (1987),
but essential details were left out.
The Titertek Multiskan is equipped with a 405nm filter
and the pathlength in the instrument is measured from the
bottom of the plate through the solution rather than across
the cuvette as in conventional photometers. The accuracy of
the volume in each plate well is therefore critical.
Problems are minimized, however, by the large number of
»»
replicates (8 per dilution) that are run. The use of a
repeating pippetor capable of inoculating 8 wells
simultaneously expedites the process.
An important limitation in the use of the Multiskan
plate reader is that only 50, 100, 150, or 200 y.L may be
pippeted with the manufacturer's pippetor. This limitation
95

96
was integrated into the miniaturization procedure for the
(3-galactosidase assay as follows:
1. Premix cells and toxicant or wastewater sample in
troughs (supplied by Flow Laboratories) in a ratio of 1
part bacterial cells to 9 parts sample.
2. Inoculate this reaction mixture in 100 y.L aliguots to a
96-well plate (12 columns X 8 rows). Column 1 (with 8
replicates) serves as a control, and no toxicant is added.
The subsequent columns receive dilutions of the test
toxicant.
3. Incubate for 30 minutes at 35°C.
4. Add 100 i_lL aliquot of premixed buffer, 5X growth media,
and inducer
(mixed in a
ratio
of
8:1:1).
The control
(column 1)
receives the
same,
but
in the
mixture
the
inducer is replaced with water.
5. Incubate
for 60 minutes
at 35°C
•
6. Addition
of 50 y.L SDS
(10%;
w/v)
+ ONPG
(0.2%;
w/v)
(in a ratio of 1:1).
7. After approximately 15 minute for color development,
100 y.L of sodium carbonate (Na2CÜ3, 1M) is added to stop
the reaction.
8. Read absorbance on plate reader at 405nm.
The plate reader instrument is programmed to blank
column 1 (control; no inducer). Reading the plate takes a
few seconds and the results are printed out. Finally, the

97
assay can include an acetate film to seal the plate
minimizing evaporation of volatile test chemicals.
It is important to note that plate readers read
absorbance vertically, therefore the common usage of
chloroform or toluene to extract (3-galactosidase could
cause refractive problems. For this reason chloroform was
replaced with higher amounts of SDS (10%) to extract the
enzyme. This modification to the traditional assay for
3-galactosidase (Miller, 1972) was possible because
3-galactosidase activity is not inhibited by SDS. This does
not appear to be the case with other enzymes (e.g.,
tryptophanase).
Results for E. coli 3-galactosidase biosynthesis
inhibition obtained using the Multiskan plate reader for
cadmium, sodium dodecyl sulphate and pentachlorophenol were
comparable to previous results obtained using a
spectrophotometer (Chapter 4).

APPENDIX B
AN ALTERNATE SUBSTRATE
for (3-GALACTOSIDASE DETERMINATION
Spectrophotometric determinations of (3-galactosidase
are generally conducted using the water soluble substrate
o-nitrophenyl-p-D-galactopyranoside (ONPG). When ONPG is
hydrolyzed by (3-galactosidase it yields the yellow product
nitrophenol, which is measured at 420nm. Many wastewater
samples are inherently yellow and may interfere in
detection of enzyme activity.
This problem could be circumvented by employing
chlorophenol red-(3-D-galactopyranoside (CPRG), available
from Boehringer Mannheim Biochemicals, Indianapolis, IN.
CPRG is another water soluble substrate for [3-
galactosidase. CPRG is light sensitive, but was stable for
several weeks stored at 4°C in the dark. Hydrolyzed CPRG
is red and measured at 574 nm.
In the [3-galactosidase assay described in the previous
chapters the final concentration of ONPG used was 0.04%.
In comparative
experiments
CPRG
was
used
at a final
concentration 0.
005%. E.
coli
induced
with
IPTG for
60
minutes produced
an A42o =
4.4
+
0.43
with
ONPG, and
an
a574 = 1.4 +
0.10 with
CPRG.
As
a result CPRG
is
approximately 2.5 times more sensitive than ONPG.
98

99
Extensive experiments with toxicants were not
undertaken. However, the effect of PCP on (3-galactosidase
biosynthesis in E. coli C3000 (washed in saline) was tested
using the CPRG substrate. The IC50 for PCP using CPRG was
17 mg/L. This is a value comparable to results with ONPG
(Chapter 4).

APPENDIX C
DEVELOPMENT OF OTHER ENZYME BIOSYNTHESIS
TOXICITY TESTS
Penicillinase Biosynthesis Assay
During the course of this research several enzyme
biosynthesis assays were explored that were not reported in
earlier chapters. One of these was penicillinase
biosynthesis in Bacillus licheniformis (see Chapter 6). The
formation of penicillinase is induced in Bacillus by the
addition of penicillin or cephalosporin (Pollock (1961;
1963; Imsande, 1970).
Penicillinase ((3-lactamase) is measured by manometric,
iodometric, acidimetric, or UV spectrometric techniques
(Ross and O'Callaghan, 1975). New approaches using
chromogenic (3-lactamase substrates have greatly simplified
assay for (3-lactamase.
One of these chromophores is nitrocefin (O'Callaghan
et al. , 1972) and another is PADAC (Schindler and Huber,
1980; Jorgensen et al., 1982)., Nirocefin is a very
sensitive chromophore that in the presence of (3-lactamase
produces a red color. Unfortunately nitrocefin is
prohibitively expensive, and was not used in these studies.
The chromophore used was pyridinium-2-azo-p-dimethylanaline
(PADAC; Calbiochem, La Jolla, CA) and has a distinct violet
color (566nm) which turns yellow (450nm) when the [3-lactam
100

10
ring is hydrolyzed. Thoughâ– less expensive than nitrocefin
the cost of PADAC is still high (S69/25mg).
PADAC suffers the serious disadvantage of being
rendered colorless not only by (3-lactamases cleaving the
3-lactam ring, but by the ability of certain bacterial
species to hydrolyze PADAC at the diazo bond (Jorgensen et
al., 1982). This appeared to be the case with the test
organism in this study (e.g., B. licheniformis). This
bacterium, when induced with penicillin in the presence of
toxic chemicals, produced varying amounts penicillinase as
measured by differential loss in PADAC's violet color, but
within 20 minutes all the solutions turned colorless.
Decolorization also occurred if cells were removed by
centrifugation (2500 x g). However, if formalin was added
to the system the decolorization reaction was halted.
The protocol for penicillinase biosynthesis was as
follows:
Step 1. Grow B. licheniformis cells (see Chapter 6)
and dilute cells in fresh media, grow to
a550 = 0.6.
Step 2. Incubate with toxicant
Step 3. Induce with penicillin (2 Units/mL) for 2
hours
Step 4. Add PADAC (10 pL/mL; final concentration)
Step 5. Incubate until control is colorless, about 5
minutes, then add 0.2 mL formalin (37% w/v) to
stop the reaction

102
Step 6. Measure absorbance at 565nm.
An IC50 of 80 mg/L was derived for sodium dodecyl
sulfate (SDS), which compares reasonably to the results of
another extracellular enzyme (a-glucosidase; Chapter 6)
found in Bacillus lioheniformis. IC50s for phenol and
cadmium were about 1000 mg/L and 5 mg/L, respectively. The
relatively poor sensitivity to cadmium is probably due to
incubation in the presence of growth medium, and could be
improved if the cells were washed (see Chapter 4).
PADAC powder stored frozen at -10°C was not stable
after 1 year, despite the manufacturer's claims.
SOS-Chromotest Bioassay for Toxicity
The SOS-chromotest (Quillardet and Hofnung, 1985) is a
quantitative bacterial colorimetric assay for genotoxic
chemicals. It resembles the Ames Test except that results
are generated in a few hours versus 48 hours required in
the Ames Test. The E. coli PQ37 tester strain carries a
sf i: : lacZ fusion and has a deletion in the lac region so
that S-galactosidase activity is strictly dependent on
expression of sf i. DNA damage triggers a response (SOS
response) resulting in accelerated protein synthesis
(induction) of the sfi gene. As the lac gene is coupled to
this gene, (3-galactosidase determination reflects the
inducing ability of the genotoxic test compound. The test
has typical dose-response curve for control mutagenic
compounds (Quillardet and Hofnung, 1985).

103
A toxicity test (for .inhibition of enzyme synthesis)
was developed by introducing a known DNA damaging agent
into the SOS-Chromotest protocol. The compound chosen was
naladixic acid and the optimal concentration in the assay
was determined to be approximately 20-100 ug/mL (final
concentration). The principle of this approach is identical
to that of Naveh et al. (1984) who exposed a dark mutant
bioluminescent bacteria to antibiotics, then introduced a
SOS inducer (the genotoxin proflavine) to initiate back
mutation to light production.
Only preliminary experiments have been conducted to
date. The assay response to sodium dodecyl sulfate was 50-
100 mg/L. The tester strain (PQ37) is constitutive for
alkaline phosphatase, which the authors recommend to use to
test for protein synthesis inhibitors, thus avoiding false
positives. It would be interesting to compare the assay
with naladixic acid to constitutive alkaline phosphatase,
as experience tells us that constitutive enzyme activity
may be less sensitive than enzyme biosynthesis (Chapter 3).
Finally, the E. ooli PQ37 tester strain may be
sensitive to hydrophobic chemicals as it carries an rfa
mutation that renders its outer membrane deficient in
lipopolysaccharide.

REFERENCES
Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for
detecting carcinogens and mutagens with Salmonella/
mammalian-microsome mutagenicity test. Mutat. Res.
31:347-364.
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BIOGRAPHICAL SKETCH
Ronald John Dutton was born May 19 , 1955 , in Flin
Flon, Manitoba, Canada. He spent the following 15 years in
Jinja, Uganda, and Potrerillos, Chile. His family then
moved to Anaconda, Montana, and he {received a scholarship
to attend highschool at the Mercersburg Academy,
Mercersburg, Pennsylvania, where he graduated in 1972. He
then attended McGill University, Montreal, Quebec, for two
years, and then the University of Montana, Missoula,
Montana for two years, graduating in 1978 with a B.Sc. in
biology. Following graduation he worked for two years,
most notably for six months in Sitka, Alaska, with the U.S.
Forest Service. Then he enrolled in the University of
Florida to pursue a Master of Science degree in the
Department of Environmental Engineering, which he obtained
in 1984. He is currently aspiring to complete the
requirements for the Doctor of Philosophy degree in the
same department.
Ronald J. Dutton is a member of the Water Pollution
Control Federation, the Society of Environmental Toxicology
and Chemistry, and the American Society of Microbiology.
He is married to Nana Lopez, and together they have two
sons, Andrew and Adam.
116

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the deqree of Doctor of Philosophy.
Gatói^el Bitton,‘■''Chairman
Processor of Environmental
Engineering Sciences
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
W. Lámar Miller
Professor of Environmental
Engineering Sciences
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Environmental
Engineering Sciences

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Ben L. Koopman
Associate Professor of
Engineering Sciences
nvironmental
This dissertation was submitted to the Graduate Faculty of
the College of Engineering and to the Graduate school and
was accepted as partial fulfillment for the degree of
Doctor of Philosophy.
December, 1988 / d ' /^AjO-í-,
Dean, College of Engineering
Dean, Graduate School

54
strength. Cadmium may be complexed with LB growth medium
components (e.g. , 1% tryptone, 0.5% yeast extract, or 1%
NaCl).
E. coli C3000 washed with either NaCl or MOPS were
significantly more sensitive (p < 0.05) to SDS than
unwashed cells (Table 4-2). MOPS washed cells were also
significantly more sensitive to cadmium. The ability of
SDS to lyse cells is apparently related to the ionic
strength of the suspension medium. In a related study,
Corwin et al. (1971) found that washing E. coli K12 in
phosphate buffer (0.1M) significantly increased SDS
susceptibility, but lower ionic strength phosphate buffer
(0.01M) or Tris buffer did not.
Mutants with Possible Outer Membrane Alterations
Several E. coli strains that carry mutations that were
thought to affect permeability were screened for
sensitivity to SDS and PCP (Table 4-3). (3-galactosidase
biosynthesis assay was carried out according to the
standard protocol (Figure 4-1). The most sensitive strain
examined was EWlb with a mutation resulting in reduced
synthesis of Omp F porin protein, a major outer membrane
porin protein (Whitney, 1971; Misra and Reeves, 1987).
In strain EWlb, the Tol C mutation results in a
deficient production of Omp F porin protein. Porins . are
key hydrophilic pathways, thus a decreased sensitivity to
water soluble compounds might be expected (Hancock, 1984).

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TITLE: Enzyme biosynthesis in bacteria as a basis for toxicity testing / (record
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