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Fate and survival of genetically engineered microorganisms and their recombinant genes in the natural environment

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Fate and survival of genetically engineered microorganisms and their recombinant genes in the natural environment
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Awong, Judy, 1956-
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English
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xii, 225 leaves : ill., photos ; 29 cm.

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Bacteria ( jstor )
DNA ( jstor )
Escherichia coli ( jstor )
Fate ( jstor )
Groundwater ( jstor )
Lakes ( jstor )
Microcosms ( jstor )
Microorganisms ( jstor )
Plasmids ( jstor )
Rhizosphere ( jstor )
Dissertations, Academic -- Environmental Engineering Sciences -- UF ( lcsh )
Environmental Engineering Sciences thesis Ph. D ( lcsh )
City of Gainesville ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1990.
Bibliography:
Includes bibliographical references (leaves 206-224).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Judy Awong.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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FATE AND SURVIVAL OF GENETICALLY ENGINEERED
MICROORGANISMS AND THEIR RECOMBINANT GENES
IN THE NATURAL ENVIRONMENT











BY

JUDY AWONG


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


VIN1VERSJTy OF FLOJDA


1990






























Copyright 1990

by

Judy Awong





























To my parents,
Mary and Phillip Awong
















ACKNOWLEDGEMENTS


The author would like to acknowledge and thank the

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

his advice, support, and encouragement during the course of

this study, and in particular for his role as mentor during

her doctoral candidacy. His guidance over the years has

provided many valuable lessons that will always be greatly

appreciated. The author would also like to thank the

cochairman of her doctoral committee, Dr. G. Rasul Chaudhry,

for his support, encouragement, and enthusiasm during the

course of this study, and for his assistance in developing

the dissertation topic. The author is extremely grateful to

Dr. Chaudhry for providing her the opportunity for research

in a new and challenging area of study.

The author is also grateful to the other members of

her committee, Dr. Thomas L. Crisman, Dr. Ben L. Koopman and

Dr. Sam Farrah, for their support and help during the course

of this study. Special thanks are extended to Dr. Thomas L.

Crisman for his advice, guidance, and friendship during the

course of her academic career.

The author also wishes to thank her fellow students and

friends, in particular, Ulrike Crisman, Dr. Ronald J. Dutton

and Henry Meier, for their strong support and friendship










during the course of this study. Thanks are also due to many

of the staff members of the Department, in particular, Jo

David, Shirley Jordan, Eleanor Humphries, and Eleanor

Merritt, for their understanding and friendly assistance

throughout the years.

Special thanks are given to Stephen A. Taylor for his

love, support, and inspiration during the preparation and

writing of the dissertation.

Finally the author acknowledges with love and gratitude

her parents, Mary and Phillip Awong, for their love,

support, and encouragement during her academic endeavors.

















TABLE OF CONTENTS


page
ACKNOWLEDGEMENTS ....................................... iv

ABSTRACT ................................................ x

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

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

Part I: Recombinant DNA Technologies .............. 5
Introduction ........................... 5
Genetic Manipulation ............................ 5
Part II: Applications of Recombinant DNA Products. 9
Introduction .................................... 9
Potential Environmental Uses ...... ...... 10
Part III: Methods for Testing and Monitoring
Recombinant Organisms in Environments ........ 14
Methods for Assessing Fate and Effects ......... 14
Methods for Detection and Enumeration .......... 17
Methods for Assessing Genetic Stability ........ 21
Part IV: Survival of Genetically Engineered
Organisms and Stability of Recombinant
Plasmids in the Natural Environment .......... 22
Introduction ..................................... 22
Soil, Plant and Rhizosphere Systems ............ 24
Aquatic Systems .................................. 28
Part V: Genetic Transfer by Genetically Engineered
Organisms in the Natural Environment ......... 32
Introduction ............................. 32
Conjugation ...................................... 33
Transduction ..................................... 36
Transformation ................................... 38

3 MICROCOSM DESIGN AND PROTOCOL FOR ASSESSING THE
FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN AQUATIC ENVIRONMENTS .......................... 40

Introduction ........................................ 40
Materials and Methods .............................. 42
Survival Chambers ................................ 42










Microcosm Design .......................... 44
Static renewal system .......................... 45
Flow-through system ........................... 45
Test Protocol for Survival Studies .............. 47
Bacterial strains .............................. 47
Bacterial growth studies ...................... 49
Comparison of plating techniques .............. 50
Comparison of selective media types ........... 50
Comparison of Microcosms for Survival Studies... 51
Results and Discussion ............................ 52
Bacterial Growth Studies ........................ 52
Comparison of Plating Techniques ................ 52
Comparison of Media types ....................... 57
Comparison of Microcosms ........................ 62

4 SURVIVAL OF AND PLASMID STABILITY IN GENETICALLY
ENGINEERED AND WILDTYPE STRAINS OF ESCHERICHIA
COLI AND PSEUDOMONAS PUTIDA IN AQUATIC
ENVIRONMENTS ..................................... 65

Introduction ....................................... 65
Materials and Methods .............................. 68
Bacterial Strains ............................... 68
Aquatic Samples ................................. 69
Survival Experiments ............................ 69
Microcosm design ............................ 69
Experimental procedures ....................... 70
Effect of temperature ......................... 70
Sterile vs non-sterile conditions ............. 71
Toxicant effect ............................... 71
Plasmid stability .............................. 72
Statistical analysis .......................... 72
Results ............................................ 73
Survival and Plasmid Stability in Lake Water .... 73
Temperature dependent studies ................. 73
Sterile vs non-sterile conditions ............. 79
Survival in the presence of a herbicide ....... 81
Plasmid stability ............................. 83
Survival and Plasmid Stability in Activated
Sludge Effluent ................................ 83
Temperature dependent studies ............... 83
Plasmid stability ........................... 93
Survival and Plasmid Stability in Ground Water.. 94
Survival studies ............................... 94
Plasmid stability .............................. 98
Discussion ......................................... 98

5 FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN THE CORN RHIZOSPHERE ........................ 110

Introduction ...................................... 110


vii










Materials and Methods ............................ 112
Hydroponic Cultures ............................ 112
Bacterial Strains ............................... 113
Survival and Growth Studies .................... 114
Interaction between the Genetically Engineered
Bacteria and Rhizosphere Microorganisms ...... 114
Statistical Analysis ............................ 115
Results .......................................... 115
Effect of Root Exudates on the Growth of GEMs.. 115
Comparison of the Growth Patterns of Wildtype
and GEM strains in the Presence of Corn-root
exudates ..................................... 118
Survival of GEMs in a Simulated Rhizosphere in
the Presence of Indigenous Rhizosphere
Microorganisms ............................... 118
Discussion ......................................................... 128

6 STRUCTURAL AND PHYSIOLOGICAL ALTERATIONS OF
GENETICALLY ENGINEERED AND WILDTYPE STRAINS
OF E. COLI AFTER EXPOSURE TO AN AQUATIC
ENVIRONMENT .................................... 133

Introduction ... .o. ..... . .... 133
Materials and Methods...............................135
Bacterial Strains and Culture Media ............. 135
Aquatic Microcosm ............ .. .................. 136
Bacterial Enumeration ........................... 137
B-Galactosidase Biosynthesis .................... 137
INT-Dehydrogenase Activity ..................... 139
Cell Permeability............................... 140
Results.......................................... 141
Bacterial enumeration............................ 141
B-galactosidase biosynthesis ..................... 146
INT-dehydrogenase activity ..................... 148
Cell permeability................................ 148
Morphological changes.......................... 151
Discussion............. .. ................. 152

7 FATE OF EXTRACELLULAR DNA IN AQUATIC SYSTEMS:
IMPLICATIONS AND POTENTIAL FOR GENETIC
TRANSFORMATION................................. 159

Introduction.................................... 159
Materials and Methods ............................... 161
Aquatic Samples ................................. 161
Sample Preparation .............................. 162
Preparation of Extracellular DNA............... 162
Determination of DNA Inoculum Concentration.... 163
DNA Recovery Procedure......................... 163
Sample Background DNA.......................... 164
DNA Degradation Study.......................... 164


viii










Results .......................................... 166
Efficiency of the DNA Recovery Method .......... 166
DNA Degradation: Characterization by Gel
Electrophoresis .............................. 166
Raw sewage ................................. 166
Lake water ................................. 168
Ground water ............................... 169
Tap water .................................. 169
Kinetics of Degradation ........................ 169
Discussion ....................................... 180

8 CONSTRUCTION OF A MODEL GEM FOR USE IN
GENE TRANSFER STUDIES .......................... 190

Introduction ............................. ........ 190
Materials and Methods ............................ 191
Bacterial Strains, Plasmids and Growth
Conditions ................................... 191
Isolation of Plasmid DNA ...................... 191
Restriction Analysis of Plasmid DNA ............ 191
Cloning of Model GEM ........................... 193
Conjugation Studies .............................. 193
Results and Discussion ........................... 194
Construction of the GEM ........................ 194
Conjugation Studies ............................. 195

9 CONCLUSION ....................................... 201

REFERENCES ............................................ 206

BIOGRAPHICAL SKETCH ..................................... 225

















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


FATE AND SURVIVAL OF GENETICALLY ENGINEERED
MICROORGANISMS AND THEIR RECOMBINANT GENES
IN THE NATURAL ENVIRONMENT

By

Judy Awong

August 1990


Chairman: Gabriel Bitton
Cochairman: G. Rasul Chaudhry
Major Department: Environmental Engineering Sciences

Prospects for the routine release of genetically

engineered microorganisms (GEMs) into the environment are

becoming increasingly feasible. Deliberate release of

recombinant microorganisms has, however, raised questions

concerning potential adverse environmental effects. The

purpose of this study was to develop and utilize

laboratory-contained microcosms to study the fate and

survival of GEMs and their recombinant DNA in natural

environments.

A model aquatic microcosm that utilized membrane

diffusion chambers in a flow-through or static renewal

system was used to study the survival of genetically

engineered and wild-type strains of Escherichia coli and










Pseudomonas putida in various aquatic systems. Results

indicate that the GEMs survived better than or as well as

their wild-type counterparts in all systems tested.

The fate of GEMs and their wild-type strains was also

studied in the corn rhizosphere under hydroponic conditions.

Both strains grew well in the presence of root exudates. No

significant difference was noted in growth pattern between

the GEMs and their wild-type counterpart.

The presence of indigenous microorganisms decreased the

survival rate of the GEMs but P. putida was better able to

compete with the indigenous population than E. coli. In lake

water, the herbicide, Hydrothol-191, significantly decreased

the numbers of P. outida, but no significant difference was

observed between the GEM and wild-type strain. The

recombinant DNA of the GEMs remained fairly stable within

the host cell under all conditions tested.

While much attention has focused on the transfer of

intracellular DNA in the environment, there is little

information on the fate of extracellular DNA (eDNA) under

natural conditions. The fate of eDNA was studied in various

aquatic systems. Results indicate that chromosomal DNA is

degraded at a faster rate than plasmid DNA which undergoes a

series of structural changes prior to and during

degradation. Rapid hydrolysis of eDNA occurs in raw sewage

and lake water by cell-associated and extracellular

nucleases. Potential for genetic transformation does exist.










A model GEM was constructed for use in gene transfer

studies. In-vitro filter matings indicate that the mob gene

does cause higher transfer frequencies. However, gene

transfer was not observed in lake water.


xii















CHAPTER 1
INTRODUCTION

The past decade has provided the world with explosive

developments in the area of molecular and cellular

biotechnology. Newly constructed genotypes obtained from

recombinant DNA technology are believed to be of

considerable value for many areas of basic and applied

research. High potential of benefits and large economic

incentives have now promoted biotechnology research in over

10,000 laboratories worldwide (Jain et al., 1988).

To date, almost all research has been limited to

physically contained systems. The number of cases of

deliberate release of genetically engineered bacteria into

the environment is few and fairly recent (Halvorson et al.,

1985). Prospects for the routine release of genetically

engineered organisms (GEOs) into the environment are

becoming increasingly feasible. The potential uses of

recombinant bacteria in the environment include enhancement

in food and agriculture production, biocontrol of insects

and diseases, metal and mineral leaching, environmental

remediation and waste treatment (Gillett et al., 1985;

Halvorson et al., 1985; Johnston and Robinson, 1984).

Deliberate release of GEOs has, however, raised

considerable concern and attention within the public and

scientific sectors. Questions and issues concerning










potential adverse environmental effects are foremost in the

minds of many involved. Genetically engineered

microorganisms (GEMs) that are released may have the

capacity to reproduce, spread beyond the initial point of

release and transfer their novel genetic information to the

indigenous microbial populations. The possibility also

exists that recombinant organisms may behave differently

from their non-recombinant parental strains when released

into the environment.

The effective use of GEMs in biological control, in

improving crop yield and in controlling environmental

pollution depends upon successful colonization at the

appropriate sites. However, the inherent properties that

would allow these organisms to adequately compete, survive,

and propagate in their new environments might also produce

adverse effects on the natural ecosystem.

The concept of deliberate release of GEMs to the

environment requires a clear understanding of their behavior

and survival, their interactions with indigenous organisms,

their potential for spread, their effects on physiochemical

processes, and the ability to detect and monitor the fate of

the organisms and their recombinant genes within a natural

system (Jain et al., 1988; Keeler, 1988; Tiedje et al.,

1989).

It is obvious that many questions relevant to the

safety of GEOs in the environment still need to be addressed

and answered. There is at present a dearth of information










concerning the fate of GEOs in the natural environment.

However, research is already underway in many areas, and the

last several years has produced some useful and relevant

information that can be applied towards better understanding

the fate of GEOs in the environment.

The scientific, legal and policy issues associated with

environmental applications of biotechnology have been

addressed by several federal agencies. At present, four

regulatory agencies (USDA, EPA, FDA and OSHA) share

responsibility for controlling GEOs now covered by existing

laws. Three other agencies (USDA, NIH and NSF) are involved

in overseeing research activities (Fiksel and Covello,

1986). Current guidelines recommend research activities with

recombinant organisms and their products be confined to

contained settings within laboratories. Such guidelines have

made empirical methods such as microcosm testing,

indispensable tools for purposes of risk and fate

assessments.

This dissertation assesses the fate and survival of

GEMs and their recombinant DNA in natural environments. The

research focuses primarily on selected model GEMs developed

by transferring antibiotic or heavy-metal resistance genes

into Escherichia coli and Pseudomonas putida, two commonly

used host systems for recombinant DNA work. The overall

objective of the dissertation was accomplished by developing

and utilizing a microcosm approach to investigate specific

hypotheses.










The specific objectives of this research were as

follows:

1. To determine the fate and survival of genetically

engineered bacteria and their wild-type strains in aquatic

systems (lake water, activated sludge effluent and ground

water) and the corn rhizosphere in controlled environmental

settings.

2. To monitor the stability of the recombinant DNA

within the host organism in the above mentioned systems.

3. To determine and compare structural and

physiological alterations of GEMs and their wild-type

strains after exposure to aquatic environments.

4. To determine the fate of extracellular DNA in

aquatic samples and its potential for genetic transfer.

5. To construct a model GEM for studying genetic

transfer via conjugation.

The following null hypotheses were tested:

1. No significant difference in survival rates between

the GEMs and wild-type strains under given environmental

conditions.

2. Genetic transfer does not occur between GEMs and

other types of bacteria.

3. Recombinant DNA of the GEMs are not stable under

given environmental conditions.

4. No physiological differences between GEMs and wild-

type strains after environmental exposure.















CHAPTER 2
LITERATURE REVIEW


Part 1. Recombinant DNA Technology

Introduction

Recent developments in recombinant DNA biotechnology

offers the potential for unrestrained rearrangement of the

genetic information present in organisms. This allows the

opportunity to custom design organisms to accomplish

specific tasks.

Genetic modifications that are readily envisioned as

being useful to the biotechnology industry include 1)

amplification of the levels of specific enzymes in

microorganism; 2) the rearrangement of regulating DNA base

sequences controlling the expression of specific genes in

response to specific stimuli; 3) the introduction of genes

for new enzymatic functions into organisms which do not

normally possess them and 4) the modification of individual

genes to alter the characteristics of individual enzymes

(Johnston and Robinson, 1984).

Genetic Manipulation

During the course of the last two decades, several

techniques have been developed to aid genetic manipulation.

These are 1) in-vitro recombinant DNA technologies, 2) in-










vivo methods using transposable genetic elements, 3) normal

genetic exchange by means of conjugation, transduction or

transformation, 4) protoplast fusion and 5) generalized and

site-specific mutagenesis (Johnston and Robinson, 1984;

Gillett et al., 1985; Saunders and Saunders, 1987). New

techniques developed for genetic transformation include

electroporation, projectile insertion, nuclear micro-

injection, electroinjection, and the use of liposomes and

spheroplasts (Cocking and Davey, 1987; David et al., 1989;

Holo and Nes, 1989; Morikawa et al., 1986; Reich et al.,

1986; Tiedje et al., 1989). A brief description of these

methods is given below.

In-vitro recombinant technoloav. In-vitro recombinant

DNA technology involves the insertion of a DNA segment from

one organism into another host genome (rDNA host), by using

vector DNA molecules which can be transferred and are

capable of replicating autonomously in the host cell (Jain

et al., 1988). A generalized concept of rDNA technology is

depicted in Figure 2-1.

Transposable genetic elements. Transposons are short

DNA base sequences that are capable of inserting as discrete

nonpermuted DNA sequences into various sites within a

genome. Transposon-mediated mutations are capable of

spontaneous reversion.

Natural sexual genetic exchange. In bacteria, new

combinations of genes can be generated via normal sexual

genetic exchange. These exchange mechanisms include













Chromosofnal DNA


/Plasmid vector
D DNA







Isolate plasmid


I
0

Digestion with
restriction enzyme
6

QLigation with /
T4 DNA ligase


Recombinant
plasmid


N Foreign
genes)

/

Digestion with
restriction enzyme





/

/ Freign
ene(s)


Chromosome

1 0)




Genetically engineered
microorganism (GEM)


Figure 2-1. Generalized concept of recombinant DNA
technology.
Source: Adapted from Jain et al., 1988.










conjugation, transformation and transduction. Conjugation

involves the transfer of DNA from a donor to a recipient

bacterium and requires cell-to-cell contact. The transfer

(tra) genes required for conjugation are encoded by

conjugative plasmids. In transduction, DNA transfer is

mediated by a temperate bacteriophage. These 'transducing

phage' may introduce novel DNA from donor cells to recipient

cells. Transformation is the process by which microorganisms

take up extracellular or 'naked' DNA and subsequently

acquire an altered genotype (Smith et al., 1981; Stewart and

Carlson, 1986). In all three processes, chromosomal and

plasmid DNA can be transferred from the host to the

recipient bacterium.

ProtoDlast fusion. Induced protoplast fusion has been

reported in both prokaryotic and eukaryotic microorganisms

(Hopwood and Wright, 1978; Peberdy, 1979). In this process,

bacteria lose their cell walls, their protoplasts fuse and

the cell walls are regenerated. After fusion, genetic

rearrangements can occur to give rise to new gene

combinations.

MutaQenesis. Mutations are heritable changes in genetic

material that can occur spontaneously or can be induced.

Errors in DNA replication and misrepair of DNA damage can

influence the occurrence of spontaneous mutations (Saunders

and Saunders, 1987). Induced mutations can also be achieved

by various chemical and physical agents termed mutagens.









Recent methodologies in genetic transformation. In

recent years, new techniques have been reported for the

introduction of DNA into organisms. Electroporation is a

technique that renders cell membranes temporarily permeable

to macromolecules, such as DNA and proteins, by exposing

cells to brief electrical pulses of high field strength

(Chassy and Flickinger, 1987; Fiedler and Wirth, 1988).

Microinjection of DNA into the nuclei of protoplasts has

also been developed recently as an efficient method of gene

transfer (Reich et al., 1986). A significant development

over microinjection is the use of electric field pulses to

introduce the foreign DNA into the cells. This

'electroinjection' technique bypasses the necessity to

isolate protoplasts (Morikawa et al. 1986). The use of

artificial lipid vesicles (liposomes) and spheroplasts that

encapsule or contain plasmid DNA are also being considered

for transferring DNA to host cells (Cocking and Davey,

1987).


Part II: ADlications of Recombinant DNA Products

Introduction

The potential uses and applications of rDNA products or

genetically engineered organisms are wide and varied. Newly

constructed genotypes are believed to be of value not only

in many areas of basic and applied research, but also for

economic exploitation. Recombinant DNA technology has been

long used in the manufacture of pharmaceuticals and









industrial chemicals (Gillett et al., 1985). At present, an

estimated 10,000 laboratories are currently conducting

biotechnological research in public and private corporation,

universities, and governmental agencies world-wide, and more

than 200 companies are marketing biotechnology products

(Jain et al., 1988).

The successful use of rDNA technology in the

pharmaceutical and chemical industries has dispelled early

fears and concerns about the practice and safety of genetic

engineering in the laboratory. As a result, the potential

for application has now broadened to include agriculture,

mining, pest control, pollution control and a host of other

environmental uses (Gillett et al., 1985; Keeler, 1988;

Lindow, 1985).


Potential Environmental Uses

The proposed environmental uses of GEOs can range from

transgenic animals and modified crop plants to bacteria

designed for specific tasks such as biodegradation of

pollutants. Some potential uses of GEOs are listed in Table

2-1.

Agriculture. The application of rDNA technology to

agriculture is directed to develop crop plants that will

provide more completely balanced nutrition, tolerate

environmental stresses, photosynthesize more efficiently,

exhibit enhanced food storage and express resistance to

pests and pathogens (Gillett et al., 1985; Keeler, 1988).

Other desired applications of genetic engineering in














TABLE 2-1. Potential environmental uses of genetically
engineered organisms



** Improve nutritional quality of food crops

** Decrease dependence on chemical pesticide
by using recombinant microbial pesticides
** Increase plant tolerance and resistance
to pathogens and pests

** Decrease crop sensitivity to chemicals

** Increase plant tolerance to environmental
stresses

** Increase crop yields by manipulating
photosynthesis

** Improve soil quality

** Control weeds

** Improve food storage

** Improve nitrogen fixation

** Water pollution reduction

** Clean up oil spills

** Decomposition of organic wastes

** Cloud seeding, snow making

** Mining: bacterial retrieval of metals

** Energy production from organic biomass

** Oil Recovery


Source: adapted after Keeler (1988) and Saunders
and Saunders (1987).










agriculture include the enhancement of nitrogen fixation,

biological control of insects, and expression of toxin genes

of pathogens in food grain plants. Animals and humans

consuming such food will develop protection against the

potential pathogen, a concept that is similar to

vaccinations.

Pollution control. During the last 80-90 years, the

environment has become overloaded with human, animal, plant

and industrial wastes. Attempts to control or reverse these

adverse events have focused on developing improved waste

treatment systems and technologies that can control the

source of pollution. However, these systems are incapable of

effectively dealing with persistent toxic chemicals that

enter the environment at point sources such as dump sites,

plumes, agricultural runoffs and chemical spills.

It is this problem that has been the target of

biotechnology research whose object is either a) the

enhancement of growth and activity of indigenous organisms

at a pollution site, b) the addition of non-indigenous

'active-degraders' to a pollution site, or c) to enhance and

improve the degradative capabilities of organisms that can

degrade hazardous pollutants to innocuous products

efficiently and economically (Gillett et al., 1985).

Genetic engineering shows much promise for future use

in pollution abatement. Certain organisms, in particular the

genus Pseudomonas, have the capacity to degrade a variety of

hydrocarbons and are therefore potentially useful for









pollution control. Main approaches to the construction of

desired xenobiotic-degrading strains involve recruitment and

heterologous expression of degradative genes for gene

fusion, gene amplification and mutagenesis.

Biomass conversions. The conversion of biological raw

material (biomass) to useful products such as foods,

bioplastics, fuel and other chemicals can be accomplished by

microbial fermentation. Biotechnology offers the possibility

of improving such transformations. Areas that are currently

under investigation include the conversion of biomass into

fuels such as ethanol and methane, and enhanced biopolymer

degradation (Saunders and Saunders, 1987; Zaugg and Swarz,

1981).

Enhanced oil recovery. Conventional oil recovery

techniques are capable of extracting only one-third of the

underground oil reservoirs. The use of modified

microorganisms and/or microbial products has been suggested

for improved recovery efficiencies (Springham, 1984).

Specific modifications for enhanced oil recovery include

biopolymer and biosurfactant production, bioleaching of rock

matrices, and gas production.

Mining. The application of biotechnology in the mining

industry is almost exclusively limited to bacterial leaching

operations in which metals are solubilized from low-grade

ores. Genetic manipulation has been directed towards

enhancing the leaching capabilities of the bacteria, and

increasing its tolerance to toxic metals, heat, fluctuations









in acidity and oxygen deficiencies (Gillett et al., 1985;

Saunders and Saunders, 1987). Microorganisms are now being

used for biomining of precious metals and oil recovery.

Part III: Methods for Testing and Monitoring

Recombinant Organisms in the Environment

Testing and monitoring methodologies can be divided

into three main categories: methods for assessing fate and

effects of the organisms; methods for detecting, identifying

and enumerating the organisms; and methods for assessing

genetic stability of the organisms.


Methods for Assessing Fate and Effects

Methods for assessing fate and effects can range from

simple contained systems, such as flasks and growth

chambers, to large scale field tests. An increasingly

important technique is the use of microcosms which

incorporate both physical and biological factors within a

contained setting (Omenn, 1986). Microcosms are usually of

two types: naturally derived microcosms and synthesized or

standardized microcosms. Naturally derived microcosms

utilize the actual material, such as soil and lake water,

from the natural community. Standardized microcosms are

comprised of chemically defined media and sediments, and a

variety of algae, grazers and detritivores (Kindig et al.,

1983; Taub, 1989).

To date, the majority of research utilizing recombinant

microorganisms is carried out in naturally derived









microcosms. The US Environmental Protection Agency has

developed various terrestrial microcosms designed to

investigate the following five ecosystems 1)

rhizobium/legume/soil interactions; 2) root rhizosphere; 3)

soil/plant systems for studying GEMs capable of degrading

pesticides; 4) vegetables undergoing microbial decay; and 5)

plant leaf surfaces (Armstrong et al., 1987; Gillett et al.,

1985; Omenn, 1986).

Microcosms have previously been used to study the fate

and effects of bacteria in the environment (Bissonette et

al., 1975; Burton et al., 1987; Lessard and Sieburth, 1983;

Liang et al., 1982), but much of the existing information

pertains to naturally occurring pathogens and other

organisms of public health significance. Current studies on

the fate of GEMs in the environment have utilized a variety

of microcosm designs.

Microcosms used in soil studies range from simple

screw-cap test tubes to intact soil cores. Glass test tubes

and vials have been used to study the survival of

recombinant bacteria in soil (Devanas and Stotzky, 1986;

Devanas et al., 1986; Wang et al., 1989), soil slurries

(Walter et al., 1989), soil extract (Walter et al., 1987)

and aquifer material (Jain et al., 1987). Van Elsas et al.

(1989) utilized 70-ml flasks for studying the survival and

genetic stability of plasmid containing Pseudomonas in two

types of soil. Bleakley and Crawford (1989) utilized 150-ml

beakers for studying genetic transfer between Streptomyces









species in soil. More complex microcosms in the form of

intact soil cores have been utilized for evaluating the fate

and ecological impact of the release of GEMs (Bentjen et

al., 1989; Fredrickson et al., 1988; 1989).

Freshwater microcosms consisting of 20 liter glass

carboys were utilized by Steffan et al. (1989a; 1989b) for

monitoring specific microbial populations. Other aquatic

microcosms used for monitoring GEMs include glass bottles

(Trevors et al., 1989), culture flasks (Chaudhry et al.,

1989; Morgan et al., 1989) and mason jars (Scanferlato et

al., 1989). More detailed and complex microcosms include

laboratory-scale waste treatment plants (Mancini et al.,

1987) and model activated-sludge units (McClure et al.,

1989)

Microcosms have also been developed to study the fate

of GEMs associated with the plant system. Armstrong et al.

(1987) and Knudsen et al. (1988) utilized a complex

microcosm to assess survival and gene transfer by

recombinant bacteria associated with plants and herbivorous

insects. Yeung et al. (1989) utilized a Styrofoam cup-

membrane assembly to study growth of GEMs in soil and

rhizosphere.

Fulthorpe and Wyndham (1989) and Rochelle et al. (1989)

recently described aquatic microcosms that simulated a more

natural setting and thus provided a more rigorous test on

the introduced species. Although indigenous organisms were

utilized as the test organisms in these microcosms, they can









be potentially useful in future studies involving GEMS.

Fulthorpe and Wyndham (1989) described a flowthrough lake

microcosm for detecting survival and activity of catabolic

genotypes. Rochelle et al. (1988) developed a rotating disc

microcosm to study gene transfer in riverine systems. Both

microcosms allowed continuous flow of the test water

throughout the experimental procedure.

Although microcosms serve an important function in the

study of GEMs, they presently provide only limited insight

into the natural ecosystem. A limitation to the use of

microcosms is the fact that test results are often not

directly comparable due to the wide diversity of microcosm

systems currently in use. However, the use of controlled

laboratory conditions allows the precise study of

environmental variables on the fate of GEMs and rDNA in the

environment.


Methods for Detection and Enumeration

Methods for detection and enumeration of introduced

GEMs and their rDNA sequences usually involve the use of

markers, such as antibiotic resistance. Methodologies can be

arbitrarily divided into conventional and non-conventional

techniques. The difference between the two groups is based

on the distinction that conventional techniques are widely

and routinely used procedures for assessment of microbial

populations in nature, while non-conventional techniques are

those that (1) have been developed but have not been applied

for environmental use or (2) have been specifically










developed for monitoring rDNA products in the environment

(Jain et al., 1988).

Conventional Methods. The conventional methods of

enumerating microorganisms include 1) selective plating and

enrichment techniques, 2) Most-Probable-Number (MPN)

technique and 3) epifluorescence count technique. Selective

plating and enrichment techniques are perhaps the most

commonly used methods for enumeration. GEMs carrying

specific selectable marker genes can be selected and

enumerated on appropriate agar media. The majority of

current studies utilize GEMs carrying antibiotic resistance

(Armstrong et al., 1987; Devanas et al., 1986; Trevors et

al., 1989; Walter et al., 1989; Fredrickson et al., 1989).

Alternate markers which have been proposed include cloned

lacZY genes in fluorescent pseudomonads (Drahos et al.,

1986); lux genes enabling bacteria to emit light (Shaw and

Kado, 1986); cloned genes for prodigiosin (red pigment)

biosynthesis (Davenhauer et al., 1984); and cloned xylE gene

which encodes for 2,3-catechol dioxgenase (Lyon et al.,

1988; Morgan et al., 1989).

MPN procedures for bacterial enumeration are widely

used in the area of public health. The procedure has also

being utilized for different microbial groups such as

nitrifying bacteria (Rennie, 1978) and rhizobia (Weaver and

Frederick, 1982). Application of the MPN method for

enumeration of GEMs has been reported in few studies.

Bentjen et al. (1989) and Fredrickson et al. (1988; 1989)










used the MPN technique to enumerate Tn5 mutants in soil-core
microcosms. Steffan et al. (1989) also utilized MPN assays

for monitoring GEMs in freshwater ecosystems. Fulthorpe and

Wyndham (1989) utilized an MPN-DNA hybridization technique

to investigate the 3-chlorobenzoate catabolic genotype in

aquatic systems.

Epifluorescent direct counts are routinely used to

determine the numbers and biomass of bacteria in the natural

environment. The acridine orange direct count (AODC) is a

commonly used staining procedure for enumerating

microorganisms in environmental samples (Hobbie et al.,

1977). The lack of specificity of the AODC technique makes

the method useless for enumerating GEMs that are mixed with

a natural population.

Non-Conventional Methods. The non-conventional methods

of enumerating GEMs in the environment include 1)

immunological techniques; 2) enzyme-linked immunosorbent

assay (ELISA); 3) radioactive markers; 4) fluorescent

antibodies markers; 5) plasmid epidemiology and DNA

fingerprinting; 6) use of selectable genotypic markers; 7)

use of nucleic acid sequence analysis; 8) nucleic acid

hybridization techniques such as DNA:DNA colony

hybridization, Southern blot hybridization, nucleic acid

hybridization with DNA extracts, DNA:RNA hybridization, and

use of biotinylated probes; 9) new selective enrichments;

10) protein and enzyme analysis; 11) isozymes; 12) protein









gels and 13) in-vitro amplification of target DNA by

polymerase chain reaction (Jain et al., 1988).

The most commonly used non-conventional method for the

study of GEMs and rDNA is hybridization (Jain et al., 1988).

DNA probe methodology allows detection of specific genes

and/or the organisms containing these genes in the

environment. The methodology eliminates the requirement for

successful culture of recovered organisms. DNA hybridization

is a useful and important tool for monitoring organisms

that become non-culturable and for detecting gene transfer

to indigenous populations.

Previous studies have focused on the use of DNA

hybridization methodologies for detecting specific

indigenous microorganisms or functional groups of organisms

(Holben and Tiedje, 1988; Pettigrew and Saylor, 1986; Yates

et al., 1985). DNA probes are currently being utilized for

studying the fate of GEMs and their rDNA in the environment.

Bentjen et al. (1989) utilized both colony and dot-blot

hybridization to monitor Tn5 mutants in rhizosphere soil,

plant endorizosphere, insects and xylem exudates. The

application of DNA probes and colony hybridization has also

been reported for 1) the detection of catabolic genotypes

in sediment samples (Sayler et al., 1985), in soils

(Chaudhry et al., 1988) and freshwater systems (Amy and

Hiatt, 1989; Morgan et al., 1989; Steffan et al., 1989); 2)

study of maintenance and stability of introduced genotypes

in groundwater aquifer material (Jain et al., 1987); 3)









monitoring and study of plasmid stability in soils

(Fredrickson et al., 1988; Van Elsas et al., 1989); 4)

observing genetic transfer in soils (Bleakley and Crawford,

1989; Zeph and Stotzky, 1989) and waste water (Mancini et

al., 1987); and 5) detection of deletions in an engineered

DNA sequence in soil systems (Jansson et al, 1989). These

studies demonstrate the usefulness of DNA probe technology,

especially when used in conjunction with conventional

selective plating procedures.

In-vitro amplification of rDNA by the polymerase chain

reaction (PRC) can greatly enhance detection of recombinant

target DNA in environmental samples. Using lake water and

raw sewage, Chaudhry et al. (1989) demonstrated the

usefulness of PCR over conventional plating methods for

monitoring GEMs in the environment. The PCR method detected

the presence of the GEMs when selective plating did not.

Similar results have been reported by Steffan and Atlas

(1988).

While it is evident that a wide variety of

methodologies and approaches are available for monitoring

and detecting GEMs in the environment, further development

and refinement is necessary. At present, a combination of

protocols might serve as the best strategy for assessing the

fate of GEMs and rDNA in the environment.


Methods for AssessinQ Genetic Stability

Genetic stability studies address the concerns of

genetic transfer from engineered organisms to the natural









flora, and the maintenance of the engineered genes or

plasmid vectors within the host cell. It is conceivable that

a precisely designed recombinant organism might carry

transcription signals or coding sequences that would have

unpredictable effects on an indigenous host organism (Omenn,

1986). Furthermore, the stability of the recombinant DNA is

important if bacterial cells containing recombinant plasmids

are to be used in environmental biotechnology applications.

At present, routine selective plating and/or DNA

probing has been used to assess the stability of marker DNA

sequences in plasmids and chromosomal sites, and to check

for gene transfer (McClure et at., 1989; Richaume et al.,

1989; Saye et al., 1990; Zeph and Stotzky, 1989). A

combination of well designed microcosms and effective marker

attributes can serve as reliable and powerful tools for

investigating genetic stability.

Part IV: Survival of Genetically Engineered Organisms
and Plasmid Stability of Recombinant Plasmids
in the Natural Environment

Introduction

The survival and growth of recombinant organisms in the

natural environment will depend primarily on 1) the nature

of the bacterial host and the cloning vector, 2) the

selective advantages or disadvantages conferred on the host

by the presence of the foreign DNA and 3) the ecological

niches occupied by the recombinant hosts (Curtiss et al.,

1977).










The use of appropriate hosts and vectors has been

considered for biological containment of recombinant

organisms. The choice of hosts can range from isolated

wild-type strains to laboratory strains that are dependent

on uniquely defined laboratory conditions. Plasmid-cloning

vectors can range from conjugative and mobilizable plasmids

to non-conjugative or suicide plasmids (Molin et al., 1987).

The presence of foreign DNA may alter the survival of

the cloning vectors or the hosts that contain them. The

foreign DNA may or may not confer selective advantages to

the host cell depending on the energy requirements and

precursors for replication of additional DNA (Curtiss et

al., 1977). In addition, the synthesis of new gene products

that are specified by the foreign DNA could pose an

additional burden.

The final ecological niches occupied by recombinant

organisms will have the most direct effect on their growth

and survival. Released GEMs are likely to be dispersed and

can enter freshwater and marine environments via

agricultural run-off and drainage systems. The vast array of

environmental variables associated with soil and aquatic

systems can directly affect the growth and survival of the

GEMs. Environmental factors most affecting growth and

survival include temperature, adsorption, dessication,

nutrient availability, pH, soil/sediment type, presence of

toxicants, viruses, seasonality, predation and competition










(Beringer and Bale, 1988; Faust et al., 1975; Liang et al.,

1982; McFeters and Stuart, 1972).

Numerous studies have been conducted to study bacterial

survival in the natural environment (Bissonette et al.,

1975; Burton et al., 1987; Lessard and Sieburth, 1983; Liang

et al., 1982; McFeters and Stuart, 1972; Ohana et al., 1987;

Scheuerman et al., 1988). However much of the existing

information pertains to naturally occurring pathogens and

other microorganisms of public health significance. The

potential for the use and release of GEMs in the

environment has recently led to more specific research on

the fate of recombinant organisms in the natural

environment. The last few years have produced an array of

useful and informative material on the survival of GEMs in a

variety of natural environments.


Soil. Plant and Rhizosphere Systems

The fate of GEMs in the soil environment has been

investigated in a variety of studies. Devanas et al. (1986)

showed that the survival of genetically-engineered strains

of E. coli in soil was primarily a function of the bacterial

strain and not of the contained plasmid. The nutritional

state of the soil was also shown to influence the degree of

survival. Devanas and Stotzky (1986) also demonstrated that

recombinant genes inserted into plasmid DNA had little

effect on the survival of the bacterial host and

maintenance of the vector. Similar results were reported by

Walters et al. (1987) who utilized soil extracts to study










the survival of recombinant strains of Pseudomonas spp. and

Escherichia coli.

Van Elsas et al. (1989) investigated the effect of soil

texture on survival and plasmid stability of Pseudomonas

fluorescens. Soil type was found to significantly affect

host survival and plasmid maintenance. Higher survival and

stability were observed in heavier-textured soil. There was

no detectable effect of plasmid type (conjugative vs

nonconjugative) on host survival.

Intact soil-core microcosms have been successfully

used to study the fate and ecological impact of Tn-5 mutants

under varying conditions (Bentjen et al., 1989; Fredrickson

et al., 1988; 1989). The design of the microcosms maintains

many of the features useful for evaluating GEM transmission

through the ecosystem, including colonization of the

rhizosphere and endorhizosphere, carriage by insect vectors,

potential displacement of other rhizospheric microorganisms,

and effects on plant growth. In all cases, the population of

the introduced Tn-5 mutants declined slowly over time in the

surface soil, but colonized the rhizosphere and rhizoplane

throughout the 60-cm soil-core depth. The Tn-5 mutants were

transported through the core with percolating water and were

present in the gut of earthworms (Fredrickson et al., 1989).

Inoculation of Tn-5 mutants had no effects on neither plant

leaf nitrogen concentration nor niche displacement of

rhizospheric populations (Bentjen et al., 1989).









Several other studies have also reported on the

survival of GEMs in the rhizosphere and plant associated

systems. Armstrong et al.(1987) described a microcosm method

for assessing survival of recombinant bacteria associated

with plants and herbivorous insects. Leaf, whole insect,

foregut and frass samples were periodically assayed to

enumerate recombinant strains. The recombinant strain was

found to slowly decrease over time on leaf surfaces, but an

increase in population size was noted in foregut samples and

none was detected in frass samples.

Yeung et al. (1989) studied the growth of genetically

engineered Pseudomonas aeruainosa and Pseudomonas putida in

the soil and rhizosphere. Despite a high level of enzyme

production by the engineered strains, the presence of the

cloned genes had no effect on the growth of these strains in

the soil or rhizosphere. No significant difference was noted

between wild-type and engineered strains.

The survival of ice nucleation-active (INA) and

genetically engineered non-INA strains of Pseudomonas

svrinaae on oat seedlings was compared after subjection to

various freezing temperatures (Buttner and Amy, 1989). The

data indicated a potential competitive advantage of INA

strains over the engineered non-INA strains in mild freezing

environments.

The first account of a genetically engineered strain

having a measurable effect on a natural ecosystem was

reported by Wang et al. (1989). The survival and effects of










wild-type, mutant and recombinant StreDtomvces was studied

in a soil ecosystem. With all strains, population densities

slowly declined, though one recombinant strain survived

significantly better in nonsterile soil than its

nonrecombinant parent. One recombinant strain significantly

increased the short-term rate of soil organic carbon

turnover, while another recombinant strain temporarily

reduced carbon mineralization rates during the first days of

release. Additional studies also indicated genetic

instability in one recombinant strain.

The use of DNA probes to detect the stability of

recombinant genes and plasmid vector sequences in soils was

investigated by Jansson et al. (1989). Two Pseudomonas

strains were engineered to contain the nDtII gene and

plasmid vector sequences in their chromosomes. Incubation of

the strains in nonsterile soil, followed by total DNA

isolation and Southern blot hybridization, revealed the loss

of plasmid vector sequences from the chromosome though the

nptII gene was retained. It appeared the extreme conditions

encountered in soil systems resulted in stress-induced

deletions.

In general, it was found that the survival rates of

GEMS was significantly lower in non-sterile soils than in

sterile soils. This increased decline in non-sterile soil

was attributed to a combination of biotic factors such as

predation, antagonism and competition from the natural

population.









Acuatic Systems

Survival studies of GEMs in the aquatic environment

have been conducted using mainly closed or static

microcosms, ranging in size from simple 30-ml culture flasks

(Trevors et al., 1989) to larger 20-liter glass carboys

(Steffan et al., 1989). The potential uses of flowthrough

microcosms have also been reported (Fulthorpe and Wyndham,

1989; McClure et al., 1989). A variety of detection and

enumeration methodologies have also been employed for

studying survival and genetic stability in GEMs.

Amy and Hiatt (1989) used a combination of selective

plating and DNA probing to detect the survival of GEMs in

lake water. Survival of GEMs remained high in filtered

(0.22-um pore size) lake water but was lower in untreated

lake water and lake water filtered with 0.8-um pore size

membrane. Total recoverable bacteria were greater in the

0.8-um filter-treated samples than the untreated samples,

suggesting grazing by zooplankton and microplankton. The

recombinant plasmid was retained in all experiments

regardless of whether plasmid DNA was of benefit to the

cells.

The fate of recombinant pseudomonads released into lake

water was determined by a series of direct membrane filter

methods developed for direct phenotypic and genotypic

detection (Morgan et al., 1989). Recombinant plasmids

encoding a xy1E marker gene encoding catechol 2,3-

dioxygenase (C230) facilitated the detection and enumeration










of the recombinant strains. Microcosms consisted of 5-liter

conical flasks containing filtered or untreated lake water.

Following release of the GEMs into sterile lake water, the

organism persisted, but the marker phenotype and genotype

declined to undetectable levels. Production of a viable but

nonculturable population was not observed. In both the

sterile and untreated lake water, both the xylE gene and the

product, C230 protein, decreased over time but declined

more rapidly in the sterile lake water. The results indicate

that the metabolic burden from overexpression appears to

affect maintenance of the plasmid in released hosts in

sterile lake water.

Chaudhry et al. (1989) derived an alternative method

for directly detecting and monitoring the fate of GEMs in

the environment. In-vitro amplification of target DNAs was

achieved by polymerase chain reaction (PCR) and then

hybridized to a specific oligonucleotide or DNA probe.

Comparisons with selective plating methods indicated that

whereas no viable GEMs were detected after 6 and 10 days of

incubation in lake water and raw sewage, respectively, the

PCR amplification method detected cells for up to 10 and 14

days, respectively. This indicated the presence of cells

that had lost their ability to grow on selective media but

still carried the marker gene.

The survival of genetically engineered Erwinia

carotovora L-864, with a kanamycin resistance gene inserted

in its chromosome, was monitored in pond water and sediment










(Scanferlato et al., 1989). Aquatic microcosms consisted of

850-mi glass Mason jars containing sediment and pond water.

The density of both genetically engineered and wild-type

strains declined at the same rate, and was no longer

detectable by viable counts after 32 days. The introduction

of the GEM affected the indigenous bacterial community.

Total bacterial density significantly increased, including

the density of bacteria belonging to the proteolytic

functional group. In contrast, the density of indigenous

pectolytic and amylolytic bacteria was not affected by the

introduction of the GEM.

Steffan et al. (1989) utilized gene probe methodology

for tracking GEMs with catabolic genotypes in freshwater

(reservoir) samples. The GEMS consisted of Alcaligenes A5

and P. cepacia AC1I00 that degraded 4-chlorobiphenyl and

2,4,5-trichlorophenoxyacetic acid, respectively. Aquatic

microcosms consisted of 20-liter glass carboys filled with

water samples supplemented with glucose and a mineral salts

solution. Colony hybridization of the viable heterotrophic

bacterial populations and dot blot hybridization of total

recovered DNA showed persistence of the GEMs in the presence

and absence of the xenobitic substrates that these organisms

biodegrade. Although there was a gradual decline in

population densities, both GEMs were still detected in the

microcosm two months after their introduction into the

microcosms. Addition of the appropriate xenobiotic









substrates enhanced survival of both GEMs. The recombinant

plasmids were extremely stable under all test conditions.

Trevors et al. (1989) investigated the survival of and

plasmid stability in Pseudomonas and Klebsiella spp.

introduced into agricultural drainage water. Microcosms

consisted of 30-ml glass bottles containing 9-ml of sterile

or non-sterile drainage water. Experiments were conducted

under aerobic and anaerobic conditions and the presence or

absence of added nutrients. The two strains of Pseudomonas

survived well in sterile drainage water incubated

aerobically, with or without added nutrients. However, the

Klebsiella strain only survived in the presence of added

nutrients. The GEMs did not survive well under anaerobic

conditions without nutrients, but showed good survival in

the presence of nutrients. Maintenance of the three

plasmids was found to be host-, plasmid- and environment-

dependent. Plasmid pBR322 was not stably maintained in

Klebsiella under all conditions tested, and pRK2501 was

readily lost from P. Rutida CYM318. Maintenance of RP4 by P.

fluorescens R2f was markedly influenced by nutrients, which

caused a loss of plasmids from cells.

A more complex microcosm was utilized by McClure et al.

(1989) to study the survival of Pseudomonas putida UWC1 in a

laboratory scale activated sludge unit (ASU). The engineered

strain harbored a cloned non-self-transmissible plasmid,

pDl0, that encoded the breakdown of 3-chlorobenzoate. The

ASU maintained a healthy, diverse protozoan population









throughout the experiment, and the introduced strains did

not adversely affect the functioning of the unit. The GEM

persisted in the ASU, in the presence of the sludge

microflora containing predatory protozoa, for more than

eight weeks, although the population size did decline

gradually. Plasmid pD10 was stably maintained in the host

bacterium in the presence or absence of 3-chlorobenzoate,

but did not enhance the degradation of 3-chlorobenzoate in

the ASU.

All these survival studies indicate a significant

decrease in cell numbers of GEMs after introduction into

nonsterile aquatic samples as compared to filtered or

sterile samples. This apparently is a general phenomenon

which has been described for a variety of bacteria in

aquatic environments (Liang et al., 1982, Sinclair and

Alexander, 1984). The decrease may be attributed to biotic

factors such as predation, competition and antagonism by the

indigenous population.


Part V: Genetic Transfer by Genetically Engineered
Organisms in the Natural Environment

Introduction

Among bacteria, genetic information can be naturally

transferred from one species to another by several known

mechanisms (conjugation, transduction, transformation and

possibly by vesicles, a yet unexplained mechanism). These

mechanisms can also be expected to apply- to GEMs that are

released into the environment. The potential for gene









transfer in the environment depends on the survival and

transport of organisms in the ecosystem. To date, there is

limited information available on natural gene transfer in

the environment. The increasing feasibility for the use and

release of GEMs in the environment has focused greater

attention on the need for gene transfer studies in the

natural environment (Stotzky and Babich, 1986; Trevors et

al., 1987). The following review focuses mainly on recent

studies of genetic transfer using recombinant organisms in

the natural environment.


Coniugation

Conjugation is the most well studied of the three

mechanisms of genetic transfer. Past studies have dealt

mainly with conjugative plasmids in soil (Graham and

Istock, 1978; Trevors, 1987; Trevors and Oddie, 1986;

Weinberg and Stotzky, 1972) and aquatic systems (Altherr and

Kasweck, 1982; Gowland and Slater,1984; Mach and Grimes,

1982). Much of the aquatic studies were conducted from a

public health point of view and addressed the potential for

transfer of antibiotic resistance plasmids (R plasmids) in

waters receiving sewage effluents (Bell et al., 1983; Goyal

et al., 1979; Grabow et al., 1975).

More recent studies strongly suggest or demonstrate

plasmid gene transfer among indigenous organisms on plant

surfaces and plant tissue (Manceau et al., 1986; Talbot et

al., -1980), in cultured rainbow trout (Toranzo et al.,










1984), in soil (Krasovsky and Stotzky, 1987; Trevors, 1987;

Trevors and Oddie, 1986; Van Elsas et al., 1987) and in

various aquatic habitats (Bale et al., 1988; Gowland and

Slater, 1984; Kobori et al., 1984; Saye et al., 1987).

Recently, studies utilizing GEMs have provided

additional and useful information on genetic transfer by

conjugation. The majority of these studies focused on soil

and utilized simple microcosm settings to determine gene

transfer. Van Elsas et al. (1987) studied the transfer of

plasmids between bacilli in air-dried soil under sterile and

non-sterile conditions. Transfer frequencies in filter

matings (1 x 10-6) were much higher than those observed

for incubations in sterile loamy sand (0.7 x 10-7).

Essentially no transconjugants were obtained in non-sterile

soil suggesting a rapid decline of the recipient population.

However, in the presence of bentonite clay, plasmids were

transferred at higher frequencies, and the survival of the

recipient population was enhanced. The presence of nutrients

in the soil was also shown to stimulate plasmid transfer.

Bleakley and Crawford (1989) investigated the effects

of varying moisture and nutrient levels on the transfer of

the recombinant, conjugational plasmid pIJ303 between

Streptomyces species in sterile silt loam. Their results

suggested that nutrient-amended, relatively dry soils

possess frequent microsites where mycelial growth and

conjugationally mediated plasmid exchange can readily occur.










Richaume et al. (1989) demonstrated the influence of

soil variables such as clay, organic matter, soil pH, soil

moisture and soil temperature on the potential for

intergeneric plasmid transfer. Maximum transfer frequencies

were noted at a clay content of 15%, a soil pH of 7.25, a

soil moisture content of 8%, and incubation temperature of

280C.

Walter et al. (1989) evaluated a simple microcosm

method for measuring conjugal transfer of recombinant DNA in

soil slurries. Using a variety of environmental variables

they observed the highest numbers of transconjugants at 350C

in nutrient-enriched soil slurries. Low frequencies were

observed in low nutrient conditions or low pH values.

Transfer of recombinant plasmids has also been

demonstrated in wastewater, utilizing model laboratory-scale

waste treatment (LSWT) facilities (Mancini et al., 1987;

McClure et al., 1989). Transconjugants were detected at

different locations within the model system (Mancini et al.,

1987) suggesting that nutrients in sewage are sufficient for

plasmid mobilization. The potential for plasmid transfer via

triparental mating was also shown in the LSWT (Gealt, 1985;

Mancini et al., 1987). McClure et al. (1989) utilized a

model activated sludge unit to demonstrate the transfer of

mobilizing plasmids from indigenous populations to the

recombinant strain which carried a non-self-transmissible

plasmid pDl0. Further experiments showed that bacteria in

the activated sludge population could then act as recipients










for plasmid pD10. The implications of these studies suggest

that genetic exchange of recombinant genes placed on "safe"

(non-transmissible) vector plasmids can still be mediated by

conjugation. An interesting observation noted by McClure et

al. (1989) was the fact that some activated sludge

transconjugants showed higher rates of catabolic activities

than the original recombinant strain.

Conjugative plasmid transfer by recombinant bacteria

has also been investigated in plants (Armstrong et al.,

1987; Knudsen et al., 1988) and animals (Armstrong et al.,

1987). Armstrong et al. (1987) utilized a complex microcosm

model to assess survival and plasmid transfer of recombinant

bacteria associated with plants and herbivorous insects.

Plasmid transfer from recombinant to non-recombinant strains

was not detected in either plant, whole insect, foregut or

frass samples.

Knudsen et al. (1988) reported a predictive model for

conjugative plasmid transfer in the rhizosphere and

phyllosphere. The model was tested in microcosms planted

with radish seeds and on leaf surfaces of radish and bean

plants. Transconjugants were isolated from both rhizosphere

and phyllosphere microcosms after one day. This was followed

by an initial rapid increase and a subsequent decline in the

numbers of transconjugants.


Transduction

Generalized transduction systems have been observed in

many common soil and aquatic organisms such as Rhizobium,










Bacillus and Pseudomonas (Reanny et al., 1982). These

studies, however, were mostly conducted as in-vitro

experiments using pure cultures of bacteria (Zinder and

Lederberg, 1952). The recent interest in gene transfer in

the natural environment has again prompted research in this

area, but information is still sparse.

The first reported study of in-situ transduction was

conducted in a freshwater environment using strains of

Pseudomonas aeruginosa (Morrison et al., 1978). Transduction

of P. aeruQinosa streptomycin resistance by a generalized

transducing phage, F116, was shown to occur in flow-through

environmental test chambers submerged in a freshwater

reservoir.

In a similar experiment, Saye et al. (1987)

demonstrated the transduction of plasmid Rms149 by the

generalized transducing bacteriophage 4DSI. Plasmid DNA was

transferred from a nonlysogenic plasmid donor to a 4DS1

lysogen of P. aeruainosa that served both as the source of

the transducing phage and as the recipient of the plasmid

DNA. Transduction was observed both in the presence and

absence of the indigenous microbial population. In a later

study, transduction of single chromosomal loci and

cotransduction of closely linked loci were observed between

lysogenic and nonlysogenic strains (Saye et al., 1990).

These studies clearly demonstrated the ability to generate

and select new genetic combinations through phage-mediated

exchange.










Zeph et al., (1988) demonstrated the transduction of
bacterial resistance genes for chloramphenicol and mercury

into Escherichia coli by the transducing coliphage P1 in

nonsterile soil. In a following experiment, Zeph and Stotzky

(1989) utilized a biotinylated DNA probe to detect phage P1

transductants in nonsterile soil. Although the probe did

detect transductants of E. coji that was added to the soil,

no phage P1 transductants of indigenous bacteria were

detected.

The lack of available information clearly illustrates

the need for additional studies of this potentially,

extensively used mechanism of gene transfer in natural

environments.


Transformation

While current studies have increasingly been focused on

the potential for gene transfer via conjugation and

transduction, very little attention has been given to

transformation. Since plasmid and chromosomal DNA is

certain to be released from lysed bacterial cells, it is

important to inquire about the fate and effects resulting

from the release of the extracellular DNA in soil and

aquatic systems and in specific niches such as legume

nodules and the rhizosphere.

Few studies have been reported on natural

transformation in the environment. Graham and Istock (1978;

1979) demonstrated transformation in B. subtilis in sterile









soil. Ardema et al. (1983) found that DNase reduced

transformation frequencies of free DNA more than that of DNA

adsorbed onto quartz sand. Lorenz and Wackernagel (1987)

reported similar results using a flow-through system of sand

filled glass columns. These studies indicated that soil

components other than organic materials and clay minerals

can bind DNA and retard its enzymatic degradation.

To date, no information exists that demonstrates

genetic transfer via transformation by the DNA of GEMs.

Several studies have shown the presence of extracellular DNA

in the natural environment (DeFlaun et al., 1986; 1987;

Minear, 1972; Pillai and Ganguly, 1972) and a few have

investigated its dynamics and persistence (Paul et al.,

1987; 1988). However, very little is still known about the

fate of extracellular DNA and its implication for genetic

transfer via transformation.















CHAPTER 3
MICROCOSM DESIGN AND PROTOCOL FOR ASSESSING THE
FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN AQUATIC ENVIRONMENTS



Introduction

In general, microcosms can be described as fully-

contained laboratory ecosystems which simulate an ecological

community with its complete ensemble of interacting

microbial species. Microcosms can range from laboratory

contained flasks, mason jars and growth chambers to fully

contained greenhouses and systematically sampled natural

areas termed mesocosms. Of increasing importance is the use

of microcosms which incorporate both physical and biological

components within a contained setting (Omen, 1986).

Many types of research questions which evolve from a

consideration of biotechnology risk assessment can be

addressed by the use of microcosms. Microcosms can serve as

standard test systems that can be adapted to a variety of

organisms and environmental conditions. Testing and

monitoring of GEMs by use of microcosms allow data gathered

in one test environment to support design and justification

of tests in natural environments.

A variety of microcosm models have been utilized for

the study of GEMs in the aquatic environment. Experimental









microcosms range from simple glass bottles and mason jars

(Scanferlato et al., 1989; Trevors et al., 1989) to more

complex and detailed structures such as a laboratory scale

wastewater treatment pilot plant (Mancini et al., 1987). Two

recently described microcosm models have been proposed for

use in the study of GEMs in aquatic systems (Fulthorpe and

Wyndham, 1989; Rochelle et al., 1989). Both microcosms

functioned as flow-through systems which simulated a more

natural setting and therefore provided a more rigorous

testing of the introduced organisms.

In order to study the fate and survival of GEMs in the

aquatic environment, an aquatic microcosm was developed that

utilizes modified survival chambers in a simple laboratory

setting designed to allow the testing of a variety of

environmental variables. The microporous membrane of the

chamber allows continuous exchange of water, solutes and

nutrients between the chambers and the surrounding water. A

significant advantage of this system is the continuous

interaction of the test bacteria with their surrounding

environment. These conditions provide a more thorough test

of the fitness of the organism and/or its recombinant genes.

This section describes the design of the aquatic

microcosms that was utilized for studying the fate and

survival of GEMs in various aquatic environments. The

section also summarizes some preliminary experiments that

led to the development of the final test procedures that

were utilized in the overall study.










Materials and Methods


Survival Chambers

Survival studies were conducted using modified membrane

diffusion chambers as described by McFeters and Stuart

(1972). The survival chambers were constructed from

6.5-mm Plexiglass acrylic sheeting (Commercial Plastics,

Jacksonville, Fla.) as depicted in Figure 3-1. The internal

lumen of the chamber was 6-cm in diameter and 6.5-mm wide,

and accommodated a 20-ml sample when assembled and filled.

The total surface area of the membranes within the chambers

was 56.8-cm2, and the surface area to volume ratio was 2.84.

Durapore membrane filters (0.2-um pore size, GVWP-293-

25, Millipore Corp., Bedford, Mass.) were cut into circular

pieces with a diameter of approximately 7.5-cm. Durapore

filters were used instead of cellulosic membrane filters

since the former are stronger and more resistant to

biodegradation. During assembly, the membrane filters were

inserted on either side of the central spacer and held in

place by the two outer retainers (Figure 3-1). A thin

coating of Lubriseal (Arthur A. Thomas Co., Philadelphia)

was applied between the membrane filter and chamber walls to

ensure a watertight seal.

Two 22-gauge hypodermic needles (Becto Dickerson Co.,

Rutherford, NJ) were fitted into the top of the central

spacer to allow filling and withdrawal of samples. To ensure

a secure and watertight seal, both needles were fixed into

position by applying a seal of silicone (Dow Corning Co.,




43











60







o01
0 2 4 6 8_1_0
cm


Figure 3-1. Schematic diagram of membrane diffusion chamber.









Midland, MI) at the point at which the needles entered the

chambers. Dust cap covers for the hyperdermic needles were

made by filling the hubs of 1-ml plastic syringes with

silicone.

Prior to assembly, dust caps were sterilized by

autoclaving at 1210C for 15 minutes. The chambers and

membranes were semi-assembled by placing the membranes on

the outer retainers that were treated with Lubriseal. The

chambers and membranes were then sterilized by irradiation

with ultraviolet light (GTE Sylvania Inc, Danvers, Mass.)

for at least two hours. The chambers were assembled

aseptically under UV light using stainless steel nuts and

bolts to secure the chambers. Sterile 22-gauge needles were

then inserted into the central lumen, capped with the

sterile dust caps and sealed into position with silicone.

The silicone seal was allowed to dry at least 24 hours prior

to use of the chambers.


Microcosm DesiQn

Laboratory scale microcosms were designed to utilize

the membrane diffusion chambers in a simple setting that

would allow testing of a variety of environmental variables,

simulate a more natural environment, and yet keep the

recombinant bacteria within a contained setting. Two types

of designs were utilized during research procedures.

Initially, a static renewal system was developed and tested

for survival studies. Further improvements produced a flow-










through system that more closely mimicked the natural

environment.

Static renewal system. Static renewal microcosms

consisted of 1- or 5-gallon aquarium tanks containing the

appropriate aquatic sample. The tanks were placed on

magnetic stirrers to allow for continuous mixing and

circulation of the test water. Aeration of the water was

provided by a Challenger air pump and air stones. Membrane

diffusion chambers containing the GEMs or test organisms

were then immersed into the water and sampled at intervals

for survival studies. Water samples were collected daily or

every 48 hours and allowed to acclimatize to the appropriate

test temperature. The test water in the tanks was replaced

every 24 hours. Figure 3-2 illustrates the design of the

static renewal microcosm.


Flow-through systems. Modifications to the static

renewal system allowed for continuous flow of the test water

throughout the duration of the experiment. The design of the

microcosm was essentially the same as described above for

the static renewal system. The flow of water into and out of

the aquaria tanks was regulated by peristaltic pumps

(Buchler, Fort Lee, NJ) at a flow rate of approximately 3-4

ml min-1. Overflow water from the tanks was collected in 20

liter carboys, treated with chlorine and then discarded.

A later modification to the flow-through system

included the elimination of the peristaltic pumps by

utilizing aquarium air tubing constricted with stainless









AQUARIUM TANK


SURVIVAL CHAMBER


MAGNET


MAGNETIC STIRRER


Figure 3-2. Schematic diagram of the static-renewal
microcosm.









steel clamps that allowed the test water to be siphoned at a

fixed rate into the aquaria tanks. This modification was

similar to the microcosm described by Fulthorpe and Wyndham

(1989). Outflow from the tanks was accomplished by drilling

holes in the tanks to serve as outlet ports at a height that

was approximate to that of the chambers. The overflow water

was collected using tygon tubing attached to the outlet

ports and decontaminated before discarding. This setup

avoided the complete immersion of the needle sampling ports

which increases the chances of contamination during the

sampling procedure. Figure 3-3 illustrates the design of the

flow-through microcosm.

Both types of microcosms were utilized in either a

constant temperature room or an environmental chamber

(Percival, Boone, Iowa) at a constant temperature and light

regime (16 hours light: 8 hours dark).


Test Protocol for Survival Studies

Preliminary experiments were undertaken to determine

optimal conditions for routine sampling, enumeration and

detection of test organisms. Preliminary tests included

growth studies of the test organisms (both GEMs and their

wildtype strains); comparison of plating techniques for

enumeration of viable counts; and a comparison of the type

of selective media best suited for detection of the GEMs.

Bacterial Strains. Escherichia coli (strain HB01) and

Pseudomonas putida (strain 50014) were used as wildtype

reference strains. Genetically engineered E. coli (strain
































MAGNETIC STIRRER




COLLECTION


RESERVOIR TANK




AQUARIUM TANK

I SURVIVAL CHAMBER


Figure 3-3. Schematic diagram of the flow-through microcosm.










50008) was obtained by inserting an EcoR1 DNA fragment from

pRC10 into a derivative of pBR322. The EcoRi fragment

contained the gene for mercury resistance and the genes

involved in 2,4-D degradation (Chaudhry and Huang, 1988).

The recombinant plasmid was then cloned into HB101. Presence

of the recombinant plasmid was first tested by growth on LB

agar plates containing 40 ug/ml and 50 ug/ml of HgC12 and

ampicillin, respectively, and subsequently confirmed by

plasmid isolation and restriction analysis. The engineered

strain of P. Rutida (strain 50058) harbored the plasmid

R68.45 which conferred resistance to carbenicillin,

kanamycin and tetracycline (Haas and Holloway, 1976).


Bacterial growth studies. In order to accurately

determine the initial inoculum concentration for survival

studies, it was necessary to conduct growth studies that

would correlate optical density readings with bacterial

concentrations (CFUs/ml). Wildtype strains of _. coli and

P. Dutida were grown in Luria-Bertani (LB) broth at 350C and

280C, respectively. Genetically engineered E. coli was grown

in LB broth supplemented with HgC12 (40 ug/ml), while

engineered P. Dutida was grown in LB broth containing

tetracycline (15 ug/ml) and kanamycin (50 ug/ml). Growth

curves were constructed by inoculating 50 ml of the

appropriate media with an overnight culture of the bacterial

strain and incubating at the necessary temperature. At

specific time intervals, samples were aseptically withdrawn










for bacterial enumeration and optical density measurements

(550nm).


Comparison of Dlating techniques. Two methods were

compared for bacterial enumeration. The conventional

spread-plate method was compared to the drop-plate

technique of Hoben and Somasegaran (1982). Samples were

diluted in sterile potassium phosphate buffer (pH 7.2) and

plated on the appropriate selective or non-selective media.

For the spread-plate method, 0.1 ml aliquots were

aseptically spread onto the media. In the drop-plate method,

20 ul of the sample was carefully dropped onto the media,

and allowed to completely dry before incubation. Both

methods were carried out in triplicate.


Comparison of selective media types. It has been

suggested that bacterial cells can become physiologically

injured due to environmental stresses imposed by aquatic

environments (Bissonette et al., 1975). The extent of such

injuries can lead to a loss in ability to produce colonies

on selective media. It is possible that the aquatic

environment can induce enough stress upon the GEMs to the

extent that they become physiologically debilitated and

therefore not detectable by the selective media of choice.

To determine whether the extent of injury would lead to an

inability to grow on the selective media, the GEMs were

subjected to lake water and enumerated at specific time

intervals on varying strengths of selective media. P. putida









and E. coli were enumerated on (1) LB media; (2) full-

strength selective media (LB plus tetracycline (15 ug/ml)

and kanamycin (50 ug/ml) and LB plus HgC12 (40 ug/ml)

respectively); (3) half-strength selective medium; and (4)

quarter-strength selective medium.


Comparison of Microcosms for Survival Studies

Many survival studies, including those of GEMs, have

been conducted in simple flask microcosms (Liang et al.,

1982; Morgan et al., 1989). Although these microcosms are

simple to maintain and easy to work with, there are

potential problems and disadvantages associated with them.

Closed systems create a potential for "bottle effects" that

can arise over time. Such systems are also closed to

nutrient and chemical inputs that may be associated with

the ecosystem. The microcosm that was developed for this

research eliminates much of the problems associated with

closed microcosms.

A comparative study between a "closed" flask microcosm

and the "open" flowthrough microcosm described above was

used to determine the extent to which the two types of

microcosms could influence survival rates of introduced

organisms. Genetically engineered and wildtype P. putida

were used as the test organisms. Bacterial cultures were

grown until late logarithmic phase and harvested by

centrifugation at 8000 g for 10 minutes at 40C. The cells

were washed three times with sterile phosphate buffer (pH

7.2) and resuspended in filter-sterilized lake water for use









as the inoculum source. Triplicate survival chambers were
inoculated with 20 ml of the washed cells (approximately 107

CFU/ml), and then immersed into the above described

microcosm. Flasks microcosms consisted of 125 ml flasks

containing 20 ml of autoclaved lake water. Triplicate flasks

were inoculated with suspensions of washed cells to achieve

an inoculum concentration of approximately 107 CFU/ml. Both

microcosms were incubated at 250C. At specific time

intervals, 1.0 ml aliquots were aseptically removed to

determine bacterial numbers. Bacteria were enumerated using

the drop plate technique described above.


Results and Discussion

Bacterial Growth Studies

Bacterial growth curves and their corresponding optical

density values are depicted in Figures 3-4 to 3-7. Although

both strains of J. coli produced similar growth patterns,

the growth rate of the recombinant strain was higher than

that of the wildtype. This was not observed for the P.

putida strains. Growth studies indicate a higher growth rate

for the wildtype strain. These standard growth curves have

proven to be fairly accurate for determining bacterial

concentrations for inoculation purposes.


Comparison of Plating Technicues

Comparison between the spread-plate technique and the

drop-plate method indicates that the drop-plate method




















-.4


0 2 4 6 $ 10 12

TIME (hrs)












Figure 3-4. Growth curve and corresponding optical density
values (A50) of wild-type Escherichia coli
HBI01), grown at 35uC.
































0 2 4 6 8 10 12


TIME (HRS)












Figure 3-5. Growth curve and corresponding optical density
values (A550) of genetically engineered Escherichia coli
(50008), grown at 350C.



















0

x



LL
0


1-0


0"8

0-6 t(

0-4





0-2


0 2 4 6 8 10


TIME (HRS)








Figure 3-6. Growth curve and corresponding optical density
values (A55,) of wild-type Pseudomonas putida
150058), grown at 27C.






















00

N%
O-J




m
U.


0 2 4 6 8 10


TIME (HRS)











Figure 3-7. Growth curve and corresponding optical density
values (A550) of genetically engineered Pseudomonas putida
(50058), grown at 270C.









consistently gave higher numbers of viable counts. This

trend was observed for both wildtype ( Figure 3-8) and

recombinant (Figure 3-9) strains of E. coli plated on non-

selective and selective media, respectively. The drop-plate

method has several advantages over the traditional spread-

plate method. The methodology of the drop-plate technique

allows as much as four dilutions (three replicates each) per

plate, as compared to the spread-plate method which requires

a single plate for each replication of each dilution. The

drop-plate method therefore utilizes 1/12 of the media and

petri dishes normally required for spread plates. In

addition to its cost-effectiveness, the drop-plate method is

also faster and as reliable as the conventional spread-plate

method.


Comparison of Media Types

A comparison of four media types for selective growth

of the genetically engineered strains of E. coli and P.

Dutida indicated no significant differences between media

types for both strains (Figures 3-10 and 3-11,

respectively). An exception was observed for P. putida after

6 hours of exposure to the lake water (Figure 3-11). Viable

counts were highest on half-strength selective media and

lowest on quarter-strength media. The results of this study

suggest that exposure to lake water does not necessarily

cause physical or physiological injury to an extent that

affects the growth of cells on selective media. Since full-

strength selective media is effective for screening









CFU/rnl (Millions)


Sread Method


BDrop Method


800



600



400-



200-



0-


6 7


0 1


Time (hours)











Figure 3-8. Comparison of enumeration techniques
by the spread plate and drop plate methods for
values (A550) of genetically engineered Pseudomonas putida
wild-type Escherichia coli (HBl0l).


M


4 8










1600-

1400

1200

1000

800

600

400-

200 -

0-


6


Figure 3-9. Comparison of enumeration techniques
by the spread plate and drop plate methods for
genetically engineered Escherichia coli (50008).


CFU/ml (Millions)


* Spread Method
S Drop Method


0 I 2 4
Time (hours)










Log CFU/ml


0 6


/2LS
S112L.9 4 2OPP. NOCI


21 44 216
Time (hours)


LS 4Oppm HCI
m 11L9 0 lOPPM HgCI


Figure 3-10. Comparison of four different media types
for selective growth of genetically engineered
Escherichia coli (50008).








Log CFU/mI


0 6 21 44 216
Time (hours)


a0 LS
I 1/2 (LB Tet Kim)


ZMLB lt Km
331/4 (LB Tot + Km)


Figure 3-11. Comparison of four different media types
for selective growth of genetically engineered
Pseudomonas putida (50058).









indigenous populations for the appropriate selective

markers, and is useful for detecting plasmid or gene

stability, full strength media was therefore selected for

routine use in all consecutive experiments.


Comparison of Microcosms

The survival of wildtype and engineered strains of P.

putida in lake water was simultaneously conducted using

flask microcosms and survival chambers in a flow-through

system. This study was designed to determine whether the

type of microcosm affects the survival rates or patterns of

the test organisms. The results indicated that the design of

the microcosm does play an important function in survival

studies. Results of the flask studies showed little to no

effect on the survival of the two strains of P. putida

(Figure 3-12). However, results using the flowthrough

microcosm indicated an initial decline in numbers during the

first 48 hours, followed by growth and stabilization of the

two populations. This trend was most likely a better

indication of survival in the natural environment. The

organisms were under constant pressure to adapt to changing

nutrient conditions and toxins associated with the constant

flow of the lake water through the survival chambers.

Although it can be argued that the two microcosms cannot be

compared, it clearly suggests the importance of microcosm

design for fate and survival studies. Designing the

microcosm to simulate natural conditions as closely as




























0 50 100 150 200 250 300 360 400
Time (hours)


Flnk (50014)
-Chamber (80014)


--- Flk (50068)
-- Chamber (60088)


Figure 3-12. Comparison of survival studies of wild-type
and genetically engineered strains of Pseudomonas Dutida
in lake water at 250C, using flask microcosms
and survival chambers.






64



possible obviously provides a more rigorous test for the

introduced organisms.

The preliminary experiments described here were

basically comparative studies to determine optimal test

procedures for the following standard experiments.
















CHAPTER 4
SURVIVAL OF AND PLASMID STABILITY IN GENETICALLY
ENGINEERED AND WILDTYPE STRAINS OF E. COLI
AND P. PUTIDA IN AQUATIC ENVIRONMENTS


Introduction

At present, the number of cases of deliberate release

of genetically engineered bacteria into the environment are

few and fairly recent (Halvorson et al., 1985). Prospects

for the routine release of genetically engineered organisms

into the environment are becoming increasingly feasible. The

potential use of recombinant bacteria in the environment

include enhancement in food and agricultural production,

biocontrol of insects and diseases, metal and mineral

leaching, environmental remediation and waste treatment

(Gillett et al., 1985; Halvorson et al., 1985; Johnston et

al., 1984; Keeler, 1988).

Deliberate release of such recombinant microorganisms

has however raised considerable concern and attention among

the public and scientific sectors. Questions and issues

concerning potential adverse environmental effects are

foremost in the minds of many involved. Genetically

engineered microorganisms (GEMS) that are released may have

the capacity to reproduce, spread beyond the initial point

of release and transfer their novel genetic information to









the indigenous microbial populations. The possibility also

exists that recombinant organisms may behave differently

from their non-recombinant parental strains when released

into the environment. Such information is therefore needed

to properly evaluate and assess the potential impact of GEMs

on representative ecosystems.

Although numerous studies have been conducted on

bacterial survival in the natural environment (Bissonette et

al., 1975; Burton et al., 1987; Lessard and Sieburth, 1983;

McFeters and Stuart, 1972), much of the existing information

pertains to naturally occurring pathogens and other

microorganisms of public health significance. More recent

studies have included other species (Liang et al., 1982;

Ohana et al., 1987; Scheuerman et al., 1988), but still to

date, few studies have been conducted to determine the fate

of recombinant bacteria and their "novel" genes in natural

ecosystems. Investigations have dealt mainly with soil

systems (Bentzen et al., 1989; Devanas et al., 1986;

Fredrickson et al., 1988; 1989; Van Elsas et al., 1989;

Walter et al., 1987) and plant associated systems such as

leaf surfaces, the rhizosphere and plant-feeding insects

(Armstrong et al., 1987; Knudsen et al., 1988; Yeung et al.,

1989). Recent reports have provided additional information

on aquatic systems, including wastewater effluents (Amy and

Hiatt; 1989; McClure et al., 1989; Morgan et al., 1989;

Scanferlato et al., 1989; Trevors et al., 1989). However,

many questions still remain to be answered about the fate of









recombinant bacteria in the aquatic environment. Recently,

polymerase chain reaction (PCR) has been used to pursue such

studies with greater precision and sensitivities (Chaudhry

et al., 1989).

Laboratory-contained microcosms serve an important

function in the study of GEMs. Current regulatory standards

strongly recommend research with recombinant bacteria only

within contained settings (Johnston and Robinson, 1984).

Microcosms can serve as standard test systems that can be

adapted to a variety of organisms and environmental

conditions. In this study, a microcosm approach utilizing

membrane diffusion chambers (Altherr and Kasweck, 1982;

Fliermans and Gordon, 1977; McFeters and Stuart, 1972) was

applied for studying the survival and fate of recombinant

bacteria in the aquatic environment. The porous membranes of

the chamber allowed continuous exchange of water, solutes

and nutrients between the chambers and the surrounding

waters. A significant advantage of this system was the

continuous interaction of the environmental sample, such as

that found in natural environments, with the test bacteria.

The microcosm was designed to allow the testing of a

variety of environmental variables including the effects of

toxicants. The.discharge of toxic materials into the aquatic

environment has led to questions regarding the impacts of

these chemicals on the ecosystem. Of major concern is the

effect on aquatic life and water quality (Rand and

Petrocelli, 1985). Microbial activity has been shown to be









intrinsically affected by the presence of such toxicants,

with subsequent direct or indirect impacts on the rest of

the ecosystem. With the increasing use of chemicals in the

environment, it is important to understand how GEMs would

respond in such adverse conditions and whether they behave

differently (structurally or physiologically) from their

non-recombinant parent strains.


Materials and Methods


Bacterial Strains

Escherichia coli (strain HB101) and Pseudomonas Putida

(strain 50014) were used as wild-type reference strains.

Genetically engineered _. coli (strain 50008) was obtained

by inserting an EcoRl DNA fragment from pRC10 into a

derivative of pBR322. The EcoRl fragment contained the gene

for mercury resistance and the genes involved in 2,4-D

degradation (Chaudhry and Huang, 1988). Detailed

construction of the clone was described in Chapter 3.

Pseudomonas Putida (strain 50058) was used as the

genetically engineered strain and harbored R68.45 which

conferred resistance to carbenicillin, kanamycin and

tetracycline (Haas and Holloway, 1976).

E. coli and 2. putida were grown in LB at 350C and

280C, respectively. For the genetically engineered coli

and P. putida, LB was supplemented with HgCI2 (40 ug ml-1)

and tetracycline (15 ug ml-1) and kanamycin (50 ug ml-)

respectively. All cultures were incubated until late









logarithmic phase and harvested by centrifugation at 8000 g

for 10 minutes at 40C. The cells were washed three times

with sterile phosphate buffer (pH 7.2) and resuspended in

filter-sterilized test water to a density of 105 to 107

CFU/ml before use in survival experiments.


Aquatic Samples

Test samples included lake water, ground water and

activated sludge effluent. Lake water was collected from a

hypereutrophic lake (Lake Alice, Gainesville, Fl.) at a

depth of approximately 1 meter. Ground water was obtained

from a municipal water treatment plant (Gainesville, Fl.).

The sample was collected from a composite deep well that

tapped the Floridan Aquifer. Activated sludge effluent was

obtained from the wastewater treatment plant (University of

Florida, Gainesville, Fl.) that treats domestic wastewater

from the university campus. Samples were collected every 48

hours in 20 liter Nalgene carboys and allowed to acclimatize

to the appropriate test temperature prior to use.


Survival Experiments

Microcosm design. Survival experiments were conducted

using either static renewal or continuous flow-through

systems as described in Chapter 3. The design and assembly

of the survival chambers is also given in Chapter 3.

Temperature-dependent lake experiments were conducted using

a static renewal system. However, the modified flow-through

system was used to study survival in ground water and









activated sludge effluent. All experiments were carried out

in either a constant temperature room or an environmental

chamber (Percival, Boone, Iowa) at a constant temperature

and light regime (16 hrs light: 8hrs dark).


Experimental procedures. For all experiments,

triplicate chambers were inoculated with 20 ml of washed

cells resuspended in test water, and then immersed into the

microcosms. After certain time intervals, each chamber was

removed, shaken well to resuspend the cells, and 1.0 ml of

suspension removed to determine bacterial numbers. Viable

counts were enumerated using the drop-plate technique (Hoben

and Somasegaran, 1982) on either LB agar plates for wild-

type strains (HB101 and 50014); LB + HgCl2 (40 ug m1-1) for

E. coli (50008) and LB + tetracycline (25 ug m1-1) +

kanamycin (50 ug m1-1) for P. putida (50058). Direct counts

were determined by the acridine orange direct count (AODC)

method of Hobbie et al. (1977).


Effect of temperature. The effect of temperature on the

survival rates of wildtype and genetically engineered

strains of E. coli and F. Dutida was studied by conducting

experiments at 15, 25 and 300C. Temperature-dependent

experiments were run for lake water and activated sludge

effluent under sterile conditions i.e. in the absence of

indigenous populations. Survival studies in ground water

were conducted at 220C, since ground water maintains this

temperature year round.









Sterile vs non-sterile conditions. The survival of the

genetically engineered strains under sterile and nonsterile

conditions was studied using six chambers inoculated with

the appropriate bacterial strain and immersed in lake water

as described above. After 60 hours, during which bacterial

numbers were enumerated every 24 hrs, three chambers were

removed and each inoculated with 1 ml of lake water. The

chambers were replaced and thereafter represented nonsterile

conditions. The lake water was initially screened to ensure

that none of the indigenous population grew on the selective

media used for the engineered strains. This experiment was

conducted at 270C using a flow-through system as described

above.

Toxicant effect. The effect of toxicants on the

survival of P. putida and its engineered strain was studied

using the aquatic herbicide, Hydrothol-191 (7-oxabicyclo

[2,2,1] heptane-2,3-dicarboxylic acid). Hydrothol-191 is

commonly used throughout the state of Florida in lakes and

streams for control of aquatic weeds (Dupes and Mahler,

1982). The herbicide has a half-life of approximately 21

days (Reinert et al., 1985), and suggested application rates

of 1-5 mg/L (Pennwalt Corporation, 1980). The survival

response of the two strains of P. putida in lake water

amended with Hydrothol 191 (1 mg/L final concentration) was

studied using a static renewal process. A stock solution of

Hydrothol-191 (530mg/L) was prepared from the liquid

formulation (Pennwalt Corp., Philadelphia, Pennsylvania)










containing 53% endothall as the active ingredient. The test

water was made by adding appropriate amounts of stock

solution to acclimated lake water to produce a hydrothol

concentration of 1 mg/L based on percent active ingredient.

This water was then added to the tanks containing the

survival chambers. The test water for the microcosm was

prepared and renewed daily throughout the experiment which

was conducted at 270C under sterile conditions.


Plasmid Stability

To study the stability of the engineered genes under

the given test conditions, the genetically engineered

strains were simultaneously enumerated on both selective and

non-selective (LB) media. Plasmid stability studies were

conducted at 15, 25 and 300C for lake water and activated

sludge effluent, and at 220C for ground water. Experiments

were performed under sterile conditions.


Statistical Analysis

The data are expressed as mean values + standard

deviation from the mean. Basic computations were performed

using STAT-2 (Stat-Soft Inc., Tulsa, Oklahoma) statistical

program. Exponential decline rate models were fit to the

population data by performing linear regression on

logarithmic CFU values against time. Rates of population

change were compared by a modified t-Test (Zar, 1984) to

determine significant differences in survival rates between

bacterial strains.









Results


Survival and Plasmid Stability in Lake Water

Temperature-dependent studies. The effect of

temperature on the survival pattern of wild-type and

genetically engineered strains of E. coli and P. p2tida is

shown in Figures 4-1 to 4-3. Rates of population change are

summarized in Table 4-1 and 4-2. At 150C (Fig 4-1), the

rates of population change were similar for both wild-type

and genetically modified strains. All four strains exhibited

only a slight decrease in numbers after 19 days of

incubation at 150C. The rate of population change

(loglO(CFU)/day) for wild-type P_. utida (-0.06) was not

significantly different from that of the engineered strain,

50058 (-0.08). However, a significant difference (p<0.001)

was noted between the rates of population change for the two

strains of E. coll. Wild-type HB101 showed a greater decline

in numbers (approximately 1 log) than the engineered strain

(<0.5 log) after a period of 19 days (Figure 4-1).

Rates of decline were greater at 250C (Figure 4-2)

than at 150C. The rate of population change for wild-type F.

Putida (-0.19) was significantly different (p<0.01) from

that of the engineered strain (-0.08). No significant

difference was noted between the survival rates of the two

strains of E. coil.

The greatest decline in numbers was observed at 300C

(Figure 4-3). Rates of population change for P. Dutida 50014














TABLE 4-1. Effect of temperature on the rates of
population change (loglo/day) of genetically
engineered and wild-type _&. gj under sterile
conditions in lake water.



Temperature Media Type Escherichia coli
(0C) wild-type GEM



15 LB -0.05 -0.02
LB + HgCI2 ND -0.03

25 LB -0.20 -0.17
LB + HgCI2 ND -0.19

30 LB -0.19 -0.13
LB + HgCl2 ND -0.14


ND indicates not applicable














TABLE 4-2. Effect of temperature on the rates of
population change (log10/day) of genetically
engineered and wild-type _. putida under sterile
conditions in lake water.



Temperature Media Type Pseudomonas putida
(0C) wild-type GEM



15 LB -0.06 -0.08
LB + Tet + Km ND -0.08

25 LB -0.19 -0.09
LB + Tet + Km ND -0.09

30 LB -0.21 -0.10
LB + Tet + Km ND -0.10


ND indicates not applicable












P. putida


E. coli

......... ....


ni


DAYS


Figure 4-1. Survival of genetically engineered (GEM)
and wild-type strains of Escherichia coli and
Pseudomonas putida at 15C in lake water.
Wild-type and GEM strains recovered on non-selective
medium; ( a) and (o ), respectively. GEM strains
recovered on selective medium ( o).


























"N

0j 0 ,

0 coil








__1







0 4 12 16

Figure 4-2. Survival of geneticaj9 Aegkneered (GEM)
and wild-type strains of Escherichia coli and
Pseudomonas putida at 25;C in lake water.
Wild-type and GEM strains recovered on non-selective
medium; ( n ) and ( ) respectively. GEM strains
recovered on selective medium (o).


























LL.
i 0


__1



4.









0 I p
0 4 8 12 16 2

DAYS


Figure 4-3. Survival of genetically engineered (GEM)
and wild-type strains of Escherichia coli and
Pseudomonas putida at 30 C in lake water.
Wild-type and GEM strains recovered on non-selective
medium; (a ) and (e ), respectively. GEM strains
recovered on selective medium (o ).









and 50058 were -0.21 and -0.10, respectively. After 19 days,

wild-type F. putida decreased by almost 4 log, as compared

to the engineered strain which showed only a 2 log decrease.

The difference in the survival rates between the two strains

was highly significant (p<0.005). The two strains of Z. cou

also exhibited similar trends. Rates of population change

for E. coli HB101 and 50008 were -0.19 and -0.13,

respectively. The wild-type strain showed a greater decline

(4 log) than the engineered strain (2.5 log). This

difference was shown to be highly significant (p<0.001).


Sterile vs nonsterile conditions. The effect of the

indigenous lake water microorganisms on the population

dynamics of the genetically engineered strains is shown in

Figure 4-4. The arrows indicate the time at which the

indigenous microorganisms were introduced into the chambers.

Prior to the addition of lake water into the chambers, both

strains showed an increase in numbers from an initial

concentration of approximately 3.0x103 to 1.0x105 for P.

putida and 2.0x103 to 5.0x104 for _. coli. Addition of

indigenous microorganisms caused an initial sharp decline

in numbers for both species. However, P. putida numbers

remained stable after three days in the presence of the

indigenous organisms, unlike B. coli which showed a steady

decline and was totally eliminated after 21 days. Under

sterile conditions, rates of decline were lower and a higher

population density was maintained. The rates of population

change at 250C for E. coli under sterile (-0.07) and non-
























E

LI.
E o i ,co

0 6
..I



4




2



0
0 10 20 30

DAYS


Figure 4-4. Survival of genetically engineered strains
of Escherichia coli and Pseudomonas putida in
filter-sterilized (0.2 um) and non-sterilized lake water
at 250C. All chambers initially contained the GEMs
suspended in filter-sterilized (0.2 um) lake water.
Arrows indicate the time at which non-sterile lake water
was introduced into the chambers in triplicate.










sterile (-0.17) conditions were significantly different

(p<0.001). The rates of change for P. putida were similar

under sterile (-0.06) and nonsterile (-0.07) conditions.

Statistical analysis (modified t-Test) showed no significant

difference between the two survival rates after addition of

the indigenous organisms. However, population densities

under non-sterile conditions were significantly lower

(p<0.05) than under sterile conditions. This difference is

due to the initial sharp decline in numbers immediately

following the addition of indigenous organisms. After 2-3

days, P. Dutida was able to stabilize its numbers and rate

of decline. Although the results suggest that there is no

significant difference in survival rates between sterile and

non-sterile conditions, it is important to note that the

indigenous population did have a significant effect on

population density.


Survival in the presence of a herbicide. The effect of

the herbicide Hydrothol-191 (1 mg/L final concentration) on

the survival of wild-type P. iutida (50014) and its

engineered strain (50058) is shown in Figure 4-5. Both

strains showed significantly (p<0.05) higher rates of

decline in the presence of 1 ug/mL hydrothol as compared to

the control with no toxicant. The rates of change for wild-

type P. putida however, were not significantly different

from that of the engineered strain (50058) in the presence

or absence of the toxicant.























0
.0


10 20 30


DAYS













Figure 4-5. Survival of genetically engineered (GEM)
and wild-type strains of Pseudomonas putida at 270C
in the presence and absence of the herbicide,
Hydrothol-191 (1 mg/L). Wild-type and GEM strains
incubated in the absence (o--o-) and (o---c); as well as
presence of hydrothol (e.-) and (u-), respectively.










Plasmid stability. The stability of the engineered

genes was determined by plating the genetically engineered

strains on both selective and nonselective media. The

results (Figures 1-1 to 4-3) indicated that the engineered

genes were relatively stable, with no loss occurring up to

19 days of incubation at 15, 25 or 300C. There was no

significant difference between the rates of population

change (Tables 4-1 and 4-2) for strains grown on either the

selective or non-selective media, at either of the three

temperature regimes.


Survival and Plasmid Stability in Activated Sludge Effluent

Temperature-dependent studies. The effect of

temperature on the survival pattern of wild-type and

genetically engineered strains of E. coli and P. Dutida in

activated sludge effluent is shown in Figures 4-6 to 4-11.

Rates of population change (loglO(CFU)/day) for _. coli. and

P. putida are summarized in Tables 4-3 and 4-4,

respectively. At 150C, E. coli exhibited similar rates of

decline for both wild-type (-0.04) and engineered (-0.03)

strains. Both strains survived well at 150C and showed only

a slight decrease in numbers after 14 days of incubation

(Figure 4-6). Higher rates of decline were noted for P.

putida strains at 150C (Figure 4-7). The rate of population

decline for the wild-type strain, 50014 (-0.13) was not

significantly greater than that of the engineered strain,

50058 (-0.10). The survival patterns of the two strains,

however, indicate that while the wild-type strain survived














TABLE 4-3. Effect of temperature on the rates of
population change (loglo/day) of genetically
engineered and wild-type &. coli under sterile
conditions in activated sludge effluent.



Temperature Media Type Escherichia coli
(0C) wild-type GEM



15 LB -0.04 -0.01
LB + HgCI2 ND -0.03

25 LB -0.07 -0.04
LB + HgCI2 ND -0.06

30 LB -0.09 -0.11
LB + HgCI2 ND -0.24


ND indicates not applicable















TABLE 4-4. Effect of temperature on the rates of
population change (log,,/day) of genetically
engineered and wild-type-. Dutida under sterile
conditions in activated sludge effluent.



Temperature Media Type Pseudomonas putida
(0C) wild-type GEM



15 LB -0.13 -0.10
LB + Tet + Km ND -0.10

25 LB -0.12 -0.10
LB + Tet + Km ND -0.10

30 LB -0.10 -0.14
LB + Tet + Km ND -0.14


ND indicates not applicable









Log CFU/mI


6


50 100


180 200
Time (hours)


260


300


Figure 4-6. Survival of genetically engineered (GEM)
and wild-type strain of Escherichia coli at 150C in
activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; (-*) and (--),
respectively. GEM strains recovered on
selective medium (- -).


-.-B1 -'m+- 60006 (LB)


380


--" $000 (LB H2Cl)










Log CFU/mI


a 100 200 300 400 500 8O
Time (hours)


Figure 4-7. Survival of genetically engineered (GEM)
and wild-type strain of Pseudomonas putida at 150C
in activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; (-.-) and (-+-),
respectively. GEM strains recovered on
selective medium (-*).










as well as the engineered strain during the first 4 days

of incubation, a sharp decline did occur before it could

stabilize its population (Figure 4-7).

At 250C, both E. coli and P. Dutida strains exhibited

survival patterns similar to those shown at 150C. JE. coli

populations remained stable for up to 14 days with little

decrease in numbers (Figure 4-8). No significant difference

was noted between the survival rates of the wild-type

(-0.07) and recombinant (-0.06) strains. P. putida strains

also exhibited similar rates of decline at both 15 and 250C

(Table 4-4). The rate of population change for the wild-type

strain (-0.12) was not significantly greater than that of

the engineered strain (-0.10). Unlike the trend noted at

150C, the wild-type strain did not survive as well as its

engineered counterpart at the start of the experiment but

instead showed a steady decline throughout the experiment

(Figure 4-9).

The greatest rates of decline were observed at 300C.

Both strains of E. coli decreased in numbers after 14 days

incubation (Figure 4-10), with the engineered strain showing

a greater decline (3 log) than the wild-type strain (1 log).

The rate of population change was significantly higher

(p<0.01) for the engineered strain (-0.24) than that of the

wild-type strain (-0.09). The recombinant strain of P.

putida also exhibited a greater decline in numbers at 300C

(Figure 4-11) as compared to 15 and 250C. Both strains

showed a 2 log decrease in numbers after 21 days, and their




Full Text
19
used the MPN technique to enumerate Tn5 mutants in soil-core
microcosms. Steffan et al. (1989) also utilized MPN assays
for monitoring GEMs in freshwater ecosystems. Fulthorpe and
Wyndham (1989) utilized an MPN-DNA hybridization technique
to investigate the 3-chlorobenzoate catabolic genotype in
aquatic systems.
Epifluorescent direct counts are routinely used to
determine the numbers and biomass of bacteria in the natural
environment. The acridine orange direct count (AODC) is a
commonly used staining procedure for enumerating
microorganisms in environmental samples (Hobbie et al.,
1977). The lack of specificity of the AODC technique makes
the method useless for enumerating GEMs that are mixed with
a natural population.
Non-Conventional Methods. The non-conventional methods
of enumerating GEMs in the environment include 1)
immunological techniques; 2) enzyme-linked immunosorbent
assay (ELISA); 3) radioactive markers; 4) fluorescent
antibodies markers; 5) plasmid epidemiology and DNA
fingerprinting; 6) use of selectable genotypic markers; 7)
use of nucleic acid sequence analysis; 8) nucleic acid
hybridization techniques such as DNA:DNA colony
hybridization, Southern blot hybridization, nucleic acid
hybridization with DNA extracts, DNA:RNA hybridization, and
use of biotinylated probes; 9) new selective enrichments;
10) protein and 'enzyme analysis; 11) isozymes; 12) protein


200
potential for conjugation in the presence and/or absence of
indigenous organisms, genetic transfer between gram-positive
and gram-negative organisms, and the effect of environmental
variables such as temperature, pH and toxicants on the
potential for genetic transfer in lake water.


90
Log CFU/ml
Time (hours)
Figure 4-9. Survival of genetically engineered (GEM)
and wild-type strain of Pseudomonas putida at 25C
in activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; (*) and (u-) ,
respectively. GEM strains recovered on
selective medium (-&-) .


61
Log CFU/ml
lZj
LS
1/2 (LB Tet Km)
LB Tet Km
Wh 1/4 (LS Tet Km)
Figure 3-11. Comparison of four different media types
for selective growth of genetically engineered
Pseudomonas putida (50058).


15
microcosms. The US Environmental Protection Agency has
developed various terrestrial microcosms designed to
investigate the following five ecosystems 1)
rhizobium/legume/soil interactions; 2) root rhizosphere; 3)
soil/plant systems for studying GEMs capable of degrading
pesticides; 4) vegetables undergoing microbial decay; and 5)
plant leaf surfaces (Armstrong et al., 1987; Gillett et al.,
1985; Omenn, 1986).
Microcosms have previously been used to study the fate
and effects of bacteria in the environment (Bissonette et
al., 1975; Burton et al., 1987; Lessard and Sieburth, 1983;
Liang et al., 1982), but much of the existing information
pertains to naturally occurring pathogens and other
organisms of public health significance. Current studies on
the fate of GEMs in the environment have utilized a variety
of microcosm designs.
Microcosms used in soil studies range from simple
screw-cap test tubes to intact soil cores. Glass test tubes
and vials have been used to study the survival of
recombinant bacteria in soil (Devanas and Stotzky, 1986;
Devanas et al., 1986; Wang et al., 1989), soil slurries
(Walter et al., 1989), soil extract (Walter et al., 1987)
and aquifer material (Jain et al., 1987). Van Elsas et al.
(1989) utilized 70-ml flasks for studying the survival and
genetic stability of plasmid containing Pseudomonas in two
types of soil. Bleakley and Crawford (1989) utilized 150-ml
beakers for studying genetic transfer between Streptomvces


CHAPTER 1
INTRODUCTION
The past decade has provided the world with explosive
developments in the area of molecular and cellular
biotechnology. Newly constructed genotypes obtained from
recombinant DNA technology are believed to be of
considerable value for many areas of basic and applied
research. High potential of benefits and large economic
incentives have now promoted biotechnology research in over
10,000 laboratories worldwide (Jain et al., 1988).
To date, almost
all research
has been
limited to
physically
contained
systems. The
number
of
cases of
deliberate
release of
genetically engineered
bacteria into
the environment is few and fairly recent (Halvorson et al.,
1985). Prospects for the routine release of genetically
engineered organisms (GEOs) into the environment are
becoming increasingly feasible. The potential uses of
recombinant bacteria in the environment include enhancement
in food and agriculture production, biocontrol of insects
and diseases, metal and mineral leaching, environmental
remediation and waste treatment (Gillett et al., 1985;
Halvorson et al., 1985; Johnston and Robinson, 1984).
Deliberate release of GEOs has, however, raised
considerable concern and attention within the public and
scientific sectors. Questions and issues concerning
1


17
be potentially useful in future studies involving GEMs.
Fulthorpe and Wyndham (1989) described a flowthrough lake
microcosm for detecting survival and activity of catabolic
genotypes. Rochelle et al. (1988) developed a rotating disc
microcosm to study gene transfer in riverine systems. Both
microcosms allowed continuous flow of the test water
throughout the experimental procedure.
Although microcosms serve an important function in the
study of GEMs, they presently provide only limited insight
into the natural ecosystem. A limitation to the use of
microcosms is the fact that test results are often not
directly comparable due to the wide diversity of microcosm
systems currently in use. However, the use of controlled
laboratory conditions allows the precise study of
environmental variables on the fate of GEMs and rDNA in the
environment.
Methods for Detection and Enumeration
Methods for detection and enumeration of introduced
GEMs and their rDNA sequences usually involve the use of
markers, such as antibiotic resistance. Methodologies can be
arbitrarily divided into conventional and non-conventional
techniques. The difference between the two groups is based
on the distinction that conventional techniques are widely
and routinely used procedures for assessment of microbial
populations in nature, while non-conventional techniques are
those that (1) have been developed but have not been applied
for environmental use or (2) have been specifically


27
wild-type, mutant and recombinant Streptomvces was studied
in a soil ecosystem. With all strains, population densities
slowly declined, though one recombinant strain survived
significantly better in nonsterile soil than its
nonrecombinant parent. One recombinant strain significantly
increased the short-term rate of soil organic carbon
turnover, while another recombinant strain temporarily
reduced carbon mineralization rates during the first days of
release. Additional studies also indicated genetic
instability in one recombinant strain.
The use of DNA probes to detect the stability of
recombinant genes and plasmid vector sequences in soils was
investigated by Jansson et al. (1989). Two Pseudomonas
strains were engineered to contain the notII gene and
plasmid vector sequences in their chromosomes. Incubation of
the strains in nonsterile soil, followed by total DNA
isolation and Southern blot hybridization, revealed the loss
of plasmid vector sequences from the chromosome though the
nptll gene was retained. It appeared the extreme conditions
encountered in soil systems resulted in stress-induced
deletions.
In general, it was found that the survival rates of
GEMS was significantly lower in non-sterile soils than in
sterile soils. This increased decline in non-sterile soil
was attributed to a combination of biotic factors such as
predation, antagonism and competition from the natural
population.


TABLE 8-2. Transconjugant production using in vitro
membrane-filter mating between genetically engineered
strains of ]5. coli and recipient strain of £. putida.
No. of
Bacterial Mating No. of cells/ml Transconjugante
Strains Temperature Donor Recipient per ml
pDugll
x 50479
28C
1.8
E9
3.0
E8
u

o
E2
pJAl x
50479
O
0
03
CN
3.4
E9
3.5
E8
4.8
E4
pDugll
X 50479
3 5C
1.8
E9
3.0
E8
3.3
E2
pJAl x
50479
3 5C
3.4
E9
3.5
E8
1.8
E4
197


2
potential adverse environmental effects are foremost in the
minds of many involved. Genetically engineered
microorganisms (GEMs) that are released may have the
capacity to reproduce, spread beyond the initial point of
release and transfer their novel genetic information to the
indigenous microbial populations. The possibility also
exists that recombinant organisms may behave differently
from their non-recombinant parental strains when released
into the environment.
The effective use of GEMs in biological control, in
improving crop yield and in controlling environmental
pollution depends upon successful colonization at the
appropriate sites. However, the inherent properties that
would allow these organisms to adeguately compete, survive,
and propagate in their new environments might also produce
adverse effects on the natural ecosystem.
The concept of deliberate release of GEMs to the
environment requires a clear understanding of their behavior
and survival, their interactions with indigenous organisms,
their potential for spread, their effects on physiochemical
processes, and the ability to detect and monitor the fate of
the organisms and their recombinant genes within a natural
system (Jain et al., 1988; Keeler, 1988; Tiedje et al.,
1989).
It is obvious that many questions relevant to the
safety of GEOs in the environment still need to be addressed
and answered. There is at present a dearth of information


204
8. In the root rhizosphere, no significant differences were
noted between the survival rates of GEMs and their wild-type
counterparts. This would indicate that the presence of
additional DNA has no effect on the survival of the organism
in the rhizosphere.
9. In the presence of root exudates as a sole source of
carbon, both GEM and wild-type strains exhibited a
substantial increase (1-2 log) in cell numbers. The GEMs
were therefore capable of utilizing root products for growth
and maintenance in the root vicinity.
10. The presence of indigenous rhizosphere microorganisms
caused a decline in numbers of both strains of GEMs. The
decline in numbers were attributed to both competition and
predation. Genetically engineered outida was better able
to compete with the indigenous population than the
engineered E_¡_ coli. Unlike E. coli. pseudomonads are
commonly found in the soil and root environment, which would
account for their better survival and competitive ability.
11. The presence of root exudates allowed the GEMs to
compete better with the indigenous rhizosphere population,
but only for a limited period of time. After 40 hours, the
survival rates of the GEMs declined.
12. Physiological changes were observed in both engineered
and wild-type strains of E^. coli after exposure to lake
water. Changes were observed in dehydrogenase activities,
B-galactosidase biosynthesis and cell permeability. However,


219
Pennwalt Corp. 1980. Technical information manual: the uses
and properties of endothall. AGCHEM. Pennwalt,
Philadelphia, Pennsylvania.
Pettigrew, C.A., and G.S. Sayler. 1986. The use of DNA:DNA
colony hybridization in the rapid isolation of 4-
chlorobiphenyl degradative bacterial phenotypes. J.
Microbiol. Methods 5:205-213.
Phillips, S.J., D.S. Dalgarn, and S.K. Young. 1989.
Recombinant DNA in wastewater: pBR322 degradation
kinetics. J. Water Pollut. Control Fed. 61:1588-1595.
Pillai, T.N.V., and A.K. Ganguly. 1972. Nucleic acid in the
dissolved constituents of seawater. J. Mar. Biol. Ass.
India 14:384-390.
Rand, G.M., and S.R. Petrocelli. 1985. Fundamentals of
Toxicology: Methods and Applications. Hemisphere
Publishing Corp., Washington.
Reich, T.J., V.N. Iyer, and B.L. Miki. 1986. Efficient
transformation of alfalfa protoplasts by the
intranuclear microinjection of Ti plasmids.
Bio/Technology 4:1001-1004.
Reinert, K.H., J.H. Rodgers Jr., M.L. Hinman, and T.J.
Leslie. 1985. Compartmentalization and persistence of
endothall in experimental pools. Ecotoxicol.
Environmen. Safety 10:86-96.
Reinhartz, A., I. Lampert, M. Herzberg, and F. Fish. 1987.
A new, short-term, sensitive, bacterial assay kit for
the detection of toxicants. Toxicity Assess. 2:193-
206.
Rhodes, M.W., I.C. Anderson, and H.I. Kator. 1983. In situ
development of sublethal stress in Escherichia coli:
effects on enumeration. Appl. Environ. Microbiol.
45:1870-1876.
Richaume, A., J.S. Angle, and M.J. Sadowsky. 1989. Influence
of soil variables on in situ plasmid transfer from
Escherichia coli to Rhizobium fredii. Appl. Environ.
Microbiol. 55:1730-1734.
Rochelle, P.A., J.C. Fry, and M.J. Day. 1989. Plasmid
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Ecol. 62:127-136.


106
hydrothol for the control of weeds is generally higher (1-5
mg/L) (Pennwalt Corp., 1980), the potential impact on
bacterial activity can be even greater. The LC50 value for
hydrothol has been reported to be as low as 0.49 mg/L at
25C
(Keller et
al.,
1988) NO
significant difference was
noted
between
the
engineered
and parental
strains,
indicating that
the
presence
of
extraneous DNA
did not
afford any advantage or disadvantage to the organisms. Dwyer
at al. (1988) utilized an activated sludge microcosm to
determine the fate of a GEM capable of utilizing a mixture
of 3-chlorobenzoate (3-CB) and 4-methylbenzoate (4-MB).
Although the GEM survived well in the microcosm, it did not
perform well under additional shockloads of 3-CB and 4-MB.
While the GEM never adequately recovered, a new population
of a 4-MB degrader did develop and thrived under the new
conditions.
With the increasing amounts and varieties of pollutants
that are being released into the environment it is important
to understand how genetically engineered strains behave
under the added stress. Palmer et al. (1984) detected subtle
biochemical and genetic changes in £. coli after exposure to
bay water containing toxic chemicals. In addition to having
to deal with the toxicant, engineered organisms will also
have to compete with indigenous organisms including those
that have already adapted to the environment and are capable
of degrading existing toxicants.


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
FATE AND SURVIVAL OF GENETICALLY ENGINEERED
MICROORGANISMS AND THEIR RECOMBINANT GENES
IN THE NATURAL ENVIRONMENT
By
Judy Awong
August 1990
Chairman: Gabriel Bitton
Cochairman: G. Rasul Chaudhry
Major Department: Environmental Engineering Sciences
Prospects for the routine release of genetically
engineered microorganisms (GEMs) into the environment are
becoming increasingly feasible. Deliberate release of
recombinant microorganisms has, however, raised questions
concerning potential adverse environmental effects. The
purpose of this study was to develop and utilize
laboratory-contained microcosms to study the fate and
survival of GEMs and their recombinant DNA in natural
environments.
A model aquatic microcosm that utilized membrane
diffusion chambers in a flow-through or static renewal
system was used to study the survival of genetically
engineered and wild-type strains of Escherichia coli and
x


170
ABCDEFGH I J KLMNOPQR
Figure 7-1. Degradation of spiked eDNA in four aquatic
samples after 15 minute incubation at 25C.
Lane A: extracellular DNA innoculum at time 0;
Lane B: lambda standard;
Lanes C-F: Tap Water (U, A, F and C, respectively);
Lanes G-J: Groundwater (U, A, F and C,
respectively);
Lanes K-N: Lake Water (U, A, F and C,
respectively);
Lanes O-R: Raw Sewage (U, A, F and C,
respectively).
U, A, F and C represent treatments Untreated, Autoclaved,
Filter-sterilized and Control, respectively.


50
for bacterial enumeration and optical density measurements
(550nm).
Comparison of plating techniques. Two methods were
compared for bacterial enumeration. The conventional
spread-plate method was compared to the drop-plate
technique of Hoben and Somasegaran (1982). Samples were
diluted in sterile potassium phosphate buffer (pH 7.2) and
plated on the appropriate selective or non-selective media.
For the spread-plate method, 0.1 ml aliquots were
aseptically spread onto the media. In the drop-plate method,
20 ul of the sample was carefully dropped onto the media,
and allowed to completely dry before incubation. Both
methods were carried out in triplicate.
Comparison of selective media types. It has been
suggested that bacterial cells can become physiologically
injured due to environmental stresses imposed by aquatic
environments (Bissonette et al., 1975). The extent of such
injuries can lead to a loss in ability to produce colonies
on selective media. It is possible that the aquatic
environment can induce enough stress upon the GEMs to the
extent that they become physiologically debilitated and
therefore not detectable by the selective media of choice.
To determine whether the extent of injury would lead to an
inability to grow on the selective media, the GEMs were
subjected to lake water and enumerated at specific time
intervals on varying strengths of selective media. P. putida


57
consistently gave higher numbers of viable counts. This
trend was observed for both wildtype ( Figure 3-8) and
recombinant (Figure 3-9) strains of E. coli plated on non-
selective and selective media, respectively. The drop-plate
method has several advantages over the traditional spread-
plate method. The methodology of the drop-plate technique
allows as much as four dilutions (three replicates each) per
plate, as compared to the spread-plate method which requires
a single plate for each replication of each dilution. The
drop-plate method therefore utilizes 1/12 of the media and
petri dishes normally required for spread plates. In
addition to its cost-effectiveness, the drop-plate method is
also faster and as reliable as the conventional spread-plate
method.
Comparison of Media Types
A comparison of four media types for selective growth
of the genetically engineered strains of E. coli and P.
putida indicated no significant differences between media
types for both strains (Figures 3-10 and 3-11,
respectively). An exception was observed for P. putida after
6 hours of exposure to the lake water (Figure 3-11). Viable
counts were highest on half-strength selective media and
lowest on quarter-strength media. The results of this study
suggest that exposure to lake water does not necessarily
cause physical or physiological injury to an extent that
affects the growth of cells on selective media. Since full-
strength selective media is effective for screening


36
for plasmid pD10. The implications of these studies suggest
that genetic exchange of recombinant genes placed on "safe"
(non-transmissible) vector plasmids can still be mediated by
conjugation. An interesting observation noted by McClure et
al. (1989) was the fact that some activated sludge
transconjugants showed higher rates of catabolic activities
than the original recombinant strain.
Conjugative plasmid transfer by recombinant bacteria
has also been investigated in plants (Armstrong et al.,
1987; Knudsen et al., 1988) and animals (Armstrong et al.,
1987). Armstrong et al. (1987) utilized a complex microcosm
model to assess survival and plasmid transfer of recombinant
bacteria associated with plants and herbivorous insects.
Plasmid transfer from recombinant to non-recombinant strains
was not detected in either plant, whole insect, foregut or
frass samples.
Knudsen et al. (1988) reported a predictive model for
conjugative plasmid transfer in the rhizosphere and
phyllosphere. The model was tested in microcosms planted
with radish seeds and on leaf surfaces of radish and bean
plants. Transconjugants were isolated from both rhizosphere
and phyllosphere microcosms after one day. This was followed
by an initial rapid increase and a subsequent decline in the
numbers of transconjugants.
Transduction
Generalized transduction systems have been observed in
many common soil and aquatic organisms such as Rhizobium.


153
selective or non-selective media. This indicates that the
plasmid was stably maintained within the cells throughout
the duration of exposure to lake water. Based on viable
counts, there .was no significant difference in survival
rates between the engineered and wild-type strains. However,
AODC direct counts indicate significantly higher numbers of
the wild-type strain (HB101) than the engineered strain
(50008) after nine days. This suggests possible injury or
sublethal effects to the engineered strain that would not
allow growth on non-selective or selective media. It has
been suggested that sublethal stress can cause death in
standard enumeration media (Bissonnette et al., 1975).
The morphological changes that occurred in both strains
of E. coli after their introduction into lake water have
also been reported by others (Baker et al., 1983; Lopez-
Torres et al., 1988; Sjogren and Gibson, 1981; Xu et al.,
1982). Lopez-Torres reported that 50% of the cells had
transformed to micrococci after 24 hours of in-situ exposure
to tropical marine waters. It has been suggested that these
small, rounded forms are transitional forms that occur in
oligotrophic environments from richer environments, such as
agricultural runoff or wastewater effluents (Sieburth,
1979) These cells are thought to be in the process of
starvation in the aguatic environment. Novitsky and Morita
(1978) suggested that a stress response occurs in the
bacteria that causes several reductive divisions, which
eventually causes an increase in cell number without a


138
B-GALACTOSIDASE
BIOSYNTHESIS ASSAY
STEP 1. CELL GROWTH:
grow E. coli in LB medium
overnight at 35C
STEP 2. CELL PREPARATION:
dilute cells with fresh media to A55Q = 0.2
1 r\t.t 4- t.t A = O ^
STEP 3. EXPOSURE TO TOXICANT:
add 0.9 ml toxicant to 0.1 ml cells
incubate reaction mixture for 30 min.
STEP 4. 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 5. B-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 1ml cold Na2C03
measure absorbance at 420nm
Figure 6-1. Protocol for B-galactosidase biosynthesis
activity. (Adapted from Dutton et al., 1988).


140
10 minutes and optical density of the supernatants
determined at 490nm. Results are expressed as a percentage
of dehydrogenase activity relative to time zero.
Cell Permeability
Changes in outer membrane permeability were indirectly
determined by measuring changes in the inhibition of B-
galactosidase biosynthesis to the toxicant,
pentachlorophenol (PCP) over time. The envelope of gram
negative bacteria is a complex structure that consists of
cytoplasmic membrane, a rigid peptidoglycan cell wall and an
outer membrane (Nikaido and Vaara, 1985). The outer membrane
is an effective diffusion barrier to hydrophobic substances.
Changes in outer membrane permeability can lead to increased
or decreased suspectibility of the cell to hydrophobic
toxicants. This in turn directly affects B-galactosidase
biosynthesis (Cenci et al., 1985; Dutton et al., 1988;
Reinhartz et al., 1987). The inhibition of B-galactosidase
biosynthesis is therefore a direct function of outer
membrane permeability to toxicants and is reflective of
changes in cell outer membrane permeability.
B-galactosidase inhibition assays were determined by
the method of Dutton et al. (1988). Triplicate 1-ml aliquots
were exposed to different concentrations of PCP for 30
minutes at 35C. The cells were then induced for B-
galactosidase by adding 0.01% (final concentration) IPTG and
incubating at 35C for 30 minutes. B-galactosidase activity


ACKNOWLEDGEMENTS
The author would like to acknowledge and thank the
chairman of her doctoral committee, Dr. Gabriel Bitton, for
his advice, support, and encouragement during the course of
this study, and in particular for his role as mentor during
her doctoral candidacy. His guidance over the years has
provided many valuable lessons that will always be greatly
appreciated. The author would also like to thank the
cochairman of her doctoral committee, Dr. G. Rasul Chaudhry,
for his support, encouragement, and enthusiasm during the
course of this study, and for his assistance in developing
the dissertation topic. The author is extremely grateful to
Dr. Chaudhry for providing her the opportunity for research
in a new and challenging area of study.
The author is also grateful to the other members of
her committee, Dr. Thomas L. Crisman, Dr. Ben L. Koopman and
Dr. Sam Farrah, for their support and help during the course
of this study. Special thanks are extended to Dr. Thomas L.
Crisman for his advice, guidance, and friendship during the
course of her academic career.
The author also wishes to thank her fellow students and
friends, in particular, Ulrike Crisman, Dr. Ronald J. Dutton
and Henry Meier, for their strong support and friendship
IV


147
SPECIFIC ACTIVITY (% OF TIME ZERO)
H8101 (WT)
M 50003 (GEM)
TIME (DAYS)
Figure 6-5. Changes in B-galactosidase biosynthesis of
wild-type (HB101) and genetically engineered (50008)
strains of Escherichia coli after exposure to lake
water at 25C. Results are expressed as percent
reduction in specific activity relative to time zero.


24
(Beringer and Bale, 1988; Faust et al., 1975; Liang et al.,
1982; McFeters and Stuart, 1972).
Numerous studies have been conducted to study bacterial
survival in the natural environment (Bissonette et al.,
1975; Burton et al., 1987; Lessard and Sieburth, 1983; Liang
et al., 1982; McFeters and Stuart, 1972; Ohana et al., 1987;
Scheuerman et al., 1988). However much of the existing
information pertains to naturally occurring pathogens and
other microorganisms of public health significance. The
potential for the use and release of GEMs in the
environment has recently led to more specific research on
the fate of recombinant organisms in the natural
environment. The last few years have produced an array of
useful and informative material on the survival of GEMs in a
variety of natural environments.
Soil. Plant and Rhizosohere Systems
The fate of GEMs in the soil environment has been
investigated in a variety of studies. Devanas et al. (1986)
showed that the survival of genetically-engineered strains
of Ej. coli in soil was primarily a function of the bacterial
strain and not of the contained plasmid. The nutritional
state of the soil was also shown to influence the degree of
survival. Devanas and Stotzky (1986) also demonstrated that
recombinant genes inserted into plasmid DNA had little
effect on the survival of the bacterial host and
maintenance of the vector. Similar results were reported by
Walters et al. (1987) who utilized soil extracts to study


104
capabilities of the GEMs. Whether these populations are
active, or static but physiologically active is not clear.
Similar results have been reported by Jain et al. (1989) for
introduced genotypes in groundwater aquifer material. Stable
populations of plasmid containing strains of Pseudomonas
putida were maintained for up to 8 weeks in groundwater
aquifer microcosms. Caldwell et al. (1989) demonstrated that
under long-term starvation conditions, the same plasmid in
different hosts affected the survival rates of the host
cells in well water. Some species were better able to
maintain a stable population as compared to other species
which rapidly declined and disappeared from the system. The
ability of cells to remain viable under starvation
conditions for a long period is probably the result of
metabolic arrest (Morita, 1988). This type of dormancy is
due to a lack of available energy and during this period the
cells can reorganize its genome and structure.
The ability of the genetically engineered strains to
survive and compete in the presence of indigenous
microorganisms was tested by adding lake water to survival
chambers containing GEMs and comparing their survival rates
to those of GEMs under sterile conditions. A significant
decline in numbers was noted for both species in the
presence of indigenous microorganisms. Similar results have
been reported in soil studies (Bentjen et al., 1989; Devanas
et al., 1986; Fredrickson et al., 1989; Walter et al., 1987;
Wang et al., 1989; Yeung et al., 1989) and aquatic systems


142
Table 6-1. Comparison of rates of population change
(logiQ/day) of genetically engineered and
wild-type £. coli enumerated by viable plate counts
and acridine orange direct counts (AODC).
Viable Counts
AODC
LB LB + HgCl2
Escherichia coli
Wild-type HB101
-0.23A*
-0.04a*
Escherichia coli
GEM 50008
-0.30A -0.31
I
o

o
00
tr
*
Within a column, rates with the same letters were not
significantly different at P<0.05.


163
in Tris-EDTA (TE) buffer (pH 8.0) and used as the eDNA
inoculum.
Determination of DNA Inoculum Concentration
Preliminary experiments were performed to determine the
amount of NA inoculum to be added to the reaction tubes
that would allow optimum recovery and detection of the added
DNA from the aquatic samples. Triplicate tubes containing
0.5 ml deionized distilled water were inoculated with
varying quantities of the isolated DNA inoculum. The tubes
were then immediately subjected to a DNA recovery procedure
(described below) and the recovered DNA subjected to gel
electrophoresis. Results indicated that a DNA inoculum of
10.4 ug/ml was a suitable concentration that would allow
good recovery and visual detection throughout the duration
of the experiment.
DNA Recovery Procedure
The amount of added eDNA remaining at any given time
during the experiment was determined as follows. Aliquots
(0.5 ml) of the appropriate sample were transferred to
sterile Eppendorf tubes and extracted with 1:1 chloroform
phenol (200 ul) for two minutes. The samples were then
centrifuged at 7000 rpm for five minutes and exactly 450 ul
of the top layer was transferred to new eppendorf tubes. The
sample was further extracted with ether (200 ul) for 1
minute, and the DNA was precipitated by adding sodium
acetate and absolute alcohol (Chaudhry and Huang, 1988). The


101
GEM, the presence of the foreign DNA did not affect its
growth and survival in soil or the rhizosphere. Scanferlato
et al. (1989) reported that the densities of genetically
engineered and wild-type Erwinia carotovora strains declined
at the same rate in pond water and sediment. He concluded
that the superior ability of the wild-type strain to rot
living plant tissue did not confer the wild-type with a
selective adaptation for survival. Other studies have shown
that recombinant strains can persist as well as their non
recombinant strains without specific selective pressures for
the organisms or the plasmid (Amy and Hiatt, 1989; McClure
et al., 1989; Steffan et al., 1989).
However, other reports have shown contradictory results
on the effect of the addition of extra DNA to the bacterium.
The additional metabolic load of maintaining and replicating
plasmid DNA has been reported to possibly decrease bacterial
survival and competiveness (Curtiss, 1976; Helling et al.,
1981). This may explain the higher rate of decline that was
observed for the recombinant strain of £. coli in activated
sludge effluent at 30C. Buttner and Amy (1989) reported
similar results in a study designed to assess the survival
of ice nucleation-active and genetically engineered
non-ice-nucleating Pseudomonas svrinaae strains after
freezing. They observed no significant differences between
the two strains at various freezing temperatures, but in
mild freezing environments, the wildtype strain had a
competitive advantage over the engineered strain. Trevors et


145
% SURVIVAL
0 13 6 9
TIME (DAYS)
Figure 6-4. Percent survival of wild-type (HB101) and
genetically engineered (50008) strains of Escherichia coli
after exposure to lake water at 25C,
using acridine orange direct counts (AODC).


Results 166
Efficiency of the DNA Recovery Method 166
DNA Degradation: Characterization by Gel
Electrophoresis 166
Raw sewage 166
Lake water 168
Ground water 169
Tap water 169
Kinetics of Degradation 169
Discussion 180
8 CONSTRUCTION OF A MODEL GEM FOR USE IN
GENE TRANSFER STUDIES 190
Introduction 190
Materials and Methods 191
Bacterial Strains, Plasmids and Growth
Conditions 191
Isolation of Plasmid DNA 191
Restriction Analysis of Plasmid DNA 191
Cloning of Model GEM 193
Conjugation Studies 193
Results and Discussion 194
Construction of the GEM 194
Conjugation Studies 195
9 CONCLUSION 201
REFERENCES 206
BIOGRAPHICAL SKETCH 225
ix


171
BACDEFGHI JK LMNOPQR
V
Figure 7-2. Degradation of spiked eDNA in four aquatic
samples after 1 hour incubation at 25C.
Lane designations same as in Figure 1.


CHAPTER 6
STRUCTURAL AND PHYSIOLOGICAL ALTERATIONS OF
GENETICALLY ENGINEERED AND WILDTYPE STRAINS OF E. COLI
AFTER EXPOSURE TO AN AQUATIC ENVIRONMENT
Introduction
In the natural environment, the fate of introduced
organisms ultimately depends on a variety of factors.
Explanations for the decline of non-indigenous organisms
include predation by protozoan and zooplankton, inability to
compete for available nutrients, harmful effects of
toxicants, solar irradiation and starvation associated with
a lack of organic nutrients (Curds and Fey, 1969; Henis et
al., 1989; Jannasch, 1968; Klein and Cassidy, 1967; Klein
and Alexander, 1986; Tate, 1978) .
However, estimations of die-off and decline may be
overestimated due to the existence of sublethal stress in
bacteria exposed to unfavorable conditions such as those
found in aquatic environments (Bissonnette et al., 1975;
Dawe and Penrose, 1978; Hoadley and Cheng, 1974; Klein and
Wu, 1974). Sublethally stressed or damaged cells have been
identified by their ability to grow on a nonselective, but
not on a selective media (Bissonnette et al., 1975; Klein
and Wu, 1974). More recent investigations have emphasized
the possible structural and metabolic adaptations that occur
133


135
alterations as adaptive mechanisms for survival in
nutrient-poor or starvation conditions (Novitsky and Morita,
1976; 1978; Tabor et al., 1981; Torella and Morita, 1981).
Similar alterations have also been observed for organisms
introduced to new environments (Anderson et al., 1979; Chai,
1983; Palmer et al., 1984; Tamplin and Colwell, 1986; Walsh
and Bissonnette, 1989). It has been suggested that these
alterations are adaptations to stressful environmental
conditions such as temperature, salinity, pH and the
presence of toxic compounds that may be encountered in the
natural environment. This has important practical
implications for the release of GEMs into the environment
since the fitness of the genetically engineered bacteria may
ultimately determine their value as biotechnological tools.
The purpose of this study was to determine the extent
to which genetic manipulations affect the adaptive ability
of the organism after exposure to the environment. This was
accomplished by comparing structural and physiological
alterations of the GEM with its wild-type strain after
exposure to lake water.
Materials and Methods
Bacterial Strains and Culture Media
Escherichia coli (strain HB101) was used as the wild-
type reference strain. Genetically engineered E. coli
(strain 50008) was obtained by inserting an EcoRl DNA
fragment from pRCIO into a derivative of pBR322. The EcoRl


119
Figure 5-3. Effect of corn-root exudates on the growth
of genetically engineered Escherichia coli. Initial
inoculum concentration was approximately 3.0 x 10.
( ), no root exudates; (), presence of root exudates.


33
transfer in the environment depends on the survival and
transport of organisms in the ecosystem. To date, there is
limited information available on natural gene transfer in
the environment. The increasing feasibility for the use and
release of GEMs in the environment has focused greater
attention on the need for gene transfer studies in the
natural environment (Stotzky and Babich, 1986; Trevors et
al., 1987). The following review focuses mainly on recent
studies of genetic transfer using recombinant organisms in
the natural environment.
Conjugation
Conjugation is the most well studied of the three
mechanisms of genetic transfer. Past studies have dealt
mainly with conjugative plasmids in soil (Graham and
Istock, 1978; Trevors, 1987; Trevors and Oddie, 1986;
Weinberg and Stotzky, 1972) and aquatic systems (Altherr and
Kasweck, 1982; Gowland and Slater,1984; Mach and Grimes,
1982) Much of the aquatic studies were conducted from a
public health point of view and addressed the potential for
transfer of antibiotic resistance plasmids (R plasmids) in
waters receiving sewage effluents (Bell et al., 1983; Goyal
et al., 1979; Grabow et al., 1975).
More recent studies strongly suggest or demonstrate
plasmid gene transfer among indigenous organisms on plant
surfaces and plant tissue (Manceau et al., 1986; Talbot et
al., -1980), in cultured rainbow trout (Toranzo et al.,


16
species in soil. More complex microcosms in the form of
intact soil cores have been utilized for evaluating the fate
and ecological impact of the release of GEMs (Bentjen et
al., 1989; Fredrickson et al., 1988; 1989).
Freshwater microcosms consisting of 20 liter glass
carboys were utilized by Steffan et al. (1989a; 1989b) for
monitoring specific microbial populations. Other aquatic
microcosms used for monitoring GEMs include glass bottles
(Trevors et al., 1989), culture flasks (Chaudhry et al.,
1989; Morgan et al., 1989) and mason jars (Scanferlato et
al., 1989). More detailed and complex microcosms include
laboratory-scale waste treatment plants (Mancini et al.,
1987) and model activated-sludge units (McClure et al.,
1989)
Microcosms have also been developed to study the fate
of GEMs associated with the plant system. Armstrong et al.
(1987) and Knudsen et al. (1988) utilized a complex
microcosm to assess survival and gene transfer by
recombinant bacteria associated with plants and herbivorous
insects. Yeung et al. (1989) utilized a Styrofoam cup-
membrane assembly to study growth of GEMs in soil and
rhizosphere.
Fulthorpe and Wyndham (1989) and Rochelle et al. (1989)
recently described aquatic microcosms that simulated a more
natural setting and thus provided a more rigorous test on
the introduced species. Although indigenous organisms were
utilized as the test organisms in these microcosms, they can


205
these changes were not similar for the GEM and wild-type
strains. Whether these alterations facilitate survival or
enhance the capability for adaptation in unfavorable
environments is presently not known.
13. Morphological changes were also noted in both engineered
and wild-type strains of E. coli after exposure to lake
water. Cells became smaller and more rounded. These changes
are an adaptation to starvation or stress conditions.
14. The dynamics of extracellular DNA degradation as
determined via gel electrophoresis and spectrophometric
methods indicate that chromosomal DNA is degraded at a
faster rate than plasmid DNA. The plasmid DNA undergoes a
series of structural changes prior to and during
degradation.
15. Degradation of eDNA occurred rapidly in the
environmental samples, but the degradation rates differed
among samples.
16. Rapid hydrolysis occurred in raw sewage and lake water
by cell-associated and extracellular nucleases.
17. Abiotic degradation is more important in raw sewage and
lake water than groundwater and tap water.
18. The potential for genetic transformation does exists
since the molecular size range of eDNA and their degradation
products falls within the range required for transformation.


187
Novitsky (1986) recently reported a turnover time of 19.6
days for DNA in marine sediments.
These reports suggest a wide range of degradation rates
in environmental samples. It is difficult, however, to
compare these rates due to the different methodologies and
samples used in each study. Also, since sediment may protect
DNA from degradation (Lorenz and Wackernagel, 1987) it is
difficult to compare sediment turnover times with those
found in aquatic systems. In addition, the calculated
degradation rate or turnover times does not necessarily
provide a complete picture of DNA degradation. This is
evident in the study done on groundwater. The calculated
half-life of 1.25 days suggests rapid degradation of DNA but
gives no indication of the initial stability of the DNA
during the first 24 hours. Compared to lake water, which
also shows a similar half-life (1.42 days), DNA degradation
was observed within 1 hour of incubation and is no longer
detected on the gel after 24 hours. Therefore, even though
the calculated rates were similar, the mechanisms and
kinetics of degradation were found to be somewhat different
(Figures 7-1 to 7-4).
Natural transformation is a process by which a cell
takes up naked DNA from the surrounding environment and can
possibly acquire an altered genotype that is heritable
(Smith et al., 1981). Although studies suggest different
mechanisms for natural transformation between gram positive
and gram negative bacteria, as a rule all bind double


48
Figure 3-3. Schematic diagram of the flow-through microcosm.


195
The new recombinant plasmid, pJAl, therefore contains
several antibiotic resistance markers, an EcoRl DNA fragment
of Napier grass, the mob gene and the transposable element,
Tn 5 (Figure 8-1). The combination of markers allows the
plasmid to be easily detected or traced during studies
involving environmental samples. The presence of the Napier
grass DNA makes the plasmid unique for detection purposes
because it does not hybridize with DNA isolated from
microbial communities of soil, lake and sewage waters
(Chaudhry et al., 1989).
Conjugation Studies
The presence of the mob gene in pJAl, should
theoretically increase transfer frequencies rates between
donor and recipient cells. This was studied by comparing
transfer frequency rates of the plasmids pJAl and pDugll by
in vitro membrane-filter mating. Additional studies using
lake water microcosms were also conducted to determine the
potential for genetic transfer via conjugation in natural
environments.
Results of the in vitro membrane-filter mating study
are given in Table 8-2. The results indicate higher numbers
of transconjugants in the mating cross between pJAl and
50479, than the cross between pDugll and 50479, at both
temperatures tested. An almost 2 log difference in the
number of transconjugants per ml was observed between the


137
biosynthesis, INT-dehydrogenase activity and changes in cell
permeability. Morphological changes were also noted.
Bacterial Enumeration
Viable counts were enumerated using the drop-plate
technique (Hoben and Somasegaren, 1982) on either LB or LB
plus HgCl2 (40 ug ml1) agar plates for wild-type and
engineered strains, respectively. Direct counts were
determined by the acridine orange direct count (AODC) method
of Hobbie et al. (1977). Samples were stained with 0.01%
(final concentration) acridine orange for 2 minutes and
filtered through Nucleopore filters (0.2u) that were
prestained with Irgalan Black (0.2% solution in 2% acetic
acid). Bacterial counts were made using an epifluoresence
microscope. Results are expressed as % survival relative to
time zero.
B-Galactosidase Biosynthesis
B-galactosidase biosynthesis was determined by the
method of Dutton et al. (1988). Triplicate 1-ml aliquots
were removed from the survival chambers and transferred to
small test tubes. The bacterial cells were then induced with
0.01% (final concentration) IPTG (Sigma Chemical Co., St.
Louis, MO) for 30 minutes at 35C. Beta-galactosidase was
measured using the o-nitrophenyl-B-D-galactopyranoside
(ONPG) (Sigma) method (Figure 6-1). This substrate is a
colorless compound that is converted to galactose and o-
nitrophenol in the presence of B-galactosidase. The o-


158
can provide a stressful environment at any given time. Munro
et al. (1987) also observed that cells grown in a
wastewater-seawater mixture present physiological
modifications that altered their resistance to heavy metals,
antibiotics, colicins, and bacteriophages. Increased
sensitivities were noted to heavy metals and some
antibiotics such as polymixin and aminosides. On the other
hand, sensitivity to B-lactams, tetracycline and nalidixic
acid was lowered or was zero. Increased resistance to
bacteriophage was also noted.
The implications of this study are that bacteria can
undergo structural and physiological changes in any natural
environment, and such adaptations are not necessarily
related to starvation conditions only. Structural changes
can lead to modification of properties which in turn can
directly affect cell survival, nutrient uptake, expression
of virulence and pathogenicity, and potential for in-vivo
repair. Theses changes are important factors to consider in
view of the intentional release of genetically engineered
microorganisms into the environment. As shown in this study,
both recombinant and non-recombinant strains show
adaptations after exposure to lake water, but more important
is the fact that these changes were not identical for both
strains. Whether these alterations facilitate survival or
enhance the capability for adaptation in unfavorable
environments is presently not known, and additional studies
are necessary to illustrate and confirm such hypotheses.


162
from a laboratory faucet on the University of Florida.
Samples were collected in sterile, 300 ml plastic bottles
and stored at 4C prior to sample preparation. All samples
were collected within 24 hours before the start of the
experiment.
Sample Preparation
Each sample was split into three equal aliquots before
sample treatment. Treatments included autoclaving, filter-
sterilization or no treatment of the samples. Autoclaving
was undertaken at 121C for 15 minutes. Filter-sterilization
was carried out by passing the sample through a 0.22 urn
membrane filter (Acrodisc, Gelman). Lake water and raw
sewage samples were prefiltered (Whatman) to remove large
particles prior to filtration through the 0.22 urn membrane
filter. The third aliquot received no treatment and was
designated as the non-treated sample. Triplicate 5-ml
subsamples of each treatment (autoclaved, filter-sterilized
and non-treated) were then aseptically added to large (60
ml), sterile glass tubes that served as the experimental
microcosms for the duration of the experiment. All tubes
were refrigerated at 4C for 8 hours prior to addition of
eDNA.
Preparation of Extracellular DNA
Total cellular and plasmid pBR322 DNAs of E. coli were
amplified and isolated by the alkaline lysis procedures of
Chaudhry and Huang (1988). The isolated DNA was resuspended


TABLE 4-5. Rates of population change (log10/day) of
genetically engineered and wild-type strains at 22C
under sterile conditions in ground water.
Media Type
Escherichia
coli
Pseudomonas
Dutida
wild-type
GEM
wild-type
GEM
LB
-0.09
-0.08
NA
NA
LB +
HgCl2
NA
-0.08
NA
NA
LB
NA
NA
-0.10
-0.04
LB +
Tet + Km
NA
NA
NA
-0.03
NA indicates not applicable


CHAPTER 8
CONSTRUCTION OF A MODEL GEM FOR USE
IN GENE TRANSFER STUDIES
Introduction
The increasing feasibility for the use and release of
GEMs in the environment is a cause of concern, due in part,
from a lack of knowledge of intra- and interspecies
interactions in the natural environment. The ability of
bacteria to transfer or mobilize genetic information via
conjugation has been demonstrated both in vitro and in situ
(Altherr and Kasweck, 1982; Bale et al., 1988; Bell et al.,
1983; Mach and Grimes, 1982; Trevors, 1987; Van Elsas et
al., 1987; Weinberg and Stotzky, 1972). While much of these
studies were conducted from a public health point of view
(Bell et al., 1983; Goyal et al., 1979; Grabow et al.,
1975), recent studies utilizing GEMs have provided more
specific information on the potential for genetic transfer
by GEMs in the natural environment (Bleakley and Crawford,
1989; Knudsen et al., 1988; Mancini et al., 1987; McClure et
al., 1989; Richaume et al., 1989; Van Elsas et al., 1987;
Walter et al., 1989).
The purpose of this study was to construct a model GEM
for genetic transfer studies. The GEM was constructed by
inserting the mob gene that encodes for the mobilization of
190


To my parents,
Mary and Phillip Awong


20
gels and 13) in-vitro amplification of target DNA by
polymerase chain reaction (Jain et al., 1988).
The most commonly used non-conventional method for the
study of GEMs and rDNA is hybridization (Jain et al., 1988).
DNA probe methodology allows detection of specific genes
and/or the organisms containing these genes in the
environment. The methodology eliminates the requirement for
successful culture of recovered organisms. DNA hybridization
is a useful and important tool for monitoring organisms
that become non-culturable and for detecting gene transfer
to indigenous populations.
Previous studies have focused on the use of DNA
hybridization methodologies for detecting specific
indigenous microorganisms or functional groups of organisms
(Holben and Tiedje, 1988; Pettigrew and Saylor, 1986; Yates
et al., 1985). DNA probes are currently being utilized for
studying the fate of GEMs and their rDNA in the environment.
Bentjen et al. (1989) utilized both colony and dot-blot
hybridization to monitor Tn5 mutants in rhizosphere soil,
plant endorizosphere, insects and xylem exudates. The
application of DNA probes and colony hybridization has also
been reported for 1) the detection of catabolic genotypes
in sediment samples (Sayler et al., 1985), in soils
(Chaudhry et al., 1988) and freshwater systems (Amy and
Hiatt, 1989; Morgan et al., 1989; Steffan et al., 1989); 2)
study of maintenance and stability of introduced genotypes
in groundwater aquifer material (Jain et al.,
1987); 3)


34
1984), in soil (Krasovsky and Stotzky, 1987; Trevors, 1987;
Trevors and Oddie, 1986; Van Elsas et al., 1987) and in
various aquatic habitats (Bale et al., 1988; Gowland and
Slater, 1984; Kobori et al., 1984; Saye et al., 1987).
Recently, studies utilizing GEMs have provided
additional and useful information on genetic transfer by
conjugation. The majority of these studies focused on soil
and utilized simple microcosm settings to determine gene
transfer. Van Elsas et al. (1987) studied the transfer of
plasmids between bacilli in air-dried soil under sterile and
non-sterile conditions. Transfer frequencies in filter
matings (1 x 10~6) were much higher than those observed
for incubations in sterile loamy sand (0.7 x 107) .
Essentially no transconjugants were obtained in non-sterile
soil suggesting a rapid decline of the recipient population.
However, in the presence of bentonite clay, plasmids were
transferred at higher frequencies, and the survival of the
recipient population was enhanced. The presence of nutrients
in the soil was also shown to stimulate plasmid transfer.
Bleakley and Crawford (1989) investigated the effects
of varying moisture and nutrient levels on the transfer of
the recombinant, conjugational plasmid pIJ303 between
Streptomyces species in sterile silt loam. Their results
suggested that nutrient-amended, relatively dry soils
possess frequent microsites where mycelial growth and
conjugationally mediated plasmid exchange can readily occur.


191
plasmid DNA, into the shuttle vector pRC92. The plasmid
pRC92 can be transferred between gram-positive and gram
negative bacteria, and contains an EcoRl DNA fragment of
Napier grass. The Napier grass DNA insert serves as a useful
marker or tracer for studies utilizing natural environmental
samples.
Materials and Methods
Bacterial Strains. Plasmids and Growth Conditions
The bacterial strains and plasmids used in this study
are listed in Table 8-1. Strains of E. coli and P. putida
were grown in Luria broth (LB) at 35C and 28C,
respectively. Antibiotics (Sigma Chemical Co.) were used at
the following concentrations (ug/ml): ampicillin, 100;
tetracycline, 15; chloramphenicol, 40; erythromycin, 75; and
nalidixic acid, 40; kanamycin, 40.
Isolation of Plasmid DNA
The plasmids pRC92 and pDugll were isolated by the
alkaline lysis procedure of Birnboim and Doly (1979) and
purified by banding in caesium chloride-ethidium bromide
gradients (Maniatis et al., 1982).
Restriction Analysis of Plasmid DNA
Restriction endonucleases were purchased from U.S.
Biochemical Corp., Cleveland, Ohio. Enzyme digestions were
carried out as specified by the manufacturer. Digested


92
Log CFU/ml
Time (hours)
Figure 4-11. Survival of genetically engineered (GEM)
and wild-type strain of Pseudomonas outida at 30C
in activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; ( ) and (() /
respectively. GEM strains recovered on
selective medium .


38
Zeph et al., (1988) demonstrated the transduction of
bacterial resistance genes for chloramphenicol and mercury
into Escherichia coli by the transducing coliphage PI in
nonsterile soil. In a following experiment, Zeph and Stotzky
(1989) utilized a biotinylated DNA probe to detect phage PI
transductants in nonsterile soil. Although the probe did
detect transductants of E_j_ coli that was added to the soil,
no phage PI transductants of indigenous bacteria were
detected.
The lack of available information clearly illustrates
the need for additional studies of this potentially,
extensively used mechanism of gene transfer in natural
environments.
Transformation
While current studies have increasingly been focused on
the potential for gene transfer via conjugation and
transduction, very little attention has been given to
transformation. Since plasmid and chromosomal DNA is
certain to be released from lysed bacterial cells, it is
important to inquire about the fate and effects resulting
from the release of the extracellular DNA in soil and
aquatic systems and in specific niches such as legume
nodules and the rhizosphere.
Few studies have been reported on natural
transformation in the environment. Graham and Istock (1978;
1979) demonstrated transformation in JL_ subtilis in sterile


26
Several other studies have also reported on the
survival of GEMs in the rhizosphere and plant associated
systems. Armstrong et al.(1987) described a microcosm method
for assessing survival of recombinant bacteria associated
with plants and herbivorous insects. Leaf, whole insect,
foregut and frass samples were periodically assayed to
enumerate recombinant strains. The recombinant strain was
found to slowly decrease over time on leaf surfaces, but an
increase in population size was noted in foregut samples and
none was detected in frass samples.
Yeung et al. (1989) studied the growth of genetically
engineered Pseudomonas aeruginosa and Pseudomonas putida in
the soil and rhizosphere. Despite a high level of enzyme
production by the engineered strains, the presence of the
cloned genes had no effect on the growth of these strains in
the soil or rhizosphere. No significant difference was noted
between wild-type and engineered strains.
The survival of ice nucleation-active (INA) and
genetically engineered non-INA strains of Pseudomonas
svrinaae on oat seedlings was compared after subjection to
various freezing temperatures (Buttner and Amy, 1989) The
data indicated a potential competitive advantage of INA
strains over the engineered non-INA strains in mild freezing
environments.
The first account of a genetically engineered strain
having a measurable effect on a natural ecosystem was
reported by Wang et al. (1989). The survival and effects of


164
recovered DNA was resuspended in 20 ul TE buffer (pH 8.0)
and stored at 4C until analyzed. The efficiency of the
recovery procedure was checked by subjecting known
concentrations of DNA inoculum to the recovery procedure
and comparing the recovered DNA with the actual
concentrations by gel electrophoresis and optical density
measurements.
Sample Background DNA
All samples were initially screened to determine
whether background levels of extracellular DNA were
sufficiently high to cause interference with the
experimental procedure. Samples were subjected to the
recovery method described above and analyzed by gel
electrophoresis to determine the presence or absence of
background extracellular DNA. No background DNA was detected
in any of the samples tested.
DNA Degradation Study
Triplicate tubes containing the treated samples
(autoclaved, filter-sterilized and non-treated) were
inoculated with eDNA (10.4 ug/ml) and incubated at room
temperature (25C) for the duration of the experiment.
Controls for each sample consisted of triplicate tubes
containing 5 ml of untreated sample without the spiked eDNA.
At specific time intervals (0.25, 1, 5, 8, 12, 24, and 48
hours), 0.5 ml aliquots were removed from each tube and
immediately subjected to the recovery procedure described


208
Burton, G. A., D. Gunnison, and G. R. Lanza. 1987. Survival
of pathogenic bacteria in various freshwater
sediments. Appl. Environ. Microbiol. 53:633-638.
Buttner, M.P., and P.S. Amy. 1989. Survival of ice
nucleation-active and genetically engineered non
ice-nucleating Pseudomonas svrinaae strains after
freezing. Appl. Environ, microbiol. 55:1690-1694.
Caldwell, B.A., C. Ye, R.P. Griffiths, C.L. Moyer, and R.Y.
Morita. 1989. Plasmid expression and maintenance during
long-term starvation-survival of bacteria in well
water. Appl. Environ. Microbiol. 55:1860-1864.
Carlson, C.A., L.S. Pierson, J.J. Rosen, and J.L.
Ingraham. 1983. Pseudomonas stutzeri and related
species undergo natural transformation. J. Bacteriol.
153:93-99.
Cenci, J.C., P.B. Taylor, and F.R. Leach. 1981. Use of the
Microtox assay system for environmental samples. Bull.
Environm. Contam. Toxicol. 26:150-156.
Chai, T. 1983. Characteristics of Escherichia coli grown in
bay water as compared with rich medium. Appl. Environ.
Microbiol. 45:1316-1323.
Chassy, B.M., and J.L. Flickinger. 1987. Transformation of
Lactobacillus casei by electroporation. FEMS Microbiol.
Letter 44:173-177.
Chaudhry, G.R., and G. H. Huang. 1988. Isolation and
characterization of a new plasmid from a Flavobacterium
sp. which carries the genes for degradation of 2,4-
dichlorophenoxyacetate. J. Bacteriol. 170:3897-3902.
Chaudhry, G.R., G.A. Toranzos, and A.R. Bhatti. 1989. Novel
method for monitoring genetically engineered
microorganisms in the environment. Appl. Environ.
Microbiol. 55:1301-1304.
Cocking, E.C., and M.R. Davey. 1987. Gene transfer in
cereals. Science 236:1259-1262.
Conway, T., M.O.K. Byun, and L.O. Ingram. 1987. Expression
vector for Zvmomonas mobilis. Appl. Environ.
Microbiol. 53:235-241.
Curds, C.R., G.J. Fey. 1969. Effect of ciliated protozoa on
the fate of Escherichia coli in the activated sludge
process. Water Res. 3:853-867.


Ill
microorganisms has been considered for the biocontrol of
soil-borne microbial pathogens, improving plant nutrition,
providing plant growth factors and for enhancing yields of
agricultural crops (Davidson, 1988). The most serious
candidates for these tasks are nitrogen-fixing bacteria,
phosphate solubilizers, plant growth-promoting micro
organisms, mycorrhizal fungi, and microbial insecticides
(Keeler, 1988; Okon and Hadar, 1987).
The potential use of genetically engineered
microorganisms (GEMs) in the area of agriculture is
extremely promising. There are, however, potential problems
and concerns associated with the deliberate release of GEMs
in the environment (Gillett, et al., 1985; Halvorson et al.,
1985). Such concerns have led to recent investigations into
the fate and survival of GEMs in the natural environment.
Several studies have dealt with survival and gene transfer
by GEMs in soils, soil slurries or soil extracts (Bentjen et
al., 1989; Devanas and Stotzky, 1986; Fredrickson et al.,
1988; 1989; Van Elsas et al., 1989; Walter et al., 1987;
1989; Wang et al., 1989). However, only a few reports have
been published on the fate of GEMs in plant associated
systems or the rhizosphere (Armstrong et al., 1987; Knudsen
et al., 1988; Yeung et al., 1989).
The objective of the present research was to study the
influence of growing roots on the survival of two
genetically engineered strains in a simulated rhizosphere
microcosm. The study was conducted under hydroponic


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.
Samuel R. Farrah
Associate Professor of Microbioloqy
and Cell Science
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.
August, 1990
¡jjbJh Cl
Winfred M. Phillips, Dean
College of Engineering
Madelyn M. Lockhart
Dean, Graduate School


120
TIME (hours)
Figure 5-4. Effect of corn-root exudates on the growth
of genetically engineered Pseudomonas putida. Initial
inoculum concentration was approximately 2.0 x 10.
( ), no root exudates; ( ), presence of root exudates.


146
strain when grown on selective (-0.30) and non-selective
(-0.31) media.
Bacterial enumeration by acridine orange direct counts
indicated higher numbers of bacterial cells (Figure 6-3)
than viable plate counts. By day 6, over 40% of the cells
were still present (Figure 6-4), as compared to less than
10% recovered by viable counts (Figure 6-2). The rate of
population change for the engineered strain (-0.09) was
significantly higher (p<0.05) than that of the wild-type
strain (-0.04) when compared by direct counts (Table 6-1).
B-qalactosidase biosynthesis
The two strains of E. coli showed a rapid reduction in
B-galactosidase biosynthesis within 24 hours of exposure to
lake water (Figure 6-5). Enzyme biosynthesis was reduced by
almost 60% in wild-type HB101 after 24 hours, but remained
fairly stable thereafter. After 9 days of exposure, specific
activity was reduced by almost 80% relative to the initial
enzyme activity. The engineered strain exhibited a
significantly different trend over time with respect to B-
galactosidase biosynthesis. Specific activity was rapidly
reduced within the first 24 hours of exposure (Figure 6-5)
and continued to steadily decline over time. After 9 days,
only 1% of the initial specific activity was detected for
the engineered strain as compared to over 20% for the wild-
type strain. The rates of change in B-galactosidase


72
containing 53% endothall as the active ingredient. The test
water was made by adding appropriate amounts of stock
solution to acclimated lake water to produce a hydrothol
concentration of 1 mg/L based on percent active ingredient.
This water was then added to the tanks containing the
survival chambers. The test water for the microcosm was
prepared and renewed daily throughout the experiment which
was conducted at 27C under sterile conditions.
Plasmid Stability
To study the stability of the engineered genes under
the given test conditions, the genetically engineered
strains were simultaneously enumerated on both selective and
non-selective (LB) media. Plasmid stability studies were
conducted at 15, 25 and 30C for lake water and activated
sludge effluent, and at 22C for ground water. Experiments
were performed under sterile conditions.
Statistical Analysis
The data are expressed as mean values standard
deviation from the mean. Basic computations were performed
using STAT-2 (Stat-Soft Inc., Tulsa, Oklahoma) statistical
program. Exponential decline rate models were fit to the
population data by performing linear regression on
logarithmic CPU values against time. Rates of population
change were compared by a modified t-Test (Zar, 1984) to
determine significant differences in survival rates between
bacterial strains.


108
host type, and environmental factors such as temperature and
nutrient conditions.
It has been shown in this study that survival chambers
can be used in association with microcosm models to study
the fate and survival of genetically engineered organisms
in aquatic environments. The advantages of the model
described here are it's simplicity and ease in adjusting for
environmental variables and the fact that the organisms can
be continuously exposed to the test water. The membrane
filters allow rapid exchange between the content of the
chambers and its surroundings. Additionally, the membranes
would not exclude complex organic molecules that may serve
as food for the microorganisms.
Currently used microcosm models are usually confined to
closed systems such as flasks, mason jars and test-tubes. A
serious disadvantage of such a closed system is the
potential for "bottle effects" that can arise over time.
Accumulation of cell debris and excretory by-products can
result in changes that are not truly representative of the
natural test system. This problem is eliminated in my system
by the constant exchange of materials between the chambers
and the surrounding water which is also constantly being
renewed. Such a design allows the organisms to be studied
under conditions similar to those found in nature. Using a
similarly designed microcosm model, McFeters et al. (1972)
showed that the survival rates of various pathogens in
groundwater were similar to those found under in-situ


167
to the OC and linear forms. Similar results were obtained
with filter-sterilized raw sewage (Figure 7-1, Lane Q). The
degradation rate, however, was slower in the filter-
sterilized sample since some CCC forms were still present
as compared to the untreated sample which showed no
indication of CCC forms after fifteen minutes. The
concentration of OC and linear forms was also noticeably
greater in the filter-sterilized sample than in the
untreated sample, indicating that degradation of these forms
were already occurring in the untreated sample after fifteen
minutes.
After one hour of incubation (Figure 7-2, Lanes 0-R),
eDNA was almost completely degraded in untreated raw sewage
(Figure 7-2, Lane 0). Some linear form of the plasmid was
still present as well as trace amounts of chromosomal DNA.
The heavy smear indicates a high concentration of
degradation by-products. The filter-sterilized sample
(Figure 7-2, Lane Q) still showed a large amount of plasmid
DNA in the OC and linear forms. The autoclaved sample
(Figure 7-2, Lane P) still showed no signs of DNA
degradation at this time.
The eDNA was completely degraded within five hours in
both the untreated and filter-sterilized raw sewage (gel not
shown). Degradation by-products (< 2.3 kb) were still
visible in the filter-sterilized sample at this time, but
totally disappeared after 12 hours. The autoclaved raw
sewage showed no indication of degradation until 12 hours of


127
Figure 5-10. Survival of genetically engineered Pseudomonas
outida in the presence of rhizospheric microorganisms (RM),
with corn roots present.
( ) putida in the absence of RM;
( ) P_5_ putida in the presence of RM.


FATE AND SURVIVAL OF GENETICALLY ENGINEERED
MICROORGANISMS AND THEIR RECOMBINANT GENES
IN THE NATURAL ENVIRONMENT
BY
JUDY AWONG
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
1990
ima>SITY OF nom Ulus
m


109
conditions. Flow-through rates can also be regulated
depending on the aquatic system being tested. This model can
also be easily adapted to study other aspects of GEM-
associated environmental release such as genetic transfer
and persistence.


23
The use of appropriate hosts and vectors has been
considered for biological containment of recombinant
organisms. The choice of hosts can range from isolated
wild-type strains to laboratory strains that are dependent
on uniguely defined laboratory conditions. Plasmid-cloning
vectors can range from conjugative and mobilizable plasmids
to non-conjugative or suicide plasmids (Molin et al., 1987).
The presence of foreign DNA may alter the survival of
the cloning vectors or the hosts that contain them. The
foreign DNA may or may not confer selective advantages to
the host cell depending on the energy requirements and
precursors for replication of additional DNA (Curtiss et
al., 1977). In addition, the synthesis of new gene products
that are specified by the foreign DNA could pose an
additional burden.
The final ecological niches occupied by recombinant
organisms will have the most direct effect on their growth
and survival. Released GEMs are likely to be dispersed and
can enter freshwater and marine environments via
agricultural run-off and drainage systems. The vast array of
environmental variables associated with soil and aquatic
systems can directly affect the growth and survival of the
GEMs. Environmental factors most affecting growth and
survival include temperature, adsorption, dessication,
nutrient availability, pH, soil/sediment type, presence of
toxicants, viruses, seasonality, predation and competition


183
Comparisons between the two methods indicate that
large, intact sequences of DNA (such as chromosomal and
plasmid DNAs) are quickly broken down or denatured to ssDNA.
Since the presence of the smaller degradative by-products
are detectable with ultraviolet absorbance but not with the
gel electrophoresis method used here, the sizes of these
decay products could range from 200 base pairs or less. No
attempt was made to characterize the decay products.
The high rate of degradation of eDNA in raw sewage can
be attributed to a variety of factors including a large
number of bacteria, high concentrations of nucleases, and
chemical degradation. This was confirmed by the different
degradation rates of the filter-sterilized and autoclaved
treatments. The filter-sterilized treatment represents
abiotic raw sewage, and indicates the extent of degradation
by extracellular nucleases. The results indicated that a
significant proportion of DNA degradation did occur via
nuclease attack. The results also suggested that biotic or
cell-associated degradation was also important since
degradation in the filter-sterilized treatment occurred at a
slower rate than in the untreated sample. The difference in
degradation rates between untreated and filter-sterilized
treatments can be attributed to the biological elements of
degradation. Since autoclaving destroys both living cells
and extracellular nucleases, this treatment represents the
non-biological component of DNA degradation. It is apparent


66
the indigenous microbial populations. The possibility also
exists that recombinant organisms may behave differently
from their non-recombinant parental strains when released
into the environment. Such information is therefore needed
to properly evaluate and assess the potential impact of GEMs
on representative ecosystems.
Although numerous studies have been conducted on
bacterial survival in the natural environment (Bissonette et
al., 1975; Burton et al., 1987; Lessard and Sieburth, 1983;
McFeters and Stuart, 1972), much of the existing information
pertains to naturally occurring pathogens and other
microorganisms of public health significance. More recent
studies have included other species (Liang et al., 1982;
Ohana et al., 1987; Scheuerman et al., 1988), but still to
date, few studies have been conducted to determine the fate
of recombinant bacteria and their "novel" genes in natural
ecosystems. Investigations have dealt mainly with soil
systems (Bentzen et
al. ,
1989; Devanas
et
al.,
Fredrickson et al.,
1988;
1989; Van Elsas
et
al.,
Walter et al., 1987) and plant associated systems such as
leaf surfaces, the rhizosphere and plant-feeding insects
(Armstrong et al., 1987; Knudsen et al., 1988; Yeung et al.,
1989). Recent reports have provided additional information
on aquatic systems, including wastewater effluents (Amy and
Hiatt; 1989; McClure et al., 1989; Morgan et al., 1989;
Scanferlato et al., 1989; Trevors et al., 1989). However,
many questions still remain to be answered about the fate of


150
% INHIBITION OF B-GAL ACTIVITY*
TIME (DAYS)
Inhibition at 45 ppm POP
Figure 6-7. Changes in cell permeability of
wild-type (HB101) and genetically engineered (50008)
strains of Escherichia coli after exposure to lake
water at 25C. Results are expressed in terms of
percent inhibition of B-galactosidase activity after
exposure to the toxicant, Pentachlorophenol (PCP),
at a concentration of 45 ppm.


202
be continuously exposed to the test water. The survival
chambers allow rapid exchange between the content of the
chambers and its surroundings. Additionally, the membranes
would not exclude complex organic molecules that may serve
as food for the microorganisms.
2. Temperature-dependent studies of genetically engineered
and wild-type strains of Escherichia coli and Pseudomonas
putida in lake water indicated that the GEMs survived better
or at least as well as their wild-type counterparts at 15,
25 and 30C. This suggests that the presence and/or addition
of plasmids or 'foreign' DNA to a host bacteria is not
necessarily a burden for the organism, and may actually
improve the fitness of the cell.
3. The genetically engineered strain of P_¡_ cutida survived
better than it's parent strain at 22C in groundwater. No
significant difference was noted between the two strain of
E. coli. These results are similar to those observed in lake
water.
4. Temperature-dependent studies in activated sludge
effluent indicated no significant differences between the
GEMs and wild-type strains at 15 and 25C. However, at 30C,
the engineered strain of E. coli exhibited a significantly
higher rate of decline than its parent strain. This was the
only condition under which the GEM did not survive as well
as it wild-type strain. It would appear that the
combination of the higher temperature and unfavorable


172
Figure 7-3. Degradation of spiked eDNA in four aquatic
samples after 24 hours incubation at 25C.
Lane designations same as in Figure 1.


97
Time (hours)
Figure 4-13. Survival of genetically engineered (GEM)
and wild-type strains of Pseudomonas putida at 22C
in groundwater. Wild-type and GEM strains recovered
on non-selective medium; (--) and (H ; respectively.
GEM strains recovered on selective medium (-^) .


80
DAYS
Figure 4-4. Survival of genetically engineered strains
of Escherichia coli and Pseudomonas outida in
filter-sterilized (0.2 urn) and non-sterilized lake water
at 25C. All chambers initially contained the GEMs
suspended in filter-sterilized (0.2 urn) lake water.
Arrows indicate the time at which non-sterile lake water
was introduced into the chambers in triplicate.


41
microcosms range from simple glass bottles and mason jars
(Scanferlato et al., 1989; Trevors et al., 1989) to more
complex and detailed structures such as a laboratory scale
wastewater treatment pilot plant (Mancini et al., 1987). Two
recently described microcosm models have been proposed for
use in the study of GEMs in aquatic systems (Fulthorpe and
Wyndham, 1989; Rochelle et al., 1989). Both microcosms
functioned as flow-through systems which simulated a more
natural setting and therefore provided a more rigorous
testing of the introduced organisms.
In order to study the fate and survival of GEMs in the
aquatic environment, an aquatic microcosm was developed that
utilizes modified survival chambers in a simple laboratory
setting designed to allow the testing of a variety of
environmental variables. The microporous membrane of the
chamber allows continuous exchange of water, solutes and
nutrients between the chambers and the surrounding water. A
significant advantage of this system is the continuous
interaction of the test bacteria with their surrounding
environment. These conditions provide a more thorough test
of the fitness of the organism and/or its recombinant genes.
This section describes the design of the aquatic
microcosms that was utilized for studying the fate and
survival of GEMs in various aquatic environments. The
section also summarizes some preliminary experiments that
led to the development of the final test procedures that
were utilized in the overall study.


Copyright 1990
by
Judy Awong


199
between GEMs and other bacteria, via conjugation, in aquatic
systems.
Reported studies suggest that environmental variables
play an important part in the production of transconjugants.
In soil, variables such as nutrient content, moisture
levels, pH, temperature, clay content, and organic matter
strongly influence transfer frequency rates (Bleakley and
Crawford, 1989; Richaume et al., 1989; Walter et al., 1989;
Van Elsas et al.,1987). Transfer of recombinant plasmids in
wastewater is also attributed to the relatively high
nutrient content of sewage (Mancini et al., 1987).
Several studies have investigated conjugative plasmid
transfer by recombinant bacteria in plants (Armstrong et
al., 1987; Knudsen et al., 1988) and animals (Armstrong et
al., 1987). Knudsen et al. (1988) reported the presence of
transconjugants in the rhizosphere and phyllosphere,
however, plasmid transfer was not observed in the other
studies.
It is apparent that more information is needed on the
potential for genetic transfer by GEMs in the natural
environment. This study describes preliminary experiments
for studying genetic transfer via conjugation. Microcosms
can also be used to provide information concerning genetic
transfer via transduction and transformation. The microcosm
can be adapted to test a variety of environmental
conditions. Potential areas for future research include:


222
Tiedje, J.M., R.K. Colwell, Y.L. Grossman, R.E. Hodson, R.E.
Lenski, R.N. Mack, and P.J. Regal. 1989. The planned
introduction of genetically engineered organisms:
ecological considerations and recommendations. Ecology
70:298-315.
Toranzo, A.E., P. Combarro, L.M. Lemos, and J.L. Barja.
1984. Plasmid coding for transferable drug resistance
in bacterial isolated from cultured rainbow trout.
Appl. Environ. Microbiol. 48:872-877.
Torrella, F., and R.Y. Morita. 1981. Microcultural study of
bacterial size changes and microcolony and ultra
microcolony formation by heterotrophic bacteria in
seawater. Appl. Environ. Microbiol. 41:518-527.
Trevors, J.T., T. Barkay, and A.W. Bourquin. 1987. Gene
transfer among bacteria in soil and aquatic
environments: a review. Can. J. Microbiol. 33:191-198.
Trevors, J.T., and K.M. Oddie. 1986. R-plasmid transfer in
soil and water. Can. J. Microbiol. 32:610-613.
Trevors, J.T., and J.D. Van Elsas. 1989. A review of
selected methods in environmental microbial genetics.
Can. J. Microbiol. 35:895-902.
Trevors, J.T., J.D. Van Elsas, M.E. Starodub, and L.S. Van
Overbeek. 1989. Survival of and plasmid stability in
Pseudomonas and Klebsiella spp. introduced into
agricultural drainage water. Can. J. Microbiol.
35:675-680.
Van Elsas, J.D., J.M. Govaert, and J.A. Van Veen. 1987.
Transfer of plasmid pFT30 between bacilli in soil as
influenced by bacterial population dynamics and soil
conditions. Soil Biol. Biochem. 19:639-647.
Van Elsas, J.D., J.T. Trevors, L.S. Van Overbeek, and M.E.
Starodub. 1989. Survival of Pseudomonas fluorescens
containing plasmids RP4 or pRK2501 and plasmid
stability after introduction into two soils of
different texture. Can. J. Microbiol. 35:951-959.
Walsh, S.M., and G.K. Bissonnette. 1989. Survival of
chlorine-injured enterotoxigenic Escherichia coli in an
in vitro water system. Appl. Environ. Microbiol.
55:1298-1300.
Walter, M.V., K. Barbour, M. McDowell, and R.J. Seidler.
1987. A method to evaluate survival of genetically
engineered bacteria in soil extracts. Curr. Microbiol.
15:193-197.


115
with 40 ug/ml HgCl2, was found to inhibit growth of
rhizospheric microflora and yet allow the selective growth
of the mercury resistant, recombinant E. coli. However, one
rhizospheric bacterial strain grew slowly on this selective
media, forming small colonies that were distinctly different
from £. coli. Furthermore, transfer of these colonies to
eosin methylene blue (EMB) agar did not show the
characteristic metallic sheen of E|. coli colonies.
The survival and growth of P. putida was monitored with
appropriate selective growth medium, as described above.
Statistical Analysis
Data were subjected to analysis of variance, and means
were compared by use of the Newman Keuls test. Computations
were performed with the STAT-ITCF program provided by the
Institut Technique des Cereales et Fourrages, France.
Results
Effect of Root Exudates on the Growth of GEMs
The genetically engineered strains of £. coli and P.
putida both exhibited similar growth patterns in the
presence and absence of root exudates (Figure 5-1 and 5-2).
In the absence of the plant, both strains showed a slight
increase in growth during the first 24 hours following
inoculation. The number of cells remained constant
thereafter until the end of the experiment. In the presence
of the corn plants, however, a substantial increase in cell


130
organic acids, sugars, and amino acids, as well as complex
substances such as vitamins and plant hormones (Foster et
al., 1983; Gaskins and Hubbell, 1979; Russell, 1980).
Specific carbohydrates include glucose, fructose, sucrose,
xylose, maltose, rhamnose, arabinose, raffinse and
oligosaccharide (Russell, 1980).
It appeared that the GEMs, as well as their wildtype
strains, were able to utilize root products and thus grow in
the root vicinity. Similar results were also noted by
Bentjen et al. (1989) and Fredrickson et al. (1989). Both
studies utilized soil-core microcosms, planted with wheat
and/or maize seedlings, to follow the fate of GEMs in the
rhizosphere and rhizoplane. Bentjen et al. (1989) noted
colonization of the rhizosphere by the GEMs 33 days after
their addition to the microcosm. The potential for niche
displacement of rhizospheric populations of nitrifying
bacteria and P. leauminosarum by the GEMs was also shown to
be negligible. No significant differences were noted between
control and inoculated treatments in Rhizobium populations
or nitrifying bacteria in the rhizosphere. Fredrickson et
al. (1989) also noted successful colonization of the wheat
rhizosphere and rhizoplane by recombinant strains of
Pseudomonas spp.
The presence of additional DNA and/or plasmid in the
host cell has little affect on the survival of the organism
in the rhizosphere. Results of this study indicate no
significant differences in growth patterns between the GEM


CHAPTER 7
FATE OF EXTRACELLULAR DNA IN AQUATIC ENVIRONMENTS:
IMPLICATIONS AND POTENTIAL FOR GENETIC TRANSFORMATION
Introduction
The biotechnology revolution and the potential benefits
derived from it, have over the years created an explosion of
new recombinant DNA (rDNA) products (Gillet et al., 1985;
Halvorson et al., 1985; Johnston and Robinson, 1984). The
scope of such technology has now broadened to environmental
applications (Halvorson et al., 1985), but at the same time
also raises the question of the safety of deliberate release
of genetically engineered microorganisms (GEMs) to the
environment. Concern has been raised over the spread of
these GEMs and the possible genetic transfer of their novel
DNA.
Numerous studies have been conducted to study bacterial
survival and genetic transfer among indigenous or naturally
occurring bacteria (Altherr et al., 1982; Krasovsky and
Stotzky, 1987; McFeters and Stuart, 1972; Saye et al., 1987;
Trevors et al., 1987). More recently, studies using GEMs
have provided new and useful information concerning their
survival and the fate of their recombinant genes in various
environments, including soil and aquatic systems (Armstrong
et al., 1987; Bentzen et al., 1989; Devanas and Stotzky,
159


160
1986; Dwyer et al., 1988; Knudsen et al., 1988; Mancini et
al., 1987; Walter et al., 1987). These investigations
indicate that both chromosomal and plasmid DNA can be
transferred among bacteria via conjugation or transduction
(Bleakley and Crawford, 1989; Richaume et al., 1989; Walter
et al., 1989; Zeph et al., 1988).
While current studies have focused on the survival of
recombinant bacteria and the transfer of the intracellular
DNA in the environment, there is very little information on
the fate of extracellular DNA (eDNA) under various
environmental conditions. Although studies have reported the
presence of eDNA in the environment (DeFlaun et al., 1986;
1987; Minear, 1972; Pillai and Ganguly, 1972), few have
investigated its dynamics and persistence (Paul et al.,
1987; 1988).
The presence of eDNA in the aquatic environment is
important for several reasons. DNA can serve as a source of
nutrients for microbial growth and metabolism. Dissolved DNA
can also be a source of nucleic precursors that may
otherwise be too energetically expensive for the organism to
synthesize de novo (Nygaard, 1983). However, the persistence
of extracellular recombinant DNA (erDNA) in the environment
also raises the question of whether rDNA could contribute to
acquisition of new genes by genetic transformation. The
process of transformation involves the uptake and
incorporation of free DNA by microorganisms. Transformation
in the natural environment has been demonstrated in soils


93
rates of population change were found to be not
significantly different. A comparison of the survival
patterns indicate that the wildtype strain does not survive
as well as its engineered counterpart during the first 4
days of exposure to the wastewater effluent.
Plasmid stability. Genetic stability was determined by
plating the recombinant strain on both selective and
nonselective media. Loss of plasmid or genetic stability is
indicated by a significant difference between bacterial
counts on the two types of media. With one exception, the
engineered genes of both recombinant strains of E. coli and
P. putida were relatively stable in activated sludge
effluent at 15, 25 and 30C. The stability of the
recombinant DNA and/or plasmid in E. coli. 50008 is
apparently affected at high temperatures. Significantly
lower numbers (p<0.025) were obtained using selective media
(LB plus 40 ug ml-1 mercuric chloride) than nonselective
media (LB) following incubation in activated sludge effluent
at 30C (Figure 4-10). This difference was evident within
48 hours of exposure to the effluent, as indicated by a 1
log difference in numbers between the two media types. After
14 days, a greater than two log difference was noted between
growth media. No significant differences were noted when the
strain was tested at 15 or 25C.
No significant differences were noted between the rates
of population change for P'. putida. 50058 grown on either


128
whereas the numbers of rhizospheric microorganisms were only
slightly affected.
The survival/growth patterns of P. putida in the corn
rhizosphere in the presence or absence of rhizospheric
microorganisms are shown in Figures 5-9 and 5-10. In the
absence of corn roots (Figure 5-9), £. putida was relatively
stable, and its numbers declined slightly in the presence of
rhizospheric microorganisms. However, in the presence of
corn plants and root exudates (Figure 5-10), cell densities
actually increased over time. In the absence of rhizospheric
microorganisms, this increase was similar to the growth
pattern observed in Figure 5-2. In the presence of
rhizospheric microorganisms, cell densities of P. putida
increased during the first 48 hours, then showed a slight
decline thereafter. P. putida appeared to survive better
than S* coli in the presence of indigenous rhizospheric
microorganisms under simulated rhizospheric conditions.
Discussion
The release of GEMs and traditional microbial
inoculants in soils to improve plant productivity is
becoming increasingly attractive. However, the ability of a
modified microorganism to produce specific improvements in
the soil-plant system depends primarily on its capacity to
survive in the specific environment. Several studies dealing
with the fate of GEMs in the environment have shown that
GEMs can survive in surface waters and wastewater effluents


Materials and Methods 112
Hydroponic Cultures 112
Bacterial Strains 113
Survival and Growth Studies 114
Interaction between the Genetically Engineered
Bacteria and Rhizosphere Microorganisms 114
Statistical Analysis 115
Results 115
Effect of Root Exudates on the Growth of GEMs.. 115
Comparison of the Growth Patterns of Wildtype
and GEM strains in the Presence of Corn-root
exudates 118
Survival of GEMs in a Simulated Rhizosphere in
the Presence of Indigenous Rhizosphere
Microorganisms 118
Discussion 128
6 STRUCTURAL AND PHYSIOLOGICAL ALTERATIONS OF
GENETICALLY ENGINEERED AND WILDTYPE STRAINS
OF L. COLI AFTER EXPOSURE TO AN AQUATIC
ENVIRONMENT 133
Introduction 133
Materials and Methods 135
Bacterial Strains and Culture Media 135
Aguatic Microcosm 136
Bacterial Enumeration 137
B-Galactosidase Biosynthesis 137
INT-Dehydrogenase Activity 139
Cell Permeability 140
Results 141
Bacterial enumeration 141
B-galactosidase biosynthesis 14 6
INT-dehydrogenase activity 148
Cell permeability 148
Morphological changes 151
Discussion 152
7 FATE OF EXTRACELLULAR DNA IN AQUATIC SYSTEMS:
IMPLICATIONS AND POTENTIAL FOR GENETIC
TRANSFORMATION 159
Introduction 159
Materials and Methods 161
Aquatic Samples 161
Sample Preparation 162
Preparation of Extracellular DNA 162
Determination of DNA Inoculum Concentration.... 163
DNA Recovery Procedure 163
Sample Background DNA 164
DNA Degradation Study 164
viii


179
TIME (HOURS)
Figure 7-8.
in Tap Water.
Sample;
Degradation kinetics of spiked eDNA
( ) Untreated Sample; ( O ) Autoclaved
( a ) Filter-sterilized Sample.


during the course of this study. Thanks are also due to many
of the staff members of the Department, in particular, Jo
David, Shirley Jordan, Eleanor Humphries, and Eleanor
Merritt, for their understanding and friendly assistance
throughout the years.
Special thanks are given to Stephen A. Taylor for his
love, support, and inspiration during the preparation and
writing of the dissertation.
Finally the author acknowledges with love and gratitude
her parents, Mary and Phillip Awong, for their love,
support, and encouragement during her academic endeavors.
v


43
Figure 3-
O 2 4 6 8 10
cm
1. Schematic diagram of membrane diffusion chamber.


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS iv
ABSTRACT X
CHAPTERS
1 INTRODUCTION 1
2 LITERATURE REVIEW 5
Part I: Recombinant DNA Technologies 5
Introduction 5
Genetic Manipulation 5
Part II: Applications of Recombinant DNA Products. 9
Introduction 9
Potential Environmental Uses 10
Part III: Methods for Testing and Monitoring
Recombinant Organisms in Environments 14
Methods for Assessing Fate and Effects 14
Methods for Detection and Enumeration 17
Methods for Assessing Genetic Stability 21
Part IV: Survival of Genetically Engineered
Organisms and Stability of Recombinant
Plasmids in the Natural Environment 22
Introduction 22
Soil, Plant and Rhizosphere Systems 24
Aquatic Systems 28
Part V: Genetic Transfer by Genetically Engineered
Organisms in the Natural Environment 32
Introduction 32
Conjugation 33
Transduction 3 6
Transformation 38
3 MICROCOSM DESIGN AND PROTOCOL FOR ASSESSING THE
FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN AQUATIC ENVIRONMENTS 40
Introduction 40
Materials and Methods 42
Survival Chambers 42
vi


53
Figure 3-4. Growth curve and corresponding optical density
values (Agcg) of wild-type Escherichia coli
(HB101), grown at 35C.
Optical Density


218
Novitsky, J.A., and R.Y. Morita. 1978. Possible strategy for
the survival of marine bacteria under starvation
conditions. Mar. Biol. 48:289-295.
Nygaard, P. 1983. Utilization of preformed purine bases and
nucleosides. In A. Munch-Petersen (ed), Metabolism of
Nucleotides, Nucleosides and Nucleobases in
Microorganisms, Academic Press, London, pp. 56-63.
Ohana, B., J. Margalit, and Z. Barak. 1987. Fate of Bacillus
thurinainensis subsp. israelensis under simulated field
conditions. Appl. Environ. Microbiol. 53:828-831.
Okon, Y., and H. Hadar. 1987. Microbial inoculants-as crop
yield enhancers. Crit. Rev. Biotech. 6:21-32.
Omenn, G.S. 1986. Controlled Testing and monitoring methods
for microorganisms. In J. Fiksel and V.T. Covello
(eds), Biotechnology Risk Assessment, Pergamon Books,
Inc, New York, pp. 144-163.
Palmer, L.M., D.J. Baya, D.J. Grues, and R.R. Colwell.
1984. Molecular genetic and phenotypic alteration of
Escherichia coli in natural water microcosms containing
toxic chemicals. FEMS Microbioloy Letters 21:169-173.
Paul, J.H. and D.J. Carlson. 1984. Genetic material in the
marine environment: implication for bacterial DNA.
Limnol. Oceanogr. 29:1091-1097.
Paul, J.H., and A.W. David. 1989. Production of
extracellular nucleic acids by genetically altered
bacteria in aquatic-environment microcosms. Appl.
Environ. Microbiol. 55:1865-1869.
Paul, J.H., M.F. DeFlaun, and W.H. Jeffrey. 1988. Mechanisms
of DNA utilization by estuarine microbial populations.
Appl. Environ. Microbiol. 54:1682-1688.
Paul, J.H., W.H. Jeffrey, and M.F. DeFlaun. 1985.
Particulate DNA in subtropical oceanic and estuarine
planktonic environments. Mar. Biol. 90:95-101.
Paul, J.H., W.H. Jeffrey, and M.F. DeFlaun. 1987. Dynamics
of extracellular DNA in the marine environment. Appl.
Environ. Microbiol. 53:170-179.
Paul, J.H., and B. Myers. 1982. Fluorometric determination
of DNA in aquatic microorganisms by use of Hoechst
33258. Appl. Environ. Microbiol. 43:1393-1399.
Peberdy, J.F. 1979. Fungal protoplasts: isolation, reversion
and fusion. Ann. Rev. Microbiol. 33:21-39


14
in acidity and oxygen deficiencies (Gillett et al., 1985;
Saunders and Saunders, 1987). Microorganisms are now being
used for biomining of precious metals and oil recovery.
Part III: Methods for Testing and Monitoring
Recombinant Organisms in the Environment
Testing and monitoring methodologies can be divided
into three main categories: methods for assessing fate and
effects of the organisms; methods for detecting, identifying
and enumerating the organisms; and methods for assessing
genetic stability of the organisms.
Methods for Assessing Fate and Effects
Methods for assessing fate and effects can range from
simple contained systems, such as flasks and growth
chambers, to large scale field tests. An increasingly
important technique is the use of microcosms which
incorporate both physical and biological factors within a
contained setting (Omenn, 1986). Microcosms are usually of
two types: naturally derived microcosms and synthesized or
standardized microcosms. Naturally derived microcosms
utilize the actual material, such as soil and lake water,
from the natural community. Standardized microcosms are
comprised of chemically defined media and sediments, and a
variety of algae, grazers and detritivores (Kindig et al.,
1983; Taub, 1989).
To date, the majority of research utilizing recombinant
microorganisms is carried out in naturally derived


132
the number of soil rhizospheric microorganisms over the same
period of time also revealed their greater ability to grow
under rhizospheric conditions. After 5 days of incubation,
however, the numbers of rhizospheric microorganisms declined
only slightly, whereas coli numbers dropped sharply. The
decline in numbers can be attributed to both competition and
predation by indigenous organisms. Pseudomonas putida
displayed a higher survival than £. coli in the presence of
rhizospheric microorganisms in the corn rhizosphere.
Pseudomonads are often found in the rhizosphere, and this
may explain their higher persistence in the simulated
rhizosphere. The sharper decline of £. coli is also probably
related to the limited supply of available carbon in the
medium, resulting in part from the imposed non
photo synthetic conditions. These conditions allowed for
expression of the competition between the GEMs and
rhizospheric microorganisms.
The scarcity of information on the fate of GEMs in the
rhizosphere indicates the need for future research. Of
importance is the need to investigate the potential for
genetic transfer by GEMs to indigenous populations. A
predictive model has been reported for plasmid transfer in
the rhizosphere (Knudsen et al., 1989). Although predictive
models may prove to be useful tools in risk and fate
assessments, microcosms still remain indispensable for
empirical analyses of the fate, transport, gene transfer,
and ecosystem effects of introduced organisms.


173
BACDEFGHIJKLMNOPQR
Figure 7-4. Degradation of spiked eDNA in four aquatic
samples after 48 hours incubation at 25C.
Lane designations same as in Figure 1.


156
adaptive mechanism by which the cells can enhance survival
under stressful conditions. The ability to turn off and on
particular metabolic pathways as needed allows for
conservation of energy and carbon flow and is an adaptive
feature in systems with limited nutrients (Atlas and Bartha,
1981). If such is the case then it would appear that the GEM
may be more adaptable to natural conditions than the non-
engineered strain.
The composition of the cell envelope can be altered
quantitatively and qualitatively when the cells are exposed
to different growth conditions and environmental stresses
(Lugtenberg et al., 1976). Structural modifications of the
cell envelope have been reported for bacteria exposed to
seawater (Chai, 1983; Munro et al., 1987; Palmer et al.,
1984; Rhodes et al., 1983). Results of this study also
suggest changes in cell outer membrane permeability
following exposure of the cells to lake water. Chai (1983)
reported higher sensitivity to toxicants in E. coli cells
grown in bay water.
Possible factors for this increased sensitivity were a
more efficient transport of the toxicant into the cells via
porin modification, changes in ionic charge at the cell
surface and lipopolysaccharide (LPS) alterations. Palmer et
al. (1984) also demonstrated that exposure of cells to bay
water containing toxic chemicals produced alterations of the
membrane proteins.


113
Bacterial Strains
Escherichia coli (strain HB101) and Pseudomonas putida
(strain 50014) were used as wildtype reference strains. The
modified E. coli strain (strain 50008) contained an EcoRl
DNA fragment from pRCIO containing the gene for mercury
resistance and some of the genes involved in the degradation
of 2, 4-D. Details of the construct were described in
Chapter 3. The modified £. putida strain (50058) carried a
plasmid R68.45 which conferred resistance to carbenicillin,
kanamycin, and tetracycline, and was obtained from J.
Shapiro, Department of Biology, University of Chicago.
The wildtype and recombinant strains of P. putida and
E. coli were grown overnight in 3 ml of LB (Luria's broth
containing 1% tryptone, 0.5% yeast extract, and 1% NaCl) at
27C and 37C, respectively. The cultures were then
transferred to 50 ml LB and incubated for 2 hours. LB was
supplemented with tetracycline and kanamycin at
concentrations of 15 ug/ml and 50 ug/ml, respectively, for
selective growth of P. putida 50058. For £. coli 50008, the
growth medium was amended with mercury at a final
concentration of 40 ug/ml HgCl2. Following bacterial growth
for 2 hours, the bacterial suspensions were centrifuged at
7000 g for 10 minutes, washed twice with phosphate buffer
(pH 7.2), and finally resuspended into 50 ml of phosphate
buffer.


6
vivo methods using transposable genetic elements, 3) normal
genetic exchange by means of conjugation, transduction or
transformation, 4) protoplast fusion and 5) generalized and
site-specific mutagenesis (Johnston and Robinson, 1984;
Gillett et al., 1985; Saunders and Saunders, 1987). New
techniques developed for genetic transformation include
electroporation, projectile insertion, nuclear micro
injection, electroinjection, and the use of liposomes and
spheroplasts (Cocking and Davey, 1987; David et al., 1989;
Holo and Nes, 1989; Morikawa et al., 1986; Reich et al.,
1986; Tiedje et al., 1989). A brief description of these
methods is given below.
In-vitro recombinant technology. In-vitro recombinant
DNA technology involves the insertion of a DNA segment from
one organism into another host genome (rDNA host), by using
vector DNA molecules which can be transferred and are
capable of replicating autonomously in the host cell (Jain
et al., 1988). A generalized concept of rDNA technology is
depicted in Figure 2-1.
Transposable genetic elements. Transposons are short
DNA base sequences that are capable of inserting as discrete
nonpermuted DNA sequences into various sites within a
genome. Transposon-mediated mutations are capable of
spontaneous reversion.
Natural sexual genetic exchange. In bacteria, new
combinations of genes can be generated via normal sexual
genetic exchange. These exchange mechanisms include


REFERENCES
Aardema, B.W., M.G. Lorenz, and W.E. Krumbein. 1983.
Protection of sediment-adsorbed transforming DNA
against enzymatic inactivation. Appl. Environ.
Microbiol. 46:417-420.
Alexander, M. 1977. Introduction to Soil Microbiology. John
Wiley, New York.
Altherr, M.R., and K.L. Kasweck. 1982. In situ studies with
membrane diffusion chambers of antibiotic resistance
transfer in Escherichia coli. Appl. Environ. Microbiol.
44:838-843.
Amy, P.S., and H.D. Hiatt. 1989. Survival and detection of
bacteria in an aquatic environment. Appl. Environ.
Microbiol. 55:788-793.
Anderson, I.C., M. Rhodes, and H. Kator. 1979. Sublethal
stress in Escherichia coli: a function of salinity.
Appl. Environ. Microbiol. 38:1147-1152.
Armstrong, J.L., G.R. Knudsen, and R.J. Seidler. 1887.
Microcosm method to assess survival of recombinant
bacteria associated with plant and herbivorous insects.
Curr. Microbiol. 15:229-232.
Atlas, R.M., and R. Bartha. 1981. Microbial Ecology:
Fundamentals and Applications. Addison-Wesley
Publishing Company, Inc. Ment Park, California.
Baker, R.M., F.L. Singleton, and M.A. Hood. 1983. Effects
of nutrient deprivation on Vibrio cholerae. Appl.
Environ. Microbiol. 46:930-940.
Bale, M.J., J.C. Fry, and M.J. Day. 1987. Plasmid transfer
between strains of Pseudomonas aeruginosa on membrane
filters attached to river stones. J. Gen. Microbiol.
133:3099-3107.
Bale, M.J., J.C. Fry, and M.J. Day. 1988. Transfer and
occurrence of large mercury resistance plasmid in river
epilithon. Appl. Environ. Microbiol. 54:972-978.
206


58
800
CFU/ml (Millions)
Spread Method
Drop Method
600-
400-
200-
Time (hours)
Figure 3-8. Comparison of enumeration techniques
by the spread plate and drop plate methods for
values (A550) of genetically engineered Pseudomonas putida
wild-type Escherichia coli (HB101).


CHAPTER 9
CONCLUSIONS
The purpose of this study was to determine the fate and
survival of genetically engineered microorganisms and their
recombinant DNA in natural environments. A laboratory-
contained, microcosm approach was utilized for studying the
fate and rates of population change of GEMs and wild-type
strains in various environments. Survival rates were
determined in lake water, activated sludge effluent, ground
water and the root rhizosphere. Physiological and structural
changes were also determined for GEM and wild-type strains
in lake water. The potential for genetic transfer via
transformation was investigated by observing the fate of
extracellular or free DNA in various aquatic samples.
The conclusions drawn from this study are as follows:
1. Laboratory-contained microcosms serve an important
function in the study of GEMs. Current regulatory guidelines
strongly recommend research with recombinant bacteria only
within contained settings. The microcosm that was developed
and utilized in this study is a simple but useful way to
study GEMs. The advantages of the model described in this
study are its simplicity and ease for adjusting for
environmental variables and the fact that the organisms can
201


224
Zeph, L.R., M.A. Onaga, and G. Stotzky. 1988. Transduction
of Escherichia coli by bacteriophage PI in soil. Appl.
Environ. Microbiol. 54:1731-1737.
Zeph, L.R., and G. Stotzky. 1989. Use of a biotinylated DNA
probe to detect bacteria transduced by bacteriophage PI
in soil. Appl. Environ. Microbiol. 55:661-665.


168
incubation at which time the linear and OC forms of plasmid
DNA became more apparent. By 48 hours (Figure 7-4, Lane P),
chromosomal DNA degradation occurred and much of the CCC DNA
was converted to the OC and linear forms.
Lake Water
The degradation of eDNA occurred at a slower rate in
the untreated lake water than in raw sewage. Within fifteen
minutes (Figure 7-1, Lane K), some conversion of the CCC DNA
to the OC and linear forms had already occurred, but the
intensity of the bands indicated very little degradation of
the eDNA. After one hour of incubation, chromosomal DNA was
partially degraded and over half of the CCC plasmid
converted to the OC and linear forms (Figure 7-2, Lane K).
Chromosomal DNA was totally degraded after five hours (gel
not shown) and only OC and linear forms of the plasmid were
present at this time. The eDNA was totally degraded within
twelve hours (gel not shown), but degradation by-products (<
4.0 kb) were still present. After 24 hours, no eDNA was
detected (Figure 7-3, Lane K).
The filter-sterilized lake water showed no signs of DNA
degradation after fifteen minutes and one hour of incubation
(Lane M, Figures 7-1 and 7-2, respectively). After five
hours however, intensity of the OC band increased with a
corresponding decrease in the intensity of the CCC band (gel
not shown). Some chromosomal DNA degradation also occurred
at this time. Chromosomal DNA was totally degraded after 24


LOG CFU/mi
78
DAYS
Figure 4-3. Survival of genetically engineered (GEM)
and wild-type strains of Escherichia coli and
Pseudomonas outida at 30C in lake water.
Wild-type and GEM strains recovered on non-selective
medium; ( ) and ( ), respectively. GEM strains
recovered on selective medium (o )


CHAPTER 4
SURVIVAL OF AND PLASMID STABILITY IN GENETICALLY
ENGINEERED AND WILDTYPE STRAINS OF EL COLI
AND P._ PUTIDA IN AQUATIC ENVIRONMENTS
Introduction
At present, the number of cases of deliberate release
of genetically engineered bacteria into the environment are
few and fairly recent (Halvorson et al., 1985). Prospects
for the routine release of genetically engineered organisms
into the environment are becoming increasingly feasible. The
potential use of recombinant bacteria in the environment
include enhancement in food and agricultural production,
biocontrol of insects and diseases, metal and mineral
leaching, environmental remediation and waste treatment
(Gillett et al., 1985; Halvorson et al., 1985; Johnston et
al., 1984; Keeler, 1988).
Deliberate release of such recombinant microorganisms
has however raised considerable concern and attention among
the public and scientific sectors. Questions and issues
concerning potential adverse environmental effects are
foremost in the minds of many involved. Genetically
engineered microorganisms (GEMS) that are released may have
the capacity to reproduce, spread beyond the initial point
of release and transfer their novel genetic information to
65


154
significant increase in cell biomass. This stress response
ultimately results in ultramicrobacteria or micrococci.
A variety of physiological adaptations or responses
have been reported for bacteria under starvation and low
nutrient conditions (Chai, 1983; Kurath and Morita, 1983;
Lopez-Torres et al., 1988; Novitsky and Morita, 1978; Tabor
et al., 1981; Tamplin and Colwell, 1986; Xu et al., 1982).
These include changes in respiration rates, B-galactosidase
activity, ATP content, total adenylate concentrations,
protein content and cell envelope composition. The ability
to rapidly reduce the endogenous metabolic rate may be a
primary survival requirement if bacteria are to endure
starvation conditions.
In this study, dehydrogenase activity as measured by
INT reduction showed a substantial decline over time.
Similar declines have also been reported for enteric
bacteria exposed to seawater (Lopez-Torres, 1988; Novitsky
and Morita, 1978). Lopez-Torres (1988) noted a 60% reduction
in INT-dehydrogenase activity after only 3 hours of exposure
to tropical marine waters. Results of this study suggest
that exposure to lake water causes a reduction in
respiration rates for both strains of E. coli. However, the
results indicate that the wild-type strain is more rapidly
inactivated than the engineered strain. Within the first 24
hours, dehydrogenase activity of wild-type HB101 had
declined by almost 80% as compared to the engineered strain
which showed no change in dehydrogenase activity at this


Chromosomal DNA
Recombinant
plasmid
Genetically engineered
microorganism (GEM)
Figure 2-1. Generalized concept of recombinant DNA
technology.
Source: Adapted from Jain et al.,
1988 .



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126
Figure 5-9. Survival of genetically engineered Pseudomonas
putida in the presence of rhizospheric microorganisms (RM),
without corn roots present.
( ) P_s_ putida in the absence of RM;
( ) P_ putida in the presence of RM.


60
Log CFU/ml
i i i
0 a 21 44 216
Time (hours)
LB 40 ppm HgCI
L.J 1/2LB 20ppm HgCI
3 1/4LB 10ppm HgCI
Figure 3-10. Comparison of four different media types
for selective growth of genetically engineered
Escherichia coli (50008).


94
selective or nonselective media, at either of the three
temperature regimes.
Survival and Plasmid Stability in Ground Water
Survival studies. The survival of wild-type and
genetically engineered strains of E. coli and P. putida was
studied in ground water at 22C, under sterile conditions.
Rates of population change (log10/day) are summarized in
Table 4-5. Both wild-type and engineered E. coli exhibited
similar rates of decline (-0.09 and -0.08, respectively)
that were not significantly different. Interestingly, growth
occurred during the first 48 hours of exposure to the ground
water. The engineered strain exhibited a greater increase (1
log) than the wild-type strain (0.5 log) during the first 48
hours, but both strains then declined at a similar rate
thereafter (Figure 4-12). Cell densities for wild-type HB101
were therefore consistently lower (0.5 log) than those of
the engineered strain.
Growth of wild-type P. putida was also observed during
initial exposure to ground water (Figure 4-13). A one log
increase in numbers was noted during the first 72 hours,
followed by a gradual decline after which population
densities stabilized. No growth was observed for the
engineered strain, and population densities remained fairly
constant for the duration of the test. The rate of decline
for the wild-type strain (-0.10) was significantly higher
(p<0.025) than that of the engineered strain (-0.03), even
though similar population densities were reached after 21


136
fragment contained the gene for mercury resistance and the
genes involved in 2,4-D degradation (Chaudhry and Huang,
1988). Wild-type and genetically engineered E. coli strains
were grown at 35C in LB and LB supplemented with HgCl2 (40
ug ml-1), respectively. All cultures were incubated until
late logarithmic phase and harvested by centrifugation at
8000 g for 10 minutes at 4C. The cells were washed three
times with sterile phosphate buffer (pH 7.2) and resuspended
in filter-sterilized lake water to a final density of 108
CFU/ml.
Aquatic Microcosm
Experiments were conducted using a continuous flow
through microcosm as described in Chapter 3. Lake water was
collected from a hypereutrophic lake (Lake Alice,
Gainesville, FI.) at a depth of approximately 1 meter.
Samples were collected every 48 hours in 20 liter Nalgene
carboys and allowed to acclimate to the appropriate test
temperature prior to use. Experiments were conducted in an
environmental chamber (Percival, Boone, Iowa) at 25C with a
constant light regime of 16 hours light: 8 hours dark). The
design and assembly of the survival chambers was given in
Chapter 3. Chambers were inoculated with 20 ml of the washed
cells and immersed into the microcosm. After specific time
intervals, triplicate chambers were removed and sacrificed
for the following bacterial assays: bacterial enumeration
(viable counts and direct counts), B-galactosidase


47
Steel clamps that allowed the test water to be siphoned at a
fixed rate into the aquaria tanks. This modification was
similar to the microcosm described by Fulthorpe and Wyndham
(1989). Outflow from the tanks was accomplished by drilling
holes in the tanks to serve as outlet ports at a height that
was approximate to that of the chambers. The overflow water
was collected using tygon tubing attached to the outlet
ports and decontaminated before discarding. This setup
avoided the complete immersion of the needle sampling ports
which increases the chances of contamination during the
sampling procedure. Figure 3-3 illustrates the design of the
flow-through microcosm.
Both types of microcosms were utilized in either a
constant temperature room or an environmental chamber
(Percival, Boone, Iowa) at a constant temperature and light
regime (16 hours light: 8 hours dark).
Test Protocol for Survival Studies
Preliminary experiments were undertaken to determine
optimal conditions for routine sampling, enumeration and
detection of test organisms. Preliminary tests included
growth studies of the test organisms (both GEMs and their
wildtype strains); comparison of plating techniques for
enumeration of viable counts; and a comparison of the type
of selective media best suited for detection of the GEMs.
Bacterial Strains. Escherichia coli (strain HB101) and
Pseudomonas putida (strain 50014) were used as wildtype
reference strains. Genetically engineered E. coli (strain


174
Table 1 summarizes the half lives and decay rates of
eDNA for the four aquatic samples tested. Estimated half
lives ranged from 0.88 days for untreated raw sewage to 288
days for untreated tap water.
High rates of degradation were observed in all
treatments of raw sewage with the highest rate occurring in
the untreated sample (Table 7-1). Most of the eDNA appeared
to be degraded within 10 hours of inoculation in untreated
raw sewage (Figure 7-5). This was evident in the quick
decline in absorbance during the first 10 hours followed by
a much slower decline for the rest of the incubation period.
The same was not true for the autoclaved or filter-
sterilized samples which showed most of the degradation
occurring after 24 hours (Figure 7-5). The autoclaved sample
showed a longer half life (3.24 days) than the filter-
sterilized (1.48 days) or untreated (0.88 days) samples.
The eDNA was also rapidly degraded in untreated lake
water. The results indicated an estimated half life of 1.42
days with the majority of degradation occurring after 12
hours (Figure 7-6). The filter-sterilized sample also showed
a high degradation rate (ti/2= 4-81 days), but optical
density measurements indicate that degradation did not occur
until after 48 hours of incubation (Figure 7-6). The eDNA
was relatively stable in the autoclaved lake water with a
half life of 20.63 days.
Groundwater appeared to be a relatively stable
environment for the eDNA during the first 24 hours of


176
LU
O
2
<
CG
OC
O
CO
CD
<
TIME (HOURS)
Figure 7-5. Degradation kinetics of spiked eDNA
in Raw Sewage. ( ) Untreated Sample; ( o ) Autoclaved
Sample; ( A ) Filter-sterilized Sample.


13
pollution control. Main approaches to the construction of
desired xenobiotic-degrading strains involve recruitment and
heterologous expression of degradative genes for gene
fusion, gene amplification and mutagenesis.
Biomass conversions. The conversion of biological raw
material (biomass) to useful products such as foods,
bioplastics, fuel and other chemicals can be accomplished by
microbial fermentation. Biotechnology offers the possibility
of improving such transformations. Areas that are currently
under investigation include the conversion of biomass into
fuels such as ethanol and methane, and enhanced biopolymer
degradation (Saunders and Saunders, 1987; Zaugg and Swarz,
1981).
Enhanced oil recovery. Conventional oil recovery
techniques are capable of extracting only one-third of the
underground oil reservoirs. The use of modified
microorganisms and/or microbial products has been suggested
for improved recovery efficiencies (Springham, 1984).
Specific modifications for enhanced oil recovery include
biopolymer and biosurfactant production, bioleaching of rock
matrices, and gas production.
Mining. The application of biotechnology in the mining
industry is almost exclusively limited to bacterial leaching
operations in which metals are solubilized from low-grade
ores. Genetic manipulation has been directed towards
enhancing the leaching capabilities of the bacteria, and
increasing its tolerance to toxic metals, heat, fluctuations


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.
^ / f' v Li¡,<.*/hvy
G. Rasul Chaudhry, Cochairman
Associate Professor of
Biological 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.
Be L. Kbopman^-^
Associate Professor of Environmental
Engineering Sciences


194
The potential for conjugation in lake water was also
determined by performing simultaneous experiments utilizing
membrane diffusion chambers in a microcosm setting. The
microcosm is described in detail in Chapter 3. The procedure
used for the conjugation studies was similar to that used
for the membrane-filter mating. However, instead of
transferring the donor and recipient cells to a membrane
filter, the cells were added directly to survival chambers
containing 20ml of sterile lake water. The chambers were
then immersed into the microcosm and sampled after 20 and 72
hours incubation, for the presence of transconjugants. This
study was conducted at 28C. All experiments were conducted
in triplicate.
Results and Discussion
Construction of the GEM
The recombinant plasmid, pJAl, was constructed from
plasmids pDugll and pRC92. Plasmid pDugll was constructed by
inserting a Hindlll DNA fragment from pLOI193 into the
plasmid pGS9. The Hindlll DNA fragment contained the genes
(mob) involved in plasmid mobilization during conjugation.
This plasmid also contained the transposable kanamycin
resistance element, Tn 5. Plasmid pRC92 is a hybrid plasmid
derived from pBR322 and pE194, and contains a 0.3-kilobase
(kb) EcoRl DNA fragment of Napier grass DNA. This plasmid is
capable of multiplying in both gram-positive and gram
negative bacteria.


148
biosynthesis were significantly different (p<0.05) for the
two strains of E. coli after exposure to lake water.
INT-dehvdroqenase activity
Wild-type HB101 exhibited a rapid decline (72%) in
dehydrogenase activity during the initial 24 hours of
incubation, whereas the engineered strain showed no change
in activity during the same time period (Figure 6-6).
However, after 3 days of exposure to the lake water, the
engineered strain had a reduced dehydrogenase activity that
was comparable to that of the non-engineered strain (80%
reduction). Dehydrogenase activities were maintained at
lower rates for both strains for the remainder of the
experiment.
Cell permeability
Changes in sensitivity to the toxicant PCP (45ppm) over
time suggests alterations in the cell membrane after
exposure to the lake water (Figure 6-7). Changes in cell
permeability occur in both engineered and non-engineered
strains but in significantly different ways. At the start of
the experiment, the wild-type strain (HB101) was not
inhibited by the toxicant, PCP. Instead, the chemical
produced a stimulatory effect on B-galactosidase
biosynthesis. Further exposure (1-3 days) of the cells to
lake water produced a decreased stimulatory response. After
6 and 9 days, the PCP had caused a 25% and 80% inhibition of


221
Smith, H.O., D.B. Danner, and R.A. Deich. 1981. Genetic
transformation. Annu. Rev. Biochem. 50:41-68.
Springham, D.G. 1984. Microbiological methods for the
enhancement of oil recovery. In G.F. Russell (ed.),
Biotechnology and Genetic Engineering Reviews, vol 1.
Intercept, Newcastle upon Tyne, pp. 187-221.
Steffan, R.J., and R.M. Atlas. 1988. DNA amplification to
enhance detection of genetically engineered bacteria in
environmental samples. Appl. Environ. Microbiol.
54:2185-2191.
Steffan, R.J., A. Breen, R.M. Atlas, and G.S. Sayler. 1989a.
Application of gene probe methods for monitoring
specific microbial populations in fresh water
ecosystems. Can. J. Microbiol. 35:681-685.
Steffan, R.J., A. Breen, R.M. Atlas, and G.S. Sayler. 1989b.
Monitoring genetically engineered microorganisms in
freshwater microcosms. J. Ind. Microbiol. 4:441-446.
Stewart, G.J., and C.A. Carlson. 1986. The biology of
natural transformation. Annu. Rev. Microbiol. 40:211-
235.
Stotzky, G., and H. Babich. 1986. Survival of, and genetic
transfer by, genetically engineered bacteria in natural
environments. Adv. Appl. Microbiol. 31:93-138.
Tabor, P.S., K. Ohwada, and R.R. Colwell. 1981. Filterable
marine bacteria found in the deep sea: distribution,
taxonomy and response to starvation. Microb. Ecol.
7:67-83.
Talbot,H.W., Jr., D.K. Yamamoto, M.W. Smith, and R.J.
Seidler. 1980. Antibiotic resistance and its transfer
among clinical and nonclinical Klebsiella strains in
botanical environments. Appl. Environ. Microbiol.
39:97-104.
Tamplin, M.L., and R.R. Colwell. 1986. Effects of microcosm
salinity and organic substrate concentration on
production of Vibrio cholerae enterotoxin. Appl.
Environ. Microbiol. 52:297-301.
Tate, R.L. III. 1978. Cultural and environmental factors
affecting the longevity of Escherichia coli in
histosols. Appl. Environ. Microbiol. 35:925-929.
Taub, F.B. 1989. Standardized aguatic microcosms. Environ.
Sci. Technol. 23:1064-1066.


112
conditions in order to generate information about the fate
of model GEMs in the root environment using root exudates as
the only available carbon source. The effect of indigenous
rhizospheric microorganisms on the fate of GEMs was also
investigated.
Materials and Methods
Hydroponic Cultures
Corn seeds (hybrid of 2AT98-2-1 and 3BT13 provided by
Dr. E.S. Horner from the Agronomy Department, University of
Florida) were surface sterilized by soaking for 30 minutes
in distilled water, 5 minutes in ethanol, and 30 minutes in
10% hydrogen peroxide. The seeds were then aseptically
placed on nutrient agar and incubated for 48 hours at 27C to
allow for germination. Young corn seedlings devoid of
bacterial contamination were selected according to root
length. They were aseptically introduced into autoclaved
glass tubes (Length = 15 cm; internal diameter = 2.5 cm)
containing a polyethylene support and 10 ml of a modified
Hoagland's solution medium. The seedlings were then allowed
to grow for 3 days in the dark, at 27C prior to inoculation
with the bacterial strains under investigation. Following
inoculation, the tubes were incubated at 25C and kept in the
dark until sampling. These plants could be maintained under
non-photosynthetic conditions for up to 10 days. Incubation
in the dark was considered a necessity to avoid possible
complicating effects of photosynthesis.


74
TABLE 4-1. Effect of temperature on the rates of
population change (log10/day) of genetically
engineered and wild-type E. coli under sterile
conditions in lake water.
Temperature Media Type Escherichia coli
(C) wild-type GEM
15
LB
-0.05
-0.02
LB + HgCl2
ND
-0.03
25
LB
-0.20
-0.17
LB + HgCl2
ND
-0.19
30
LB
-0.19
-0.13
LB + HgCl2
ND
-0.14
ND indicates not applicable


116
TIME (hours)
Figure 5-1. Effect of corn-root exudates on the growth
of genetically engineered Escherichia coli. Initial
inoculum concentration was approximately 3.0 x 10.
( ), no root exudates; (o), presence of root exudates.


CHAPTER 3
MICROCOSM DESIGN AND PROTOCOL FOR ASSESSING THE
FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN AQUATIC ENVIRONMENTS
Introduction
In general, microcosms can be described as fully-
contained laboratory ecosystems which simulate an ecological
community with its complete ensemble of interacting
microbial species. Microcosms can range from laboratory
contained flasks, mason jars and growth chambers to fully
contained greenhouses and systematically sampled natural
areas termed mesocosms. Of increasing importance is the use
of microcosms which incorporate both physical and biological
components within a contained setting (Omen, 1986).
Many types of research questions which evolve from a
consideration of biotechnology risk assessment can be
addressed by the use of microcosms. Microcosms can serve as
standard test systems that can be adapted to a variety of
organisms and environmental conditions. Testing and
monitoring of GEMs by use of microcosms allow data gathered
in one test environment to support design and justification
of tests in natural environments.
A variety of microcosm models have been utilized for
the study of GEMs in the aquatic environment. Experimental
40


196
l
Figure 8-1. Construction of plasmid pJAl from pRC92
and pDugll.


117
Figure 5-2. Effect of corn-root exudates on the growth
of genetically engineered Pseudomonas putida. Initial
inoculum concentration was approximately 2.0 x 10 .
(), no root exudates; (O), presence of root exudates.


81
sterile (-0.17) conditions were significantly different
(p<0.001). The rates of change for P. outida were similar
under sterile (-0.06) and nonsterile (-0.07) conditions.
Statistical analysis (modified t-Test) showed no significant
difference between the two survival rates after addition of
the indigenous organisms. However, population densities
under non-sterile conditions were significantly lower
(p<0.05) than under sterile conditions. This difference is
due to the initial sharp decline in numbers immediately
following the addition of indigenous organisms. After 2-3
days, P. putida was able to stabilize its numbers and rate
of decline. Although the results suggest that there is no
significant difference in survival rates between sterile and
non-sterile conditions, it is important to note that the
indigenous population did have a significant effect on
population density.
Survival in the presence of a herbicide. The effect of
the herbicide Hydrothol-191 (l mg/L final concentration) on
the survival of wild-type P. putida (50014) and its
engineered strain (50058) is shown in Figure 4-5. Both
strains showed significantly (p<0.05) higher rates of
decline in the presence of 1 ug/mL hydrothol as compared to
the control with no toxicant. The rates of change for wild-
type P_s_ putida however, were not significantly different
from that of the engineered strain (50058) in the presence
or absence of the toxicant.


192
TABLE 8-1. Bacterial strains and plasmids
Relevant
Strain or plasmid Traits Source or reference
Pseudomonas putida
50479
Escherichia coli
HB101
S17-1
Plasmids
pRC92
pDugll
pJAl
Nalr
recA rpsL
Mobilizing strain
Eryr
Cm^ Kmr
Cmr Kmr Eryr
G. R. Chaudhry
ATCC
R. Simon (1983)
G. R. Chaudhry
G. R. Chaudhry
This study


165
above. The actual concentration of eDNA at time 0 was
determined by adding the DNA inoculum (10.4 ug/ml) to TE
buffer (pH 8.0), and subjecting 0.5 ml aliquots to the
recovery procedure. This was done because preliminary
experiments indicated almost instantaneous degradation of
the added eDNA in some of the samples tested. In the case of
raw sewage, much of the added DNA was degraded even before
the sample could be subjected to the recovery procedure.
The recovered DNA was characterized visually by gel
electrophoresis and also by spectrophometric measurements.
For gel electrophoresis, 5 ul of the recovered DNA was
loaded onto an agarose gel (0.7%). Electrophoresis was
carried out for 90 minutes at room temperature, 60mA and
120V. The gel was stained with ethidium bromide (0.5 ug/ml)
for exactly 30 minutes for standardization purposes. The DNA
was visualized with short-wave UV light (Fotodyne) and
photographed with a Polaroid MP-4 Land Camera. DNA
concentrations were quantified by measuring optical
densities (Perkin-Elmer 320 Spectrophotometer) at 260 and
280 nm (Davis et al., 1986). A 5 ul aliquot of the recovered
DNA was added to quartz cuvettes containing 995 ul deionized
distilled water for optical density measurements. DNA
concentrations (ug/ul) were calculated as OD(260)
10 (Davis et al., 1986).
values x


193
samples were then subjected to agarose gel electrophoresis
for fractionation of the DNA. Bacteriophage lambda DNA
digested with Hindlll was used as a reference standard for
size determination of the restriction fragments.
Cloning of Model GEM
Results of restriction analysis of the two plasmids
revealed single cuts with EcoRV. Cloning of the new GEM was
accomplished by digesting the plasmids overnight with EcoRV.
followed by ligation, transformation and agarose gel
electrophoresis as described by Chaudhry and Huang (1988).
Presence of the recombinant plasmid, pJAl, was first tested
by growth on LB agar plates containing chloramphenicol and
erythromycin, and subsequently confirmed by plasmid
isolation and restriction analysis.
Conjugation Studies
Conjugation studies were determined by in vitro
membrane-filter mating as described by Chaudhry and Huang
(1988). Donor strains consisted of E. coli strain HB101
containing the plasmid, pJAl, and E. coli strain S17-1
containing the plasmid, pDugll. Pseudomonas putida (50479)
was used as the recipient in all experiments. Membrane-
filter matings were carried out at 28C and 35C.
Transconjugants were selected using LB agar plates
containing nalidixic acid (40 ug/ml) and kanamycin (20
ug/ml).


209
Curtiss, R. 1976. Genetic manipulation of microorganisms:
potential benefits and biohazards. Ann. Rev. Microbiol.
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Davenhauer, S.A., R.A. Hull, and R.D. Williams. 1984.
Cloning and expression in Escherchia coli of Serratia
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David, S., G. Simons, and W.M. De Vos. 1989. Plasmid
transformation by electroporation of Leuconostoc
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Appl. Environ. Microbiol. 55:1483-1489.
Davis, L.G., M.D. Dinner, and J.F. Battey. 1986. Basic
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Davisson, J. 1988. Plant beneficial bacteria. Biotechnology
6:282-286.
Dawe, L.L., and W.R. Penrose. 1978. "Bactericidal" property
of seawater: death or debilitation? Appl. Environ.
Microbiol. 35:829-833.
DeFlaun, M.F., and J.H. Paul. 1986. Hoechst 33258 staining
of DNA in agarose gel electrophoresis. J. Microbiol.
Meth. 5:265-270.
DeFlaun, M.F., J.H. Paul, and D. Davis. 1986. Simplified
method for dissolved DNA determination in aquatic
environments. Appl. Environ. Microbiol. 52:654-659.
DeFlaun, M.F., J.H. Paul, and W.H. Jeffrey. 1987.
Distribution and molecular weight of dissolved DNA in
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Devanas, M.A., and G. Stotzky. 1986. Fate in soil of a
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Devanas, M. A., D. Rafaeli-Eshkol, and G. Stotzky. 1986.
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Microbiol. 13:269-277.


181
fragments and degradation products. The method therefore
gives a clearer indication of eDNA degradation since it
allows determination of both spiked DNA and its degradation
by-products. When used in association with gel
electrophoresis, the optical density measurements provided a
good indication of the changes that occur during eDNA
degradation. The possibility of RNA contamination was
eliminated by RNAse treatment of the DNA used.
The eDNA innoculum (10.4 mg/liter) used in this study
was at least three orders of magnitude greater than reported
ambient concentrations. DeFlaun et al. (1986) reported eDNA
values of 1.74 to 7.8 ug/liter in various freshwater
systems. Minear (1972) detected 4-30 ug/liter of dissolved
DNA in lake water. Other studies indicated similar values
(0.5-44 ug/liter) for marine environments (Breter et al.,
1977; DeFlaun et al., 1987; Pillai and Ganguly, 1972). A
high concentration of DNA inoculum was used in this study to
facilitate recovery and detection of the added DNA. It is
possible that such a high substrate concentration may affect
the accurate determination of kinetic degradation.
Nevertheless, the results of this study indicate that
degradation of eDNA did occur rapidly in the environmental
samples, but the degradation rates differed among samples.
In addition, the DNA undergoes structural changes prior to
or simultaneously during degradation (Figures 7-1 to 7-4).
The general trend was an initial degradation of chromosomal
DNA followed by a change in the CCC form of plasmid DNA. The


35
Richaume et al. (1989) demonstrated the influence of
soil variables such as clay, organic matter, soil pH, soil
moisture and soil temperature on the potential for
intergeneric plasmid transfer. Maximum transfer frequencies
were noted at a clay content of 15%, a soil pH of 7.25, a
soil moisture content of 8%, and incubation temperature of
28C.
Walter et al. (1989) evaluated a simple microcosm
method for measuring conjugal transfer of recombinant DNA in
soil slurries. Using a variety of environmental variables
they observed the highest numbers of transconjugants at 35C
in nutrient-enriched soil slurries. Low frequencies were
observed in low nutrient conditions or low pH values.
Transfer of recombinant plasmids has also been
demonstrated in wastewater, utilizing model laboratory-scale
waste treatment (LSWT) facilities (Mancini et al., 1987;
McClure et al., 1989). Transconjugants were detected at
different locations within the model system (Mancini et al.,
1987) suggesting that nutrients in sewage are sufficient for
plasmid mobilization. The potential for plasmid transfer via
triparental mating was also shown in the LSWT (Gealt, 1985;
Mancini et al., 1987). McClure et al. (1989) utilized a
model activated sludge unit to demonstrate the transfer of
mobilizing plasmids from indigenous populations to the
recombinant strain which carried a non-self-transmissible
plasmid pDlO. Further experiments showed that bacteria in
the activated sludge population could then act as recipients


214
Kindig, A.C., L.L. Conquest, and F.B. Taub. 1983.
Differential sensitivity of new versus mature synthetic
microcosms to streptomysin sulfate treatment. In W.E.
Bishop, R.D. Cardwell and B.E. Heidolph (eds.), Aquatic
Toxicology and Harzard Assessment: Sixth Symposium.
American Society of Testing and Materials,
Philadelphia, pp. 192-203.
Klein, D.A., and L.E. Casida, Jr. 1967. Escherichia
coli die-out from normal soil as related to nutrient
availability and the indigenous microflora. Can. J.
Microbiol. 13:1461-1470.
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considered in heterotrophic microorganism enumeration
from aquatic environments. Appl. Microbiol. 27:429-431.
Klein, T.M., and M. Alexander. 1986. Bacterial inhibition in
lake water. Appl. Environ. Microbiol. 52:114-118.
Knudsen, G.R., M.V. Walter, L.A. Porteous, V.J. Prince, J.L.
Armstrong, and R.J. Seidler. 1988. Predictive model of
conjugative plasmid transfer in the rhizosphere and
phyllosphere. Appl. Environ. Microbiol. 54:343-347.
Kobori, H., C.W. Sullivan, and H. Shizuuya. 1984. Bacterial
plasmids in antarctic natural microbial assemblages.
Appl. Environ. Microbiol. 48:515-518.
Koopman, B., G. Bitton, C. Logue, J.M. Bossart, and J.M.
Lopez. 1984. Validity of tetrazolium reduction assays
for assessing toxic inhibition of filamentous bacteria
in activated sludge. In D. Liu, and B.J. Dutka (eds),
Toxicity Screening Procedures using Bacterial Systems.
Marcel Dekker, New York, pp. 132-139.
Kozyrovskaya, N.A., R.I. Gvozdyak, V.A. Muras, and V.A.
Kordyum. 1984. Changes in properties of phytopathogenic
bacteria effected by plasmid pRDl. Arch. Microbiol.
137:338-343.
Kraffczyk, I., G. Trolldenier, H. Beringer. 1984. Soluble
root exudates of maize: influence of potassium supply
and rhizosphere microorganisms. Soil Biol Biochem.
16:315-322.
Krasovsky, V.N., and G. Stotzky. 1987. Conjugation and
genetic recombination in Escherichia coli in sterile
and nonsterile soil. Soil Biol. Biochem. 19:631-638.
Kurath, G., and Y. Morita. 1983. Starvation-survival
physiological studies of a marine Pseudomonas sp. Appl.
Environ. Microbiol. 45:1206-1211.


62
indigenous populations for the appropriate selective
markers, and is useful for detecting plasmid or gene
stability, full strength media was therefore selected for
routine use in all consecutive experiments.
Comparison of Microcosms
The survival of wildtype and engineered strains of P.
putida in lake water was simultaneously conducted using
flask microcosms and survival chambers in a flow-through
system. This study was designed to determine whether the
type of microcosm affects the survival rates or patterns of
the test organisms. The results indicated that the design of
the microcosm does play an important function in survival
studies. Results of the flask studies showed little to no
effect on the survival of the two strains of P. putida
(Figure 3-12). However, results using the flowthrough
microcosm indicated an initial decline in numbers during the
first 48 hours, followed by growth and stabilization of the
two populations. This trend was most likely a better
indication of survival in the natural environment. The
organisms were under constant pressure to adapt to changing
nutrient conditions and toxins associated with the constant
flow of the lake water through the survival chambers.
Although it can be argued that the two microcosms cannot be
compared, it clearly suggests the importance of microcosm
design for fate and survival studies. Designing the
microcosm to simulate natural conditions as closely as


99
may be the case for the two bacterial species tested.
Temperature-dependent studies indicated significant
differences between the parent and genetically engineered
strains under certain conditions. In lake water, Ij¡. coli
parent strain (HB101) exhibited significantly higher rates
of population decline from it's engineered counterpart at
15C and 30C, but compared to P. putida however, similar
trends were observed at 25C and 30C. Wildtype P. putida
also displayed a significantly higher rate of decline over
it's engineered strain at 22C in groundwater. This tendency
was not observed in activated sludge effluent. On the
contrary, there were no significant differences between
rates of population change of the wild-type or modified
strains of P. putida at the three temperatures tested. The
same was observed for E. coli strains, except at 30C where
the engineered strain showed a significantly higher rate of
decline than its parent strain. This trend is the opposite
to that observed in lake water (30C) and ground water
(22).
The results of this study indicate that recombinant
strains are generally capable of surviving better or at
least as well as their parent strains, regardless of the
environment and the variables involved. In lake and ground
water, the genetically engineered strains were able to
survive better under the given conditions than their parent
strains. This would suggest that the presence and/or addi
tion of plasmids to a host bacteria is not necessarily a


105
(Amy and Hiatt, 1989; Steffan et al., 1989; Trevors et al.,
1989) using other genetically engineered strains. The
decline in numbers can be attributed to competition,
antagonism and predation by the indigenous organisms.
Several studies (Findlay et al., 1986; Scheuerman et al.,
1988; Sherr and Sherr, 1987) have shown the importance of
protozoans in controlling bacterial populations. Competition
for nutrients has also been shown to be a critical factor
for survival (Liang et al., 1982). In a study of the factors
affecting the survival and growth of introduced bacteria in
lake water, Scheuerman et al. (1988) observed that growth
of the introduced species was limited by available carbon
and sometimes nitrogen and phosphorus.
Results of this study indicate that £. coli does not
survive as well as P. outida in the presence of indigenous
organisms. E. coli had a much higher rate of decline (-0.17)
than P. putida (-0.07) and was totally eliminated after 20
days. Interestingly, P. putida was able to stabilize it's
rate of decline after an initial relatively rapid decline in
numbers. After five days there was no significant difference
between survival rates under sterile or nonsterile
conditions. It is obvious that P. putida is better adapted
to the aquatic environment than E. coli which is normally
not associated with this type of habitat.
The herbicide, hydrothol, produced a significant
decline in the numbers of both strains of P. putida at a
concentration of 1 mg/L. Since the application rates of


BIOGRAPHICAL SKETCH
Judy Awong was born December 16, 1956, in Princes Town,
Republic of Trinidad and Tobago. She received her Bachelor
of Science degree from the University of the West Indies,
St. Augustine Campus, in 1979. Following graduation, she
worked for six months as a teaching assistant in the
Agricultural Department at the University of the West
Indies, then spent a year as a Junior Research Officer at
the Institute of Marine Affairs, Trinidad. Judy joined the
graduate program in the Department of Environmental
Engineering Sciences, at the University of Florida in 1982.
She earned a Master of Science degree in 1984 and is
currently completing the reguirements for the Doctor of
Philosophy degree in the same department.
Judy Awong is a member of the American Society of
Microbiology, the Society of Environmental Toxicology and
Chemistry and the Association for the Advancement of
Science.
225


56
Figure 3-7. Growth curve and corresponding optical density
values (A550) of genetically engineered Pseudomonas putida
(50058), grown at 27C.
OPTICAL DENSITY


103
pollutants. Lake Alice serves as a receiving body for
treated waste water and storm-water runoff, and therefore
can potentially contain mercury, antibiotics and phages at
any given time. This may partly account for the better
survival rates of the recombinant strains in lake water.
Other studies of GEMs in freshwater systems indicate that
GEMs can persist for long periods of time and may in fact
become part of the autochthonous microbial community (Amy
and Hiatt, 1989; Steffan et al., 1989; Trevors et al.,
1989) .
Ground water systems have been shown to harbor
significant, physiologically stressed bacterial populations
(White et al., 1983; Wilson et al., 1983) and is usually
considered a stressful, low nutrition environment. However,
in this study the introduced organisms and their plasmids
survived well in ground water. Bitton et al. (1983) also
reported high survival rates for bacterial pathogens in
ground water. In addition to maintaining a stable
population, growth was even observed during the first 48-72
hours of exposure to the groundwater. This could likely be
due to the presence of trace amounts of nutrients that were
not removed after washing of the cells. High population
densities (104 105) were maintained after 21 days even in
the absence of selective pressure. This would indicate that
the introduced organisms adapted well to starvation
conditions and that the presence of additional DNA did not
adversely affect the metabolic and hence survival


118
numbers was observed for both strains. In a similar
experiment that utilized a higher initial concentration (6.5
x 106) of bacteria, the same trend was observed (Figure 5-3
and 5-4) for both £. coli and £. putida. respectively. It
appears that growth occurs until an optimal density
(approximately 107) is reached, regardless of the initial
inoculum concentration.
Comparison of the Growth Patterns of Wildtype and GEM
strains in the Presence of Corn-root Exudates
The growth patterns of wildtype and recombinant strains
of E. coli and P. putida were compared in the presence of
growing corn roots. The initial bacterial concentrations
were adjusted to approximately 105 CFU/ml. Figure 5-5 and
5-6 show the growth pattern of wildtype and recombinant
strains of £. coli and P. putida. respectively, following 24
hour and 92 hour incubation in the presence of root
exudates. Both wildtype and recombinant strains exhibited an
almost two-log unit increase in cell numbers after the first
24 hours of incubation. No significant difference (p<0.05)
could be observed between the growth patterns of wildtype
and recombinant strains for either E. coli and P. putida.
Survival of GEMs in a Simulated Rhizosphere in the Presence
of Indigenous Rhizospheric Microorganism
The effect of indigenous rhizospheric microorganisms on
the population dynamics of genetically engineered strains
is shown in Figures 5-7 to 5-10. The initial concentration


29
of the recombinant strains. Microcosms consisted of 5-liter
conical flasks containing filtered or untreated lake water.
Following release of the GEMs into sterile lake water, the
organism persisted, but the marker phenotype and genotype
declined to undetectable levels. Production of a viable but
nonculturable population was not observed. In both the
sterile and untreated lake water, both the xvlE gene and the
product, C230 protein, decreased over time but declined
more rapidly in the sterile lake water. The results indicate
that the metabolic burden from overexpression appears to
affect maintenance of the plasmid in released hosts in
sterile lake water.
Chaudhry et al. (1989) derived an alternative method
for directly detecting and monitoring the fate of GEMs in
the environment. In-vitro amplification of target DNAs was
achieved by polymerase chain reaction (PCR) and then
hybridized to a specific oligonucleotide or DNA probe.
Comparisons with selective plating methods indicated that
whereas no viable GEMs were detected after 6 and 10 days of
incubation in lake water and raw sewage, respectively, the
PCR amplification method detected cells for up to 10 and 14
days, respectively. This indicated the presence of cells
that had lost their ability to grow on selective media but
still carried the marker gene.
The survival of genetically engineered Erwinia
carotovora L-864, with a kanamycin resistance gene inserted
in its chromosome, was monitored in pond water and sediment


215
Lehninger, A.L. 1982. Principles of Biochemistry. Worth
Publishers, Inc. New York.
Lenski, R.E., and T.T. Nguyen. 1988. Stability off
recombinant DNA and its effects on fitness.
In J. Hodgson and A.M. Sugden (eds.), Planned Release
of Genetically Engineered Organisms. Trends in
Biotechnology/Trends in Ecology and Evolution Special
Publication. Elsevier, Cambridge, England, pp. 18-20.
Lessard, E. J., and J. M. Sieburth. 1983. Survival of
natural sewage populations of enteric bacteria in
diffusion and batch chambers in the marine environment.
Appl. Environ. Microbiol. 45:950-959.
Lewin, B. 1985. Genes III. John Wiley & Sons, Inc., New
York.
Liang, L. N., J. L. Sinclair, L. M. Mallory, and M.
Alexander. 1982. Fate in model ecosystems of microbial
species of potential use in genetic engineering. Appl.
Environ. Microbiol. 4:708-714.
Lopez-Torres, A., L. Prieto, and T.C. Hazen. 1988.
Comparison of the in situ survival and activity of
Klebsiella pneumoniae and Escherichia coli in tropical
marine environments. Microb. Ecol. 15:41-57.
Lorenz, M.G., and W. Wackernagel. 1987. Adsorption of DNA to
sand and variable degradation rates of adsorbed DNA.
Appl. Environ. Microbiol. 53:2948-2952.
Lugtenberg, B., R. Perters, H. Bernheimer, and W. Berendsen.
1976. Influence of cultural conditions and mutations on
the composition of the outer membrane proteins of
Escherichia coli. Mol. Gen. Genet. 147:251-262.
Lynch, J.M. 1982. Interactions between bacteria and plants
in the root environment. In M.E. Rhodes-Roberts and
F.A. Skinner (eds), Bacteria and plants. Academic
Press, N.Y., pp. 1-20.
Lyon, F., J. Fitzgerald, J. Brandau, and M. Walter. 1988.
Use of 2,3-catechol dioxygenase for the detection of
transconjugants recovered from environmental samples.
REGEM 1, Cardiff, p. 22. (Abstract).
Mach, P.A., and D.J. Grimes. 1982. R-plasmid transfer in a
wastewater treatment plant. Appl. Environ. Microbiol.
44:1395-1403.


157
In this study, changes in cell permeability were
indirectly determined by observing changes in sensitivities
to the toxicant PCP. Both strains showed significantly
differently trends in cell permeability over time. The
wild-type strain exhibited a steady increase in cell
permeability to a hydrophobic compound throughout the
experiment whereas the engineered strain showed an initial
decline which was then followed by increased cell
permeability after 3 days. The altered permeability of the
cell membrane might modify the nutrient uptake efficiency of
the cells and thus enhance the capacity, for survival in
natural systems. Alterations in cell permeability may
therefore be beneficial to or necessary for the survival of
cells (Chai, 1983).
As shown with other bacteria, adaptation to starvation
stress involves more or less extensive physiological
modifications and some alterations of cell envelopes. The
results of this study are noteworthy in view of the fact
that morphological and physiological alterations still
occurred in both strains of bacteria after exposure to lake
water which is not considered a nutrient poor environment.
The adaptations observed are more likely to be stress-
related rather than nutrient-related. Lake Alice is a
hypereutrophic lake that serves as a receiving body for
wastewater effluent and storm runoff, and is therefore
likely to contain a variety of chemicals and pollutants that-


198
two strains. This would suggest that the presence of the
mobilizing genes cloned into pRC92 causes an increase in the
transfer of the plasmid to recipient cells. Conway et al.
(1987) also reported increased transfer frequencies with
cloning vectors containing the mob gene.
No transconjugants were observed after 20 and 72 hours
of incubation in lake water. The lack of transconjugants,
however, does not preclude the possibility of genetic
transfer occurring in lake water. The results obtained in
this study are representative of a single experiment at two
sampling intervals. It has been shown that in vitro
conjugation studies utilizing rich media generally produce
higher transfer frequency rates than in situ studies
involving environmental samples (Gealt et al., 1985; Van
Elsas et al., 1987). Therefore, it is highly possible that
under different environmental conditions and by increasing
the sampling intervals, transconjugants could be detected in
a lake water study.
Recent studies have demonstrated genetic transfer
between GEMs and other bacteria via conjugation (Bleakley
and Crawford, 1989; Knudsen et al., 1988; Mancini et al.,
1987; McClure et al., 1989; Richaume et al., 1989; Walter et
al., 1989; Van Elsas et al., 1987). The majority of these
studies focused on soil systems and utilized simple
microcosm settings to determine gene transfer. To date,
there have been few reported studies on genetic transfer


75
TABLE 4-2. Effect of temperature on the rates of
population change (log,g/day) of genetically
engineered and wild-type P. putida under sterile
conditions in lake water.
Temperature Media Type
(C)
Pseudomonas putida
wild-type GEM
15
LB
VO
o

o
1
-0.08
LB
+
Tet
+
Km
ND
-0.08
25
LB
-0.19
-0.09
LB
+
Tet
+
Km
ND
-0.09
30
LB
-0.21
-0.10
LB
+
Tet
+
Km
ND
-0.10
ND indicates not applicable


83
Plasmid stability. The stability of the engineered
genes was determined by plating the genetically engineered
strains on both selective and nonselective media. The
results (Figures 1-1 to 4-3) indicated that the engineered
genes were relatively stable, with no loss occurring up to
19 days of incubation at 15, 25 or 30C. There was no
significant difference between the rates of population
change (Tables 4-1 and 4-2) for strains grown on either the
selective or non-selective media, at either of the three
temperature regimes.
Survival and Plasmid Stability in Activated Sludge Effluent
Temperature-dependent studies The effect of
temperature on the survival pattern of wild-type and
genetically engineered strains of E. coli and P. putida in
activated sludge effluent is shown in Figures 4-6 to 4-11.
Rates of population change (loglO(CFU)/day) for E. coli. and
P. putida are summarized in Tables 4-3 and 4-4,
respectively. At 15C, E. coli exhibited similar rates of
decline for both wild-type (-0.04) and engineered (-0.03)
strains. Both strains survived well at 15C and showed only
a slight decrease in numbers after 14 days of incubation
(Figure 4-6). Higher rates of decline were noted for P.
putida strains at 15C (Figure 4-7). The rate of population
decline for the wild-type strain, 50014 (-0.13) was not
significantly greater than that of the engineered strain,
50058 (-0.10). The survival patterns of the two strains,
however, indicate that while the wild-type strain survived


25
the survival of recombinant strains of Pseudomonas spp. and
Escherichia coli.
Van Elsas et al. (1989) investigated the effect of soil
texture on survival and plasmid stability of Pseudomonas
fluorescens. Soil type was found to significantly affect
host survival and plasmid maintenance. Higher survival and
stability were observed in heavier-textured soil. There was
no detectable effect of plasmid type (conjugative vs
nonconjugative) on host survival.
Intact soil-core microcosms have been successfully
used to study the fate and ecological impact of Tn-5 mutants
under varying conditions (Bentjen et al., 1989; Fredrickson
et al., 1988; 1989). The design of the microcosms maintains
many of the features useful for evaluating GEM transmission
through the ecosystem, including colonization of the
rhizosphere and endorhizosphere, carriage by insect vectors,
potential displacement of other rhizospheric microorganisms,
and effects on plant growth. In all cases, the population of
the introduced Tn-5 mutants declined slowly over time in the
surface soil, but colonized the rhizosphere and rhizoplane
throughout the 60-cm soil-core depth. The Tn-5 mutants were
transported through the core with percolating water and were
present in the gut of earthworms (Fredrickson et al., 1989).
Inoculation of Tn-5 mutants had no effects on neither plant
leaf nitrogen concentration nor niche displacement of
rhizospheric populations (Bentjen et al., 1989).


100
burden for the organism, and may actually improve the
fitness of the cell in certain environments (Devanas and
Stotzky, 1986). It has been shown that the nonessential cos
fragment of lambda phage actually enhances maintenance of
the host and plasmid (Edlin et al., 1984). Biel and Hartl
(1983) also reported on the ability of a Tn5 mutant E. coli
to outcompete the wild-type strain under nonselective
conditions. Wang et al. (1989) observed significantly better
survival of a recombinant strain of Streptomvces over its
corresponding nonrecombinant parent. These reports are
consistent with results obtained in this study. The presence
of foreign DNA may also cause physiological changes in the
host such as increased virulence, enhanced antibiotic
resistance and changes in the utilization of carbohydrates,
amino acids and organic acids (Kozyrovskaya et al., 1984).
More recent studies dealing with the fate of
genetically engineered microorganisms in the environment
also suggest that additional DNA has little effect on
bacterial survival. Walters et al. (1987) noted
inconsistent survival patterns between GEMs and their parent
strains in soil extracts. Their study concluded that
recombinant DNA plasmids had no significant effect on the
survival of the recombinant strains tested. Dywer et al.
(1988) also noted no significant difference in survival
between a genetically engineered Pseudomonas sp. and its
parent strain in activated sludge. Yeung et al. (1989)
observed that despite high levels of enzyme activity by the


55
Figure 3-6. Growth curve and corresponding optical density
values (A55q) of wild-type Pseudomonas putida
(50058), grown at 27C.
OPTICAL DENSITY


46
AQUARIUM TANK
Figure 3-2. Schematic diagram of the static-renewal
microcosm.


151
B-galactosidase biosynthesis, respectively. This suggests
increased cell permeability to the toxicant PCP over time.
The engineered strain shows a different trend in
sensitivity to the chemical PCP. At the onset of the
experiment, the strain was shown to be sensitive to PCP
which produced a 70% inhibition in B-galactosidase
production. After 1 and 3 days of exposure to the lake
water, the cells became less sensitive to the PCP (Figure
6-7). B-galactosidase inhibition was reduced to 25% after 24
hours and actually produced a stimulatory effect after 3
days of exposure. This trend is in direct contrast to that
observed for the non-engineered strain during the first 3
days. However, after 6 and 9 days of exposure to the lake
water, a reverse trend was noted for the engineered strain.
Sensitivity of the cells increased with time and by day 9,
the % inhibition was back to the original value of 70%. The
results of this experiment indicate an initial decrease
followed by an increase in cell permeability over time for
the engineered strain. At the end of the experiment, both
strains showed no significant difference (p<0.05) in cell
permeability with respect to % inhibition of B-galactosidase
biosynthesis by the toxicant PCP (45ppm).
Morphological changes
Direct epifluorescent microscopic observations revealed
morphological changes in cell structure. After 6 days of
exposure to lake water, approximately 50% of the cells


166
Results
Efficiency of the DNA Recovery Method
Initial studies indicated a high recovery of the added
DNA. Visual comparisons of ethidium bromide stained DNA
after gel electrophoresis indicated close similarities
between the recovered DNAs and their theoretical
concentrations. Optical density measurements (260nm) also
indicated high recoveries (89-91%) of extracellular DNA.
DNA Degradation: Characterization by Gel Electrophoresis
The degradation of eDNA was visually characterized by
gel electrophoresis (Figures 7-1 to 7-4). The figures show
structural changes in the eDNA over time. The eDNA is
characterized by five distinct bands on the gel (Figure 7-1,
Lane A). The highest band (>23.1 kb) represents chromosomal
DNA while the lower four bands represents the different
forms of plasmid pBR322. At time zero, there is very little
linear DNA (third band) as compared to the open circular
(OC; second band) and covalently closed circular (CCC;
fourth and fifth bands) forms. The majority of the plasmid
pBR322 inoculum occurred as the CCC form.
Raw Sewage
The eDNA was rapidly degraded within 15 minutes of
incubation with untreated raw sewage (Figure 7-1, Lane 0).
After fifteen minutes, chromosomal DNA and the CCC forms
were no longer present. The increased intensity of the OC
and linear bands indicate that the CCC forms were degraded


220
Roszak, D.B., D.J. Grimes, and R.R. Colwell. 1984. Viable
but nonrecoverable stage of Salmonella enteritidis in
aquatic systems. Can. J. Microbiol. 30:334-338.
Russell, R.S. 1980. Plant Root Systems: Their Function and
Interactions with the Soil. McGraw-Hill Book Company
Limited, London, U.K.
Saunders, V.A., and J.R. Saunders. 1987. Microbial Genetics
Applied to Biotechnology. Macmillan Publishing Company,
New York.
Saye, D.J., 0. Ogunseitan, G.S. Sayler, and R.V. Miller.
1987. Potential for transduction of plasmids in a
natural freshwater environment: effect of donor
concentration and a natural microbial community on
transduction in Pseudomonas aeruginosa. Appl. Environ.
Microbiol. 53:987-995.
Saye, D.J., 0. Ogunseitan, G.S. Sayler, and R.V. Miller.
1990. Transduction of linked chromosomal genes between
Pseudomonas aeruginosa strains during incubation in
situ in a freshwater habitat. Appl. Environ. Microbiol.
56:140-145.
Sayler, G.S., M.S. Shields, E.T. Tedford, A. Breen, S.W.
Hooper, K.M. Sirotkin, and J.W. Davis. 1985.
Application of DNA-DNA colony hybridization to the
detection of catabolic genotypes in environmental
samples. Appl. Environ. Microbiol. 49:1295-1303.
Scanferlato, V.S., D.R. Orvos, J. Cairns, Jr., and G.H.
Lacy. 1989. Genetically engineered Erwinia carotovora
in aquatic microcosms: survival and effects on
functional groups of indigenous bacteria. Appl.
Environ. Microbiol. 55:1477-1482.
Scheuerman, P. R., J. P. Schmidt, and M. Alexander. 1988.
Factors affecting the survival and growth of bacteria
introduced into lake water. Arch. Microbiol. 150:320-
325.
Shaw, J.J., and C.I. Kado. 1986. Development of a Vibrio
bioluminescence gene-set to monitor phytopathogenic
bacteria during the ongoing disease process in a non-
disruptive manner. Biotechnology 4:560-564.
Sherr, E.B., and B.F. Sherr. 1987. High rates of consumption
of bacteria by pelagic ciliates. Nature 325:710-711.
Sjogren, R.E., and M.J. Gibson. 1981. Bacterial survival in
a dilute environment. Appl. Environ. Microbiol.
41:1331-1336.


9
Recent methodologies in genetic transformation. In
recent years, new techniques have been reported for the
introduction of DNA into organisms. Electroporation is a
technique that renders cell membranes temporarily permeable
to macromolecules, such as DNA and proteins, by exposing
cells to brief electrical pulses of high field strength
(Chassy and Flickinger, 1987; Fiedler and Wirth, 1988).
Microinjection of DNA into the nuclei of protoplasts has
also been developed recently as an efficient method of gene
transfer (Reich et al., 1986). A significant development
over microinjection is the use of electric field pulses to
introduce the foreign DNA into the cells. This
'electroinjection' technique bypasses the necessity to
isolate protoplasts (Morikawa et al. 1986). The use of
artificial lipid vesicles (liposomes) and spheroplasts that
encapsule or contain plasmid DNA are also being considered
for transferring DNA to host cells (Cocking and Davey,
1987) .
Part II: Applications of Recombinant DNA Products
Introduction
The potential uses and applications of rDNA products or
genetically engineered organisms are wide and varied. Newly
constructed genotypes are believed to be of value not only
in many areas of basic and applied research, but also for
economic exploitation. Recombinant DNA technology has been
long used in the manufacture of pharmaceuticals and


63
PlMk (50014)
Chamber* (50014)
-+- Flaek (50058)
-9- Chamber* (50058)
Figure 3-12. Comparison of survival studies of wild-type
and genetically engineered strains of Pseudomonas putida
in lake water at 25C, using flask microcosms
and survival chambers.


144
LOG NUMBER OF CELLS
LOG NUMBER OF CELLS
TIME (DAYS)
Figure 6-3. Survival of wild-type (HB101) and genetically
engineered (50008) strains of Ecsherichia coli after
exposure to lake water at 25C, by viable plate counts and
acridine orange direct counts (AODC). A:HB101; B:50008.


149
120
100
80
60
40
20
0
DEHYOROGENASE ACTIVITY
0 13 6 9
TIME (OAYS)
Figure 6-6. Changes in INT-dehydrogenase activity of
wild-type (HB101) and genetically engineered (50008)
strains of Escherichia coli after exposure to lake
water at 25C.Results are expressed as percent
reduction in dehydrogenase activity relative to time zero.


CHAPTER 2
LITERATURE REVIEW
Part 1. Recombinant DNA Technology
Introduction
Recent developments in recombinant DNA biotechnology
offers the potential for unrestrained rearrangement of the
genetic information present in organisms. This allows the
opportunity to custom design organisms to accomplish
specific tasks.
Genetic modifications that are readily envisioned as
being useful to the biotechnology industry include 1)
amplification of the levels of specific enzymes in
microorganism; 2) the rearrangement of regulating DNA base
sequences controlling the expression of specific genes in
response to specific stimuli; 3) the introduction of genes
for new enzymatic functions into organisms which do not
normally possess them and 4) the modification of individual
genes to alter the characteristics of individual enzymes
(Johnston and Robinson, 1984).
Genetic Manipulation
During the course of the last two decades, several
techniques have been developed to aid genetic manipulation.
These are 1) in-vitro recombinant DNA technologies, 2) in-
5


49
50008) was obtained by inserting an EcoRl DNA fragment from
pRCIO into a derivative of pBR322. The EcoRl fragment
contained the gene for mercury resistance and the genes
involved in 2,4-D degradation (Chaudhry and Huang, 1988).
The recombinant plasmid was then cloned into HB101. Presence
of the recombinant plasmid was first tested by growth on LB
agar plates containing 40 ug/ml and 50 ug/ml of HgCl2 and
ampicillin, respectively, and subsequently confirmed by
plasmid isolation and restriction analysis. The engineered
strain of P. putida (strain 50058) harbored the plasmid
R68.45 which conferred resistance to carbenicillin,
kanamycin and tetracycline (Haas and Holloway, 1976).
Bacterial growth studies. In order to accurately
determine the initial inoculum concentration for survival
studies, it was necessary to conduct growth studies that
would correlate optical density readings with bacterial
concentrations (CFUs/ml). Wildtype strains of E. coli and
P. putida were grown in Luria-Bertani (LB) broth at 35C and
28C, respectively. Genetically engineered E. coli was grown
in LB broth supplemented with HgCl2 (40 ug/ml), while
engineered P. putida was grown in LB broth containing
tetracycline (15 ug/ml) and kanamycin (50 ug/ml). Growth
curves were constructed by inoculating 50 ml of the
appropriate media with an overnight culture of the bacterial
strain and incubating at the necessary temperature. At
specific time intervals, samples were aseptically withdrawn


125
Figure 5-8. Survival of genetically engineered Escherichia
coli in the presence of rhizospheric microorganisms (RM),
with corn roots present. ( )i total microflora
(E. coli + RM) ; ( ) EL. coli in the absence of RM;
(O), EL coli in the presence of RM.


155
time. This would suggest a possible competitive advantage of
the engineered strain over the non-engineered strain during
the first 24 hours of exposure to the environment.
B-galactosidase biosynthesis also showed a substantial
decline over time for both strains of E. coli. However,
rates of decline were significantly different between the
two strains. The genetically engineered strain rapidly lost
its potential for B-galactosidase biosynthesis as compared
to the wild-type strain, which still maintained 20% of its
initial activity after 9 days. Anderson et al. (1979)
observed similar declines in B-galactosidase activity with
increasing starvation time and salinity in cells exposed to
seawater. Munro et al. (1987) studied metabolic
modifications of E. coli cells during starvation in seawater
and observed that while B-galactosidase activity disappeared
gradually over time, other enzyme activities, such as
alkaline phosphatase, increased with time. The decline in
B-galactosidase biosynthesis may be accounted for by die-off
of the cells, catabolic repression, reduction in lactose
permease activity, repression of the lactose operon, or a
combination of the above (Anderson et al., 1979). Results of
this study suggest that die-off alone did not account for
the reduced specific activities since survival trends were
different from that observed for B-galactosidase
biosynthesis. No attempt was made to measure reduction in
lactose permease. The rapid decline in B-galactosidase
biosynthesis by the engineered strain may be considered an


TABLE 4-3. Effect of temperature on the rates of
population change (log10/day) of genetically
engineered and wild-type E. coli under sterile
conditions in activated sludge effluent.
Temperature
(C)
Media Type
Escherichia
wild-type
coli
GEM
15
LB
-0.04
-0.01
LB
+ HgCl2
ND
-0.03
25
LB
-0.07
-0.04
LB
+ HgCl2
ND
-0.06
30
LB
l
o

o
-0.11
LB
+ HgCl2
ND
-0.24
ND indicates not applicable


28
Aquatic Systems
Survival studies of GEMs in the aquatic environment
have been conducted using mainly closed or static
microcosms, ranging in size from simple 30-ml culture flasks
(Trevors et al., 1989) to larger 20-liter glass carboys
(Steffan et al., 1989). The potential uses of flowthrough
microcosms have also been reported (Fulthorpe and Wyndham,
1989; McClure et al., 1989). A variety of detection and
enumeration methodologies have also been employed for
studying survival and genetic stability in GEMs.
Amy and Hiatt (1989) used a combination of selective
plating and DNA probing to detect the survival of GEMs in
lake water. Survival of GEMs remained high in filtered
(0.22-um pore size) lake water but was lower in untreated
lake water and lake water filtered with 0.8-um pore size
membrane. Total recoverable bacteria were greater in the
0.8-um filter-treated samples than the untreated samples,
suggesting grazing by zooplankton and microplankton. The
recombinant plasmid was retained in all experiments
regardless of whether plasmid DNA was of benefit to the
cells.
The fate of recombinant pseudomonads released into lake
water was determined by a series of direct membrane filter
methods developed for direct phenotypic and genotypic
detection (Morgan et al., 1989). Recombinant plasmids
encoding a xvlE marker gene encoding catechol 2,3-
dioxygenase (C230) facilitated the detection and enumeration


182
CCC form appeared to be first converted to the OC form
which was then followed by a change into the linear form.
Chromosomal DNA disappeared rapidly and was completely
degraded in some samples long before plasmid DNA was
degraded. It appears that the most stable form of eDNA is
the OC and linear forms of plasmid DNA. The rapid
disappearance of chromosomal DNA can be attributed to it's
large size (>23 kb).
An interesting observation in this study was the fact
that the results obtained via gel electrophoresis did not
correlate or follow the trend shown using optical density
measurements. The gradual degradation of chromosomal and
plasmid DNAs that was observed via gel electrophoresis
(Figures 7-1 to 7-4) was actually shown as an increase in
optical density measurements that eventually decline over
time (Figures 7-4 to 7-8). Of significance is the fact that
optical density measurements were still high even after the
bands disappear from the gel. This discrepancy is due to the
fact that the absorbance value at 260nm is indicative of
total nucleic acid and is therefore reflective of smaller
DNA fragments not detectable by the gel electrophoresis
method used in this study. The initial increase in optical
density values was probably due to the denaturation of the
DNA. Denatured DNA gives higher absorbance readings at 260nm
than the same concentration of non-denatured DNA (Johnson,
1981; Lewin, 1985).


203
conditions of the activated sludge effluent decreased the
fitness of the GEM, and hence it's ability to survive.
5. The presence of indigenous lake organisms caused a
significant decline in numbers of both GEMs. The results of
this study indicate that E_¡_ coli did not survive as well as
P. putida in the presence of indigenous organisms. After an
initial decline, P_j_ put ida was able to stabilize it
population density in the presence of the indigenous
population. Ej_ coli however, was totally eliminated from the
system within 20 days.
6. The herbicide, Hydrothol-191, produced a significant
decline in the numbers of both GEM and wild-type strains of
P. putida. at a concentration of 1 mg/L. No significant
difference was noted between the GEM and wild-type strain,
indicating that the presence of extraneous DNA did not
afford any advantage or disadvantage to the organism in the
presence of a toxicant.
7. Genetic determinants of the GEMs remained stable under
most of the conditions tested. With the exception of
engineered coli in activated sludge effluent at 30C,
there was no significant difference in numbers when the test
bacteria were plated on selective and non-selective media.
This lack of difference indicated the relative stability of
the introduced genes. Stability of the recombinant genes
will depend on a variety of factors such as temperature and
nutrient conditions.


79
and 50058 were -0.21 and -0.10, respectively. After 19 days,
wild-type P. putida decreased by almost 4 log, as compared
to the engineered strain which showed only a 2 log decrease.
The difference in the survival rates between the two strains
was highly significant (p<0.005). The two strains of E¡. coli
also exhibited similar trends. Rates of population change
for E. coli HB101 and 50008 were -0.19 and -0.13,
respectively. The wild-type strain showed a greater decline
(4 log) than the engineered strain (2.5 log). This
difference was shown to be highly significant (pco.001).
Sterile vs nonsterile conditions. The effect of the
indigenous lake water microorganisms on the population
dynamics of the genetically engineered strains is shown in
Figure 4-4. The arrows indicate the time at which the
indigenous microorganisms were introduced into the chambers.
Prior to the addition of lake water into the chambers, both
strains showed an increase in numbers from an initial
concentration of approximately 3.0xl03 to 1.0x10s for P.
putida and 2.0xl03 to 5.0xl04 for E. coli. Addition of
indigenous microorganisms caused an initial sharp decline
in numbers for both species. However, P. putida numbers
remained stable after three days in the presence of the
indigenous organisms, unlike E. coli which showed a steady
decline and was totally eliminated after 21 days. Under
sterile conditions, rates of decline were lower and a higher
population density was maintained. The rates of population
change at 25C for E. coli under sterile (-0.07) and non-


TABLE 4-4. Effect of temperature on the rates of
population change (log^/day) of genetically
engineered and wild-type P. putida under sterile
conditions in activated sludge effluent.
Temperature Media Type
(C)
Pseudomonas putida
wild-type GEM
15
LB
-0.13
-0.10
LB
+
Tet
+
Km
ND
-0.10
25
LB
-0.12
-0.10
LB
+
Tet
+
Km
ND
-0.10
30
LB
-0.10
-0.14
LB
+
Tet
+
Km
ND
-0.14
ND indicates not applicable


51
and E. coli were enumerated on (1) LB media; (2) full-
strength selective media (LB plus tetracycline (15 ug/ml)
and kanamycin (50 ug/ml) and LB plus HgCl2 (40 ug/ml)
respectively); (3) half-strength selective medium; and (4)
quarter-strength selective medium.
Comparison of Microcosms for Survival Studies
Many survival studies, including those of GEMs, have
been conducted in simple flask microcosms (Liang et al.,
1982; Morgan et al., 1989). Although these microcosms are
simple to maintain and easy to work with, there are
potential problems and disadvantages associated with them.
Closed systems create a potential for "bottle effects" that
can arise over time. Such systems are also closed to
nutrient and chemical inputs that may be associated with
the ecosystem. The microcosm that was developed for this
research eliminates much of the problems associated with
closed microcosms.
A comparative study between a "closed" flask microcosm
and the "open" flowthrough microcosm described above was
used to determine the extent to which the two types of
microcosms could influence survival rates of introduced
organisms. Genetically engineered and wildtype P. putida
were used as the test organisms. Bacterial cultures were
grown until late logarithmic phase and harvested by
centrifugation at 8000 g for 10 minutes at 4C. The cells
were washed three times with sterile phosphate buffer (pH
7.2) and resuspended in filter-sterilized lake water for use


18
developed for monitoring rDNA products in the environment
(Jain et al., 1988) .
Conventional Methods. The conventional methods of
enumerating microorganisms include 1) selective plating and
enrichment techniques, 2) Most-Probable-Number (MPN)
technique and 3) epifluorescence count technique. Selective
plating and enrichment techniques are perhaps the most
commonly used methods for enumeration. GEMs carrying
specific selectable marker genes can be selected and
enumerated on appropriate agar media. The majority of
current studies utilize GEMs carrying antibiotic resistance
(Armstrong et al., 1987; Devanas et al., 1986; Trevors et
al., 1989; Walter et al., 1989; Fredrickson et al., 1989).
Alternate markers which have been proposed include cloned
lacZY genes in fluorescent pseudomonads (Drahos et al.,
1986); lux genes enabling bacteria to emit light (Shaw and
Kado, 1986); cloned genes for prodigiosin (red pigment)
biosynthesis (Davenhauer et al., 1984); and cloned xvlE gene
which encodes for 2,3-catechol dioxgenase (Lyon et al.,
1988; Morgan et al., 1989).
MPN procedures for bacterial enumeration are widely
used in the area of public health. The procedure has also
being utilized for different microbial groups such as
nitrifying bacteria (Rennie, 1978) and rhizobia (Weaver and
Frederick, 1982). Application of the MPN method for
enumeration of GEMs has been reported in few studies.
Bentjen et al. (1989) and Fredrickson et al. (1988; 1989)


88
as well as the engineered strain during the first 4 days
of incubation, a sharp decline did occur before it could
stabilize its population (Figure 4-7).
At 25C, both E. coli and P. putida strains exhibited
survival patterns similar to those shown at 15C. E). coli
populations remained stable for up to 14 days with little
decrease in numbers (Figure 4-8) No significant difference
was noted between the survival rates of the wild-type
(-0.07) and recombinant (-0.06) strains. P. putida strains
also exhibited similar rates of decline at both 15 and 25C
(Table 4-4) The rate of population change for the wild-type
strain (-0.12) was not significantly greater than that of
the engineered strain (-0.10). Unlike the trend noted at
15C, the wild-type strain did not survive as well as its
engineered counterpart at the start of the experiment but
instead showed a steady decline throughout the experiment
(Figure 4-9).
The greatest rates of decline were observed at 30C.
Both strains of E. coli decreased in numbers after 14 days
incubation (Figure 4-10), with the engineered strain showing
a greater decline (3 log) than the wild-type strain (1 log).
The rate of population change was significantly higher
(p<0.01) for the engineered strain (-0.24) than that of the
wild-type strain (-0.09). The recombinant strain of P.
putida also exhibited a greater decline in numbers at 30C
(Figure 4-11) as compared to 15 and 25C. Both strains
showed a 2 log decrease in numbers after 21 days, and their


4
The specific objectives of this research were as
follows:
1. To determine the fate and survival of genetically
engineered bacteria and their wild-type strains in aquatic
systems (lake water, activated sludge effluent and ground
water) and the corn rhizosphere in controlled environmental
settings.
2. To monitor the stability of the recombinant DNA
within the host organism in the above mentioned systems.
3. To determine and compare structural and
physiological alterations of GEMs and their wild-type
strains after exposure to aquatic environments.
4. To determine the fate of extracellular DNA in
aquatic samples and its potential for genetic transfer.
5. To construct a model GEM for studying genetic
transfer via conjugation.
The following null hypotheses were tested:
1. No significant difference in survival rates between
the GEMs and wild-type strains under given environmental
conditions.
2. Genetic transfer does not occur between GEMs and
other types of bacteria.
3. Recombinant DNA of the GEMs are not stable under
given environmental conditions.
4. No physiological differences between GEMs and wild-
type strains after environmental exposure.


114
Survival and Growth Studies
The hydroponic cultures described above were inoculated
with 1 ml of the bacterial suspensions to obtain a final
bacterial concentration of 105-106 bacteria/ml. Three tubes
were inoculated per treatment and per sample. The inoculated
tubes were gently shaken with a vortex stirrer and then
incubated in the dark at 25C. Bacterial numbers were
determined after various periods of incubation by the
microdrop method of Hoben and Somassegaran (1982). All
treatments were conducted in triplicate.
Interaction between the Genetically Engineered Bacteria and
Rhizospheric Microorganisms
Corn plants were carefully removed from field plots on
the University of Florida campus and the roots gently shaken
to eliminate non-rhizospheric soil. The roots were then
gently shaken for 3 hours in sterile distilled buffer for
removal and collection of indigenous rhizospheric
microorganisms. The organisms were concentrated by
centrifugation for 15 minutes at 7000 g, washed twice with
phosphate buffer, and resuspended in Hoagland/s medium. Each
tube received 0.5 ml of the final suspension of rhizospheric
microorganisms prior to inoculation with the genetically
engineered strains of E. coli or P. putida.
Preliminary experiments were undertaken to determine
the best medium for the selective growth of E. coli Hgr in
the presence of indigenous rhizospheric microorganisms.
Among several selective media tested, LB agar supplemented


96
Time (hours)
Figure 4-12. Survival of genetically engineered (GEM)
and wild-type strains of Escherichia coli at 22C
in groundwater. Wild-type and GEM strains recovered
on non-selective medium; (--) and H) respectively.
GEM strains recovered on selective medium (-#-) .


211
Fredrickson, J.K., D.F. Bezdicek, F.J. Brockman, and S.W.
Li. 1988. Enumeration of Tn-5 mutant bacteria in soil
by using a most-probable-number-DNA hybridization
procedure and antibiotic resistance. Appl. Environ.
Microbiol. 54:446-453.
Fulthorpe, R.A., and R.C. Wyhdham. 1989. Survival and
activity of a 3-chlorobenzoate catabolic genotype in a
natural system. Appl. Environ. Microbiol. 55:1584-1590.
Gaskins, M.H., and D.H. Hubbell. 1979. Response of
nonleguminous plants to root inoculation with free
living diazotrophic bacteria. In J.L. Hartley and
R.S. Russell (eds), The Soil-Root Interface. Academic
Press, New York, pp. 177-184.
Gauthier, M.J., P.M. Munro, and V.A. Breittmayer. 1988.
Influence of prior growth conditions on low nutrient
response of Escherichia coli in seawater. Can. J.
Microbiol. 35:379-383.
Gauthier, M.J., P.M. Munro, and S. Mohajer. 1987. Influence
of salts and sodium chloride on the recovery of
Escherichia coli from seawater. Current Microbiol.
15:5-10.
Gealt, M.A., M.D. Chai, K.B. Alpert, and J.C. Boyer. 1985.
Transfer of plasmids pBR322 and pBR325 in wastewater
from laboratory strains of Escherichia coli to bacteria
indigenous to the waste disposal system. Appl. Environ.
Microbiol. 49:836-841.
Germida, J.J., and G.G. Khachatourians. 1988. Transduction
of Escherichia coli in soil. Can. J. Microbiol.
34:190-193.
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Andow, and M. Alexander. 1985. Potential Impacts of
Environmental Release of Biotechnology Products:
Assessment, Regulation and Research Needs. Ecosystem
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growth environment on the stability of a drug
resistance plasmid in Escherichia coli K12. J. Gen.
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Goodgal, S.H. 1982. DNA uptake in Haemophilus
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Gowland, P.C., and J.H. Slater. 1984. Transfer and stability
of drug resistance plasmids in Escherichia coli K12.
Microb. Ecol. 10:1-13.


89
Log CFU/ml
Time (hours)
Figure 4-8. Survival of genetically engineered (GEM)
and wild-type strain of Escherichia coli at 25C
in activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; ( ) and (() >
respectively. GEM strains- recovered on
selective medium .


223
Walter, M.V., L.A. Porteous, and R.J. Seidler. 1989.
Evaluation of a method to measure conjugal transfer of
recombinant DNA in soil slurries. Curr. Microbiol.
19:365-370.
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Measuring genetic stability in bacteria of potential
use in genetic engineering. Appl. Environ. Microbiol.
53:105-109.
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Survival and effects of wild-type, mutant, and
recombinant Streptomvces in a soil ecosystem. Can. J.
Microbiol. 35:535-543.
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107
Genetic determinants of the GEMs remained stable under
most of the conditions tested. With the exception of
engineered E. coli. 50008, in activated sludge effluent at
30C, there was no significant difference in numbers when
the test bacteria were plated on selective and non-selective
media. This lack of difference would indicate the relative
stability of the introduced genes. Several studies have
reported no apparent loss of plasmids or recombinant DNA
from genetically engineered strains in environmental samples
(Amy and Hiatt, 1989; Caldwell et al., 1989; Devanas et al.,
1986; Jain et al., 1989; Trevors et al., 1989; Van Elsas et
al., 1989; Wang et all., 1989; Yeung et al., 1989;). Other
reports, however, have demonstrated loss of plasmid or
partial deletion of DNA sequences after exposure to
environmental samples. Fulthorpe and Wyndham (1989) reported
that a catabolic element, dpb, that coded for 3-
chlorobenzoate (3Cba) degradation, was a viable genotype in
lake water. However, in the absence of chlorobenzoate
selection, the catabolic genotype rapidly declined to very
low levels. Morgan et al. (1989) observed a loss of
plasmid-associated phenotypes in approximately 99% of the
introduced population and attributed the loss of expression
to partial deletion of the plasmid. Jansson et al. (1989)
also detected deletion of an engineered DNA sequence in
Pseudomonas sp. after exposure to soil. It is obvious from
these studies that the stability of the recombinant genes
will depend on a variety of factors including plasmid and


A model GEM was constructed for use in gene transfer
studies. In-vitro filter matings indicate that the mob
does cause higher transfer frequencies. However,
transfer was not observed in lake water.
gene
gene
xii


123
of the GEMs (approximately 105 CFU/ml) was higher than that
of the indigenous rhizospheric microorganisms (approximately
5 x 104 CFU/ml) The number of surviving GEMs and total
microbial numbers (i.e., GEMs plus rhizospheric
microorganisms) were determined with selective and
nonselective LB agar, respectively.
Figure 5-7 and 5-8 show £. coli survival for up to 10
days in the presence and absence of corn-root exudates and
rhizospheric microorganisms. In the absence of corn plants
(Figure 5-7), the growth of £. coli was affected by the
presence of rhizospheric microorganisms. The presence of the
indigenous organisms produced a decrease in cell density
after 36 hours. However, in the absence of indigenous
rhizospheric microorganisms cell densities remained fairly
constant for up to 10 days. In the presence of corn-root
exudates (Figure 5-8) and in the absence of rhizospheric
microorganisms, E. coli displayed a growth pattern similar
to that obtained in previous experiments: an almost two
log-unit increase in growth was observed. When E. coli and
rhizospheric microorganisms were incubated together in the
presence of the plant, their numbers increased up to 107 and
108, respectively, during the first stages of incubation,
representing a larger gain of indigenous microorganisms than
of the GEMs. However, E. coli growth was significantly lower
(p<0.05) in the presence of rhizospheric microorganisms.
After 5 days, the number of E. coli cells dropped sharply,


175
TABLE 7-1. Decay rate constants (k) and estimated
half-lives of extracellular DNA
in various aquatic samples.
Sample
Treatment
k
Tl/2 (days)
Raw Sewage
Untreated
-0.0327
0.88
Autoclaved
-0.0089
3.24
Filter-sterile
-0.0195
1.48
Lake Water
Untreated
-0.0203
1.42
Autoclaved
-0.0014
20.63
Filter-sterile
-0.0060
4.81
Ground Water
Untreated
-0.0231
1.25
Autoclaved
0.0018
*
Filter-sterile
0.0014
*
Tap Water
Untreated
-0.0001
288.75
Autoclaved
0.0025
*
Filter-sterile
0.0011

* indicates no degradation


Pseudomonas outida in various aquatic systems. Results
indicate that the GEMs survived better than or as well as
their wild-type counterparts in all systems tested.
The fate of GEMs and their wild-type strains was also
studied in the corn rhizosphere under hydroponic conditions.
Both strains grew well in the presence of root exudates. No
significant difference was noted in growth pattern between
the GEMs and their wild-type counterpart.
The presence of indigenous microorganisms decreased the
survival rate of the GEMs but putida was better able to
compete with the indigenous population than E^ coli. In lake
water, the herbicide, Hydrothol-191, significantly decreased
the numbers of P_¡_ putida. but no significant difference was
observed between the GEM and wild-type strain. The
recombinant DNA of the GEMs remained fairly stable within
the host cell under all conditions tested.
While much attention has focused on the transfer of
intracellular DNA in the environment, there is little
information on the fate of extracellular DNA (eDNA) under
natural conditions. The fate of eDNA was studied in various
aquatic systems. Results indicate that chromosomal DNA is
degraded at a faster rate than plasmid DNA which undergoes a
series of structural changes prior to and during
degradation. Rapid hydrolysis of eDNA occurs in raw sewage
and lake water by cell-associated and extracellular
nucleases. Potential for genetic transformation does exist.
xi


77
Figure 4-2. Survival of geneticair^eh^ineered (GEM)
and wild-type strains of Escherichia coli and
Pseudomonas outida at 25C in lake water.
Wild-type and GEM strains recovered on non-selective
medium; ( ) and ( ), respectively. GEM strains
recovered on selective medium (o ).


152
appeared smaller and more rounded. Observations made after 9
days indicated that approximately 75% of cells had become
more condensed and were actually curved (comma shaped) in
form. These morphological changes were apparent in both the
engineered and wild-type strains.
Discussion
The extent of sublethal stress and the changes that
occur upon introduction to the natural environment is
crucial for understanding how well genetically engineered
strains can adapt to their new environments. Of equal
importance is the need to determine whether genetic
engineering provides a competitive advantage under stress
conditions in terms of physiological and morphological
adaptations.
The results obtained from this study are consistent
with those reported in the literature with regard to the
morphological and physiological changes that occur after
exposure of the cells to the natural environment. The
apparent rapid die-off of cells within the first 24 hours of
exposure, as demonstrated by direct counts and viable
counts, has also been reported by others for both in-situ
and laboratory experiments (Lopez-Torres, 1988; Munro et
al., 1987; Palmer et al., 1984; Rhodes et al., 1983). Direct
counts were generally higher than numbers obtained from
viable counts. No significant difference was noted between
viable counts of the engineered strain when grown on either


139
nitrophenol is quantified by measuring its absorption at
420nm. Beta-galactosidase measurements were made by adding
0.8 ml Z-buffer, 50 ul SDS (0.1%, w/v), 50 ul chloroform and
0.2 ml ONPG (0.4%, w/v) to the cell suspensions. After color
development, the reaction was stopped with 1 ml cold Na2C03
(1M), and the absorbance measured at 420nm. 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) (Dutton et
al., 1988). Results were expressed as a percentage of enzyme
biosynthesis relative to time zero.
INT-Dehvdrogenase Activity
Dehydrogenase activities were determined by the general
method of Koopman et al. (1984). A 0.2% (w/v) solution
of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium
chloride (INT) (Eastman Kodak Co., Rochester, NY) was
prepared using deionized, filter-sterilized water.
Triplicate, 1-ml samples were first amended with 0.1 ml 10X
LB and incubated for 15 minutes to allow the cells to
resuscitate. The samples were then amended with 0.1 ml of
INT solution, incubated in the dark at room temperature for
90 minutes and fixed with 70 ul of 37% formalin. Treated
samples were stored in the dark at 4C prior to extraction.
The treated samples were centrifuged at 1500 K for 10
minutes and excess water removed. Pellets were extracted
with 1 ml 4 + 6 tetrachloroethylene/acetone for 30 minutes


45
through system that more closely mimicked the natural
environment.
Static renewal system. Static renewal microcosms
consisted of 1- or 5-gallon aquarium tanks containing the
appropriate aquatic sample. The tanks were placed on
magnetic stirrers to allow for continuous mixing and
circulation of the test water. Aeration of the water was
provided by a Challenger air pump and air stones. Membrane
diffusion chambers containing the GEMs or test organisms
were then immersed into the water and sampled at intervals
for survival studies. Water samples were collected daily or
every 48 hours and allowed to acclimatize to the appropriate
test temperature. The test water in the tanks was replaced
every 24 hours. Figure 3-2 illustrates the design of the
static renewal microcosm.
Flow-throuah systems. Modifications to the static
renewal system allowed for continuous flow of the test water
throughout the duration of the experiment. The design of the
microcosm was essentially the same as described above for
the static renewal system. The flow of water into and out of
the aquaria tanks was regulated by peristaltic pumps
(Buchler, Fort Lee, NJ) at a flow rate of approximately 3-4
ml min-1. Overflow water from the tanks was collected in 20
liter carboys, treated with chlorine and then discarded.
A later modification to the flow-through system
included the elimination of the peristaltic pumps by
utilizing aquarium air tubing constricted with stainless


217
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69
logarithmic phase and harvested by centrifugation at 8000 g
for 10 minutes at 4C. The cells were washed three times
with sterile phosphate buffer (pH 7.2) and resuspended in
C *7
filter-sterilized test water to a density of 10 to 10'
CFU/ml before use in survival experiments.
Aquatic Samples
Test samples included lake water, ground water and
activated sludge effluent. Lake water was collected from a
hypereutrophic lake (Lake Alice, Gainesville, FI.) at a
depth of approximately l meter. Ground water was obtained
from a municipal water treatment plant (Gainesville, FI.).
The sample was collected from a composite deep well that
tapped the Floridan Aguifer. Activated sludge effluent was
obtained from the wastewater treatment plant (University of
Florida, Gainesville, FI.) that treats domestic wastewater
from the university campus. Samples were collected every 48
hours in 20 liter Nalgene carboys and allowed to acclimatize
to the appropriate test temperature prior to use.
Survival Experiments
Microcosm design. Survival experiments were conducted
using either static renewal or continuous flow-through
systems as described in Chapter 3. The design and assembly
of the survival chambers is also given in Chapter 3.
Temperature-dependent lake experiments were conducted using
a static renewal system. However, the modified flow-through
system was used to study survival in ground water and


216
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corvlina and Erwinia herbicola in hazelnut tissue and
in planta. Can. J. Microbiol. 32:835-841.
Mancini, P., S. Fertels, D. Nave, and M.A. Gealt. 1987.
Mobilization of plasmid pHSV106 from Escherichia coli
HB101 in a laboratory-scale waste treatment facility.
Appl. Environ. Microbiol. 53:665-671.
Maniatis, T., E.F. Frish, and J. Sambrook. 1982. Molecular
Cloning: a Laboratory Manual. Cold Spring Harbor
Laboratory. Cold Spring Harbor, New York.
Marshall, B., P. Flynn, D. Kamely, and S.B. Levy. 1988.
Survival of Escherichia coli with and without
ColEl::Tn5 after aerosol dispersal in a laboratory and
farm environment. Appl. Environ. Microbiol. 54:1776-
1783.
McClure, N.C., A.J. Weightman, and J.C. Fry. 1989. Survival
of Pseudomonas putida UWC1 containing cloned catabolic
genes in a model activated-sludge unit. Appl. Environ.
Microbiol. 55:2627-2634.
McFeters, G.A., and D.G. Stuart. 1972. Survival of coliform
bacteria in natural waters: field and laboratory
studies with membrane-filter chambers. Appl. Microbiol.
24:805-811.
McFeters, G.A., G.K. Bissonette, J.J. Jezeski, C.A. Thomson,
and D.G. Stuart. 1974. Comparative survival of
indicator bacteria and enteric pathogens in well water.
Appl. Microbiol. 27:823-829.
McPherson, P., and M.A. Gealt. 1986. Isolation of indigenous
wastewater bacterial strains capable of mobilizing
plasmid pBR325. Appl. Environ. Microbiol. 51:904-909.


52
as the inoculum source. Triplicate survival chambers were
inoculated with 20 ml of the washed cells (approximately 107
CFU/ml), and then immersed into the above described
microcosm. Flasks microcosms consisted of 125 ml flasks
containing 20 ml of autoclaved lake water. Triplicate flasks
were inoculated with suspensions of washed cells to achieve
an inoculum concentration of approximately 107 CFU/ml. Both
microcosms were incubated at 25C. At specific time
intervals, 1.0 ml aliquots were aseptically removed to
determine bacterial numbers. Bacteria were enumerated using
the drop plate technique described above.
Results and Discussion
Bacterial Growth Studies
Bacterial growth curves and their corresponding optical
density values are depicted in Figures 3-4 to 3-7. Although
both strains of E. coli produced similar growth patterns,
the growth rate of the recombinant strain was higher than
that of the wildtype. This was not observed for the P.
putida strains. Growth studies indicate a higher growth rate
for the wildtype strain. These standard growth curves have
proven to be fairly accurate for determining bacterial
concentrations for inoculation purposes.
Comparison of Plating Techniques
Comparison between the spread-plate technique and the
drop-plate method indicates that the drop-plate method


122
Figure 5-6. Comparison of wild-type (50014) and
genetically engineered (50058) strains of Pseudomonas
putida grown in the presence of root exudates.
(SSS) wild-type strain; (s^) genetically engineered strain.


169
hours (Figure 7-3, Lane M) and by 48 hours (Figure 7-4) only
the OC and linear forms of plasmid DNA could be detected.
The autoclaved lake water (Lane L, Figures 7-1 to 7-4)
had no degradation of eDNA during the period of the
experiment.
Groundwater
The eDNA was relatively stable in groundwater for up
to 24 hours in all the treatments tested (Figure 7-3, Lanes
G-I). However, after 48 hours eDNA was completely degraded
in the untreated sample
(Figure
7-4,
Lane G).
Some
degradation of chromosomal
DNA was
also
noticed in
the
filter-sterilized samples
(Figure 7-
-4, Lane I) after
48
hours of incubation.
Tap Water
In tap water, the eDNA remained relatively unchanged in
all the samples tested (Figures 7-1 to 7-4, Lanes C-E).
Kinetics of Degradation
The natural logarithm of the optical density
measurements (260nm) were plotted against time and a linear
regression was fitted to determine the first order decay
constant. The linearized expression is:
In [OD(thr)] = In [OD(Ohr)] kt
where t is time in hours and k is the first order decay
constant. The DNA half life (t i-s calculated from the
expression: t-^^
= 0.693/k


82
s
o
o
o
DAYS
Figure 4-5. Survival of genetically engineered (GEM)
and wild-type strains of Pseudomonas outida at 27C
in the presence and absence of the herbicide,
Hydrothol-191 (1 mg/L). Wild-type and GEM strains
incubated in the absence (0----0) and (o----a); as well as
presence of hydrothol ( ) and (_), respectively.


86
Log CFU/ml
Time (hours)
Figure 4-6. Survival of genetically engineered (GEM)
and wild-type strain of Escherichia coli at 15C in
activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; (--) and (-*-),
respectively. GEM strains recovered on
selective medium (^*-) .


186
almost exclusively associated with biological activity. Gel
electrophoresis indicated that some chromosomal degradation
did occur in the filter-sterilized samples after 48 hours,
suggesting the presence of small amounts of nuclease
activity.
The rate of DNA degradation in tap water was extremely
slow 288.75 days) and no visual degradation of either
chromosomal or plasmid DNA was detected even after 48 hours
of incubation (Figure 7-4). The filter-sterilized and
autoclaved samples showed no indication of DNA degradation.
The results of this study clearly demonstrated rapid
degradation or hydrolysis of eDNA in raw sewage and lake
water by cell-associated and extracellular nucleases. The
presence of DNA-hydrolyzing bacteria and extracellular DNase
activity in seawater has also been reported (Maeda and Taga,
1974; 1976; 1981; Paul et al., 1987).
Paul et. al. (1987) concluded that dissolved DNA is
rapidly hydrolyzed in both offshore and estuarine
environments, and that the turn over rate is quite rapid
(<6.5 hours) in estuarine systems. This rate is much faster
than the rates observed in this study for both raw sewage
0.88 days) and lake water C^i/2= 1*42 days). Maeda
and Taga (1974) observed that 75% of added DNA was
hydrolyzed within 10 days when incubated in seawater, while
Bazelyan and Ayzatullin (1979) calculated a DNA turnover
time of approximately 20 days for surface ocean water.


184
that the non-biological component did exist in raw sewage
since some degradation did occur (Figure 7-4, Lane P) in the
autoclaved treatment.
In a similar study, Phillips et al. (1989) studied the
degradation kinetics of pBR322 in various stages of a
wastewater treatment facility. Using gel electrophoresis,
they determined the DNA half-life by observing the point at
which the fluorescence of OC and linear bands equaled or
exceeded the fluorescence of the CCC band. Their results
demonstrated that the average half-life of the CCC plasmid
is four minutes or less in all stages of the treatment
facility tested. They also reported complete conversion of
the CCC form to OC and linear forms within 20 minutes of
contact with untreated wastewater influent. These results
are in accordance with this study which indicated that the
CCC form was totally converted in less than 15 minutes in
untreated raw sewage (influent). The detention time of raw
sewage in the wastewater treatment facility may be up to 24
hours. This stage represents the longest retention time of
wastewater in the treatment facility, and therefore plays a
crucial part in determining whether intact DNA sequences can
survive and could be taken up by bacteria. It seems highly
unlikely that intact plasmids would survive following their
passage through the treatment facility.
The trend shown in lake water is very similar to that
shown in raw sewage, except that degradation occurred at a
slower rate. The degradation rate in the filter-sterilized


213
Holben, W.E., and J.M. Tiedje. 1988. Application of nucleic
acid hybridization in microbial ecology. Ecology
69:561-568.
Holmberg, D.J., and G.F. Lee. 1976. Effects and persistence
of endothall in the aquatic environment. Journal Water
Poll. Contr. Fed. 48:2738-2746.
Holo, H., and I.F. Nes. 1989. High-frequency transformation,
by electroporation, of Lactobacillus lactis subsp.
cremoris grown with glycine in osmotically stabilized
media. Appl. Environ. Microbiol. 55:3119-3123.
Hopwood, D.A., and H.M. Wright. 1978. Bacterial protoplast
fusion: recombination in fused protoplasts of
Streptomvces coelicolor. Mol. Gen. Genet. 162:307-317.
Jain, R.K., R.S. Burlage, and G.S. Sayler. 1988. Methods for
detecting recombinant DNA in the environment. CRC
Critical Reviews in Biotechnology 8:33-84.
Jain, R.K., G.S. Sayler, J.T. Wilson, L. Houston, and D.
Pacia. 1987. Maintenance and stability of introduced
genotypes in groundwater aquifer material. Appl.
Environ. Microbiol. 53:996-1002.
Jannasch, H.W. 1986. Competitive elimination of
Enterobacteriaceae from seawater. Appl. Microbiol.
16:1616-1618.
Jansson, J.K., W.E. Holben, and J.M. Tiedje. 1989.
Detection in soil of a deletion in an engineered DNA
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55:3022-3025.
Johnson, J.L. 1981. Genetic characterization. In P.
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Johnston, J.B., and S.G. Robinson. 1984. Genetic engineering
and the development of new pollution control
technologies. U.S. EPA Report # 600/2-84-037,
Washington, DC.
Keeler, K.H. 1988. Can we guarantee the safety of
genetically engineered organisms in the environment?
CRC Critical Reviews in Biotechnology 8:85-97.
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Bull. Environ. Contam. Toxicol. 41:233-240.


131
and its wildtype strain. Reports on the effect of the
addition of extra DNA to a bacterium are contradictory. The
additional metabolic load of maintaining and replicating
plasmid DNA has been reported to possibly decrease bacterial
competitiveness (Helling et al., 1981). However, more recent
reports suggest that in soil systems, additional DNA has
little effect on host survival (Devanas et al., 1986; Van
Elsas et al., 1989; Walters et al., 1987) or actually
increases the survivability of the GEM over its parent or
host strain (Wang et al., 1989). Yeung et al. (1989) studied
the growth of recombinant strains of Pseudomonas in the soil
and rhizosphere, and detected no differences between the
GEMs and their non-recombinant strains in the rhizosphere.
Fredrickson et al. (1989) also noted no significant
difference between the wildtype and mutant strains of a root
growth-inhibiting Pseudomonas sp. in the rhizosphere of
wheat seedlings.
The presence of indigenous rhizospheric microorganisms
affected the survival pattern of genetically engineered
Escherichia coli. especially after the first 24 hours.
During the early stages following inoculation, the presence
of rhizospheric microorganisms did not result in any decline
of E. coli. On the contrary, the numbers of both
rhizospheric microorganisms and GEMs increased. The
concentration of available carbon released by the roots was
apparently sufficient to allow for normal growth of both
GEMs and rhizospheric microorganisms. The larger increase in


121
Figure 5-5. Comparison of wild-type (HB101) and
genetically engineered (50008) strains of Escherichia
coli grown in the presence of root exudates.
(?£.') wild-type strain; (-5^), genetically engineered strain.


44
Midland, MI) at the point at which the needles entered the
chambers. Dust cap covers for the hyperdermic needles were
made by filling the hubs of 1-ml plastic syringes with
silicone.
Prior to assembly, dust caps were sterilized by
autoclaving at 121C for 15 minutes. The chambers and
membranes were semi-assembled by placing the membranes on
the outer retainers that were treated with Lubriseal. The
chambers and membranes were then sterilized by irradiation
with ultraviolet light (GTE Sylvania Inc, Danvers, Mass.)
for at least two hours. The chambers were assembled
aseptically under UV light using stainless steel nuts and
bolts to secure the chambers. Sterile 22-gauge needles were
then inserted into the central lumen, capped with the
sterile dust caps and sealed into position with silicone.
The silicone seal was allowed to dry at least 24 hours prior
to use of the chambers.
Microcosm Design
Laboratory scale microcosms were designed to utilize
the membrane diffusion chambers in a simple setting that
would allow testing of a variety of environmental variables,
simulate a more natural environment, and yet keep the
recombinant bacteria within a contained setting. Two types
of designs were utilized during research procedures.
Initially, a static renewal system was developed and tested
for survival studies. Further improvements produced a flow-


91
Time (hours)
Figure 4-10. Survival of genetically engineered (GEM)
and wild-type strain of Escherichia coli at 30C
in activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; () and (i) ,
respectively. GEM strains recovered on
selective medium (-ijf-) .


64
possible obviously provides a more rigorous test for the
introduced organisms.
The preliminary experiments described here were
basically comparative studies to determine optimal test
procedures for the following standard experiments.


54
Figure 3-5. Growth curve and corresponding optical density
values (A550) of genetically engineered Escherichia coli
(50008), grown at 35C.
OPTICAL DENSITY


39
soil. Ardema et al. (1983) found that DNase reduced
transformation frequencies of free DNA more than that of DNA
adsorbed onto quartz sand. Lorenz and Wackernagel (1987)
reported similar results using a flow-through system of sand
filled glass columns. These studies indicated that soil
components other than organic materials and clay minerals
can bind DNA and retard its enzymatic degradation.
To date, no information exists that demonstrates
genetic transfer via transformation by the DNA of GEMs.
Several studies have shown the presence of extracellular DNA
in the natural environment (DeFlaun et al., 1986; 1987;
Minear, 1972; Pillai and Ganguly, 1972) and a few have
investigated its dynamics and persistence (Paul et al.,
1987; 1988). However, very little is still known about the
fate of extracellular DNA and its implication for genetic
transfer via transformation.


71
Sterile vs non-sterile conditions. The survival of the
genetically engineered strains under sterile and nonsterile
conditions was studied using six chambers inoculated with
the appropriate bacterial strain and immersed in lake water
as described above. After 60 hours, during which bacterial
numbers were enumerated every 24 hrs, three chambers were
removed and each inoculated with 1 ml of lake water. The
chambers were replaced and thereafter represented nonsterile
conditions. The lake water was initially screened to ensure
that none of the indigenous population grew on the selective
media used for the engineered strains. This experiment was
conducted at 27C using a flow-through system as described
above.
Toxicant effect. The effect of toxicants on the
survival of P. putida and its engineered strain was studied
using the aquatic herbicide, Hydrothol-191 (7-oxabicyclo
[2,2,1] heptane-2,3-dicarboxylie acid). Hydrothol-191 is
commonly used throughout the state of Florida in lakes and
streams for control of aquatic weeds (Dupes and Mahler,
1982). The herbicide has a half-life of approximately 21
days (Reinert et al., 1985), and suggested application rates
of 1-5 mg/L (Pennwalt Corporation, 1980). The survival
response of the two strains of P. putida in lake water
amended with Hydrothol 191 (1 mg/L final concentration) was
studied using a static renewal process. A stock solution of
Hydrothol-191 (530mg/L) was prepared from the liquid
formulation (Pennwalt Corp., Philadelphia, Pennsylvania)


178
TIME (HOURS)
Figure 7-7. Degradation kinetics of
in Groundwater. ( ) Untreated Sample;
Sample; ( a ) Filter-sterilized
spiked eDNA
( O ) Autoclaved
Sample.
120


67
recombinant bacteria in the aquatic environment. Recently,
polymerase chain reaction (PCR) has been used to pursue such
studies with greater precision and sensitivities (Chaudhry
et al., 1989).
Laboratory-contained microcosms serve an important
function in the study of GEMs. Current regulatory standards
strongly recommend research with recombinant bacteria only
within contained settings (Johnston and Robinson, 1984).
Microcosms can serve as standard test systems that can be
adapted to a variety of organisms and environmental
conditions. In this study, a microcosm approach utilizing
membrane diffusion chambers (Altherr and Kasweck, 1982;
Fliermans and Gordon, 1977; McFeters and Stuart, 1972) was
applied for studying the survival and fate of recombinant
bacteria in the aquatic environment. The porous membranes of
the chamber allowed continuous exchange of water, solutes
and nutrients between the chambers and the surrounding
waters. A significant advantage of this system was the
continuous interaction of the environmental sample, such as
that found in natural environments, with the test bacteria.
The microcosm was designed to allow the testing of a
variety of environmental variables including the effects of
toxicants. The-discharge of toxic materials into the aquatic
environment has led to questions regarding the impacts of
these chemicals on the ecosystem. Of major concern is the
effect on aquatic life and water quality (Rand and
Petrocelli, 1985). Microbial activity has been shown to be


212
Goyal, S.M., C.P. Gerba, and J.L. Melnick. 1979.
Transferable drug resistance in bacteria of coastal
canal water and sediment. Water Res. 13:349-356.
Grabow, W.O.K., O.W. Prozesky, and J.S. Burger. 1975.
Behaviour in a river and dam of coliform bacteria with
transferable drug resistance. Water Res. 9:777-782.
Graham, J.B., and C.A. Istock. 1978. Genetic exchange in
Bacillus subtilis in soil. Mol. Gen. Genet. 166:287-
290.
Haas, D., and B.W. Holloway. 1976. R factor variants with
enhanced sex factor activity in Pseudomonas aeruginosa.
Mol. Gen. Genet. 144:243-251.
Halvorson, H.O., D. Pramer, and M. Rogel. 1985. Engineered
Organisms in the Environment: Scientific Issues.
American Society of Microbiology, Washington, DC.
Helling, R.B., T. Kinney, and J. Adams. 1981. The
maintenance of plasmid-containing organisms in
populations of Escherichia coli. J. Gen. Microbiol.
123:129-141.
Henis, Y., K.R. Gurijala, and M. Alexander. 1989. Factors
involved in multiplication and survival of Escherichia
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dem gebiet der boder bakteriologie und unter besonderer
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Rhizobium spp. in inoculants made from presterilized
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Environ. Microbiol. 54:703-711.


188
stranded DNA regardless of its source (Smith et al., 1981;
Stewart and Carlson, 1986). Some studies have shown
transformation of B. subtilis and S. pneumoniae by denatured
DNA under certain conditions such as low pH, but these occur
at substantially lower frequencies (Smith et al., 1981;
Stewart and Carlson, 1986). It has also been shown that high
molecular weight DNA is required for transformation (Notani
and Setlow, 1974). Carlson et al. (1983) found 10-15 kb to
be the optimal size of DNA required for transformation of
Pseudomonas stutzeri. while Smith et al. (1981) reported an
average size of 10 kb for B. subtilis. Low levels of
transformation were noted with 0.45 kb DNA (Goodgal, 1982).
The results obtained from this investigation clearly
demonstrates that the molecular weight range of eDNA and
their degradative products fall within the range required
for transformation. These degradation by-products are
sufficiently large to encode for gene sequences, given that
an average bacterial gene is 1.3 kb (Lehninger, 1982). This
suggests that the potential for genetic transformation does
exist, at least for some systems. The chances of natural
transformation occurring in raw sewage would probably be low
due to the rapid hydrolysis of eDNA. An interesting
implication for groundwater is the fact that eDNA is stable
for up to 24 hours, thereby increasing the chances of
natural transformation.
The fact that chromosomal DNA is less stable than
plasmid DNA is interesting in light of the fact that


37
Bacillus and Pseudomonas (Reanny et al., 1982). These
studies, however, were mostly conducted as in-vitro
experiments using pure cultures of bacteria (Zinder and
Lederberg, 1952). The recent interest in gene transfer in
the natural environment has again prompted research in this
area, but information is still sparse.
The first reported study of in-situ transduction was
conducted in a freshwater environment using strains of
Pseudomonas aeruginosa (Morrison et al., 1978). Transduction
of P_ aeruginosa streptomycin resistance by a generalized
transducing phage, F116, was shown to occur in flow-through
environmental test chambers submerged in a freshwater
reservoir.
In a similar experiment, Saye et al. (1987)
demonstrated the transduction of plasmid Rmsl49 by the
generalized transducing bacteriophage $DS1. Plasmid DNA was
transferred from a nonlysogenic plasmid donor to a $DSl
lysogen of P_¡_ aeruginosa that served both as the source of
the transducing phage and as the recipient of the plasmid
DNA. Transduction was observed both in the presence and
absence of the indigenous microbial population. In a later
study, transduction of single chromosomal loci and
cotransduction of closely linked loci were observed between
lysogenic and nonlysogenic strains (Saye et al., 1990).
These studies clearly demonstrated the ability to generate
and select new genetic combinations through phage-mediated
exchange.


30
(Scanferlato et al., 1989). Aquatic microcosms consisted of
850-ml glass Mason jars containing sediment and pond water.
The density of both genetically engineered and wild-type
strains declined at the same rate, and was no longer
detectable by viable counts after 32 days. The introduction
of the GEM affected the indigenous bacterial community.
Total bacterial density significantly increased, including
the density of bacteria belonging to the proteolytic
functional group. In contrast, the density of indigenous
pectolytic and amylolytic bacteria was not affected by the
introduction of the GEM.
Steffan et al. (1989) utilized gene probe methodology
for tracking GEMs with catabolic genotypes in freshwater
(reservoir) samples. The GEMS consisted of Alcaliaenes A5
and cepacia AC1100 that degraded 4-chlorobiphenyl and
2,4,5-trichlorophenoxyacetic acid, respectively. Aquatic
microcosms consisted of 20-liter glass carboys filled with
water samples supplemented with glucose and a mineral salts
solution. Colony hybridization of the viable heterotrophic
bacterial populations and dot blot hybridization of total
recovered DNA showed persistence of the GEMs in the presence
and absence of the xenobitic substrates that these organisms
biodegrade. Although there was a gradual decline in
population densities, both GEMs were still detected in the
microcosm two months after their introduction into the
microcosms. Addition of the appropriate xenobiotic


73
Results
Survival and Plasmid Stability in Lake Water
Temperature-dependent studies. The effect of
temperature on the survival pattern of wild-type and
genetically engineered strains of E. coli and P. putida is
shown in Figures 4-1 to 4-3. Rates of population change are
summarized in Table 4-1 and 4-2. At 15C (Fig 4-1), the
rates of population change were similar for both wild-type
and genetically modified strains. All four strains exhibited
only a slight decrease in numbers after 19 days of
incubation at 15C. The rate of population change
(loglO(CFU)/day) for wild-type P. putida (-0.06) was not
significantly different from that of the engineered strain,
50058 (-0.08). However, a significant difference (pco.001)
was noted between the rates of population change for the two
strains of E. coli. Wild-type HB101 showed a greater decline
in numbers (approximately 1 log) than the engineered strain
(<0.5 log) after a period of 19 days (Figure 4-1).
Rates of decline were greater at 25C (Figure 4-2)
than at 15C. The rate of population change for wild-type P.
putida (-0.19) was significantly different (p<0.01) from
that of the engineered strain (-0.08). No significant
difference was noted between the survival rates of the two
strains of E. coli.
The greatest decline in numbers was observed at 30C
(Figure 4-3). Rates of population change for P. putida 50014


207
Barder, D.A., J.K. Martin. 1976. The release of organic
substances by cereal roots in soil. N. Phytol. 76:69-
80.
Bazelyan, V.L., and T.A. Ayzatullin. 1979. Kinetics of
enzymatic hydrolysis in seawater. Oceanology 19:30-33.
Bell, J.B., G.E. Elliott, and D.W. Smith. 1983. Influence of
sewage treatment and urbanization on selection of
multiple resistance in fecal coliform populations.
Appl. Environ. Microbiol. 46:227-232.
Bentzen, S.A., J.K. Fredrickson, P. Van Voris, and S.W. Li.
1989. Intact soil-core microcosms for evaluating the
fate and ecological impact of the release of
genetically engineered microorganisms. Appl. Environ.
Microbiol. 55:198-202.
Beringer, J.E., and M.J. Bale. 1988. The survival and
persistence of genetically engineered microorganisms.
In M. Sussman, C.H. Collins, F.A. Skinner and D.E.
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London, pp. 29-46.
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Birnboim, H.C., and J. Doly. 1979. A rapid alkaline
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Appl. Microbiol. 29:186-194.
Bitton, G., S.R. Farrah, R.H. Ruskin, J. Butner, and Y.J.
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varying moisture and nutrient levels on the transfer of
a conjugative plasmid between Streptomvces species in
soil. Can. J. Microbiol. 35:544-549.
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1977. Thymine content of seawater as a measure of
biosynthetic potential. Mar. Biol. 40:1-8.


11
TABLE 2-1. Potential environmental uses of genetically
engineered organisms
** Improve nutritional quality of food crops
** Decrease dependence on chemical pesticide
by using recombinant microbial pesticides
** Increase plant tolerance and resistance
to pathogens and pests
** Decrease crop sensitivity to chemicals
** Increase plant tolerance to environmental
stresses
** Increase crop yields by manipulating
photosynthesis
** Improve soil quality
** Control weeds
** Improve food storage
** Improve nitrogen fixation
** Water pollution reduction
** Clean up oil spills
** Decomposition of organic wastes
** Cloud seeding, snow making
** Mining: bacterial retrieval of metals
** Energy production from organic biomass
** Oil Recovery
Source: adapted after Keeler (1988) and Saunders
and Saunders (1987).


180
exposure (Figure 7-7). DNA degradation was then rapid and no
eDNA could be detected after 4 days. The short half-life of
1.25 days is reflective of the quick and sudden decrease of
the added DNA in untreated groundwater. The autoclaved and
filter-sterilized treatments indicated relative stability of
the DNA under these conditions.
In tap water, degradation of eDNA occurred at an
extremely slow rate (Figure 7-8). The estimated half life in
untreated tap water was 288.75 days. Both autoclaved and
filter- sterilized samples showed no indication of DNA
degradation.
Discussion
A simple direct approach was used to determine the fate
of eDNA in a variety of aquatic samples. Gel electrophoresis
allows visual detection of the physical changes in the DNA
molecule leading to linear, OC and CCC forms.
The concentration of total DNA was measured by optical
density determinations at 260 nm. This method has
limitations in that optical density readings may be
affected by several contaminants such as RNAs, proteins, and
other organic compounds. A more specific and sensitive
method for DNA determination is the use of the fluorometric
dye Hoechst 33258 (DeFlaun and Paul, 1986; Paul and Myers,
1982). However, the spectrophometric method was adapted for
its simplicity and its ability to measure all nucleic acids
including CCC, OC and linear forms as well as smaller


210
Drahos, D.J., B.C. Hemming, and S. McPherson. 1986. Tracking
recombinant organisms in the environment: B-
galactosidase as a selectable non-antibiotic marker for
fluorescent pseudomonads. Biotechnology 4:439-444.
Dupes, J.M., and M.J. Mahler. 1982. Public funded aquatic
plant control operations in Florida. Aquatics 4:6-14.
Dutton, R.J., G. Bitton, and B. Koopman. 1988. Enzyme bio
synthesis versus enzyme activity for microbial
toxicity testing. Toxicity Assessment 3:245-253.
Dwyer, D.F., F. Rojo, and K.N. Timmis. 1988. Fate and
behaviour in an activated sludge microcosm of a
genetically engineered micro-organism designed to
degrade substituted compounds. In M. Sussman, C.H.
Collins, F.A. Skinner and D.E. Stewart-Tull
(eds), The Release of Genetically-engineered Micro
organisms. Academic Press, London, pp. 77-84.
Edlin, G., R.C. Tait, and R.L. Rodriguez. 1984. A
bacteriophage lambda cohesive ends (cos) DNA fragment
enhances the fitness of plasmid-containing bacteria in
energy-limited chemostats. BioTechnology 2:251-254.
Festl, H., W. Ludwig, and K.H. Schleifer. 1986. DNA
hybridization probe for the Pseudomonas fluorescens
group. Appl. Environ. Microbiol. 52:1190-1194.
Fiksel, J., and V.T. Covello. 1986. Biotechnology Risk
Assessment. Pergamon Books, Inc., New York.
Findlay, S., L. Carlough, M.T. Crocker, H.K. Gill, J.L.
Meyer, and P.H. Smith. 1986. Bacterial growth on
macrophyte leachate and fate of bacterial production.
.Limnol. Oceanogr. 31:1335-1341.
Fliermans, G. B., and R. W. Gordon. 1977. Modification of
membrane diffusion chambers for deep water studies.
Appl. Environ. Microbiol. 33:207-210.
Foster, R.C., A.D. Rovira, and T.W. Cock. 1983.
Ultrastructure of the Root-Soil Interface. The
American Pathological Society. St. Paul, Minnesota.
Fredrickson, J.K., S.A. Bentjen, H. Bolton, S.W. Li, and P.
Van Voris. 1989. Fate of Tn5 mutants of root growth-
inhibiting Pseudomonas sp. in intact soil-core
microcosms. Can. J. Microbiol. 35:867-873.


141
was measured by adding ONPG as described above. The results
of this assay were expressed as percent inhibition of B-
galactosidase biosynthesis relative to the control sample,
at a concentration of 45ppm PCP.
Statistical Analysis
All assays were conducted in triplicate and the data
expressed as mean values + standard deviation from the mean.
Basic computations were performed using STAT-2 (Stat-Soft
Inc., Tulsa, Oklahoma) statistical program. Exponential
decline rate models were fit to the population data by
performing linear regression on logarithmic CFU values
against time. Rates of population change were compared by a
modified t-Test (Zar, 1984) to determine significance
differences in survival rates between bacterial strains.
Results
Bacterial Enumeration
Rates of population change (log1Q/day) for the two
strains of E. coli by viable counts and direct counts are
given in Table 6-1. Results obtained from viable counts
indicated a rapid decline in cell numbers for both strains.
By day 6, less than 10% of the cells were viable (Figure 6-
2). Although the results indicated a lower rate of decline
for wild-type HB101 (-0.23), this rate was not significantly
different (p<0.05) from that of the engineered strain (-
0.30). Similar rates were also observed for the engineered


70
activated sludge effluent. All experiments were carried out
in either a constant temperature room or an environmental
chamber (Percival, Boone, Iowa) at a constant temperature
and light regime (16 hrs light: 8hrs dark).
Experimental procedures. For all experiments,
triplicate chambers were inoculated with 20 ml of washed
cells resuspended in test water, and then immersed into the
microcosms. After certain time intervals, each chamber was
removed, shaken well to resuspend the cells, and 1.0 ml of
suspension removed to determine bacterial numbers. Viable
counts were enumerated using the drop-plate technique (Hoben
and Somasegaran, 1982) on either LB agar plates for wild-
type strains (HB101 and 50014); LB + HgCl2 (40 ug ml-1) for
E. coli (50008) and LB + tetracycline (25 ug ml-1) +
kanamycin (50 ug ml-1) for P. putida (50058) Direct counts
were determined by the acridine orange direct count (AODC)
method of Hobbie et al. (1977).
Effect of temperature. The effect of temperature on the
survival rates of wildtype and genetically engineered
strains of E. coli and P. putida was studied by conducting
experiments at 15, 25 and 30C. Temperature-dependent
experiments were run for lake water and activated sludge
effluent under sterile conditions i.e. in the absence of
indigenous populations. Survival studies in ground water
were conducted at 22C, since ground water maintains this
temperature year round.


134
during sublethal stress that eventually lead to a viable but
nonculturable state (Anderson et al., 1979; Chai, 1983;
Gauthier et al., 1987; 1988; Lopez-Torres, 1988; Munro et
al., 1987; Palmer, 1984; Rhodes et al., 1983; Tamplin and
Colwell, 1986).
Adaptive features within populations contribute to both
change and stability within microbial communities.
Adaptation within microbial populations may occur as
structural or physiological features that increase the
fitness of that organism for survival within an ecosystem.
Adaptation within populations can also occur as reproductive
or behavioral survival strategies (Atlas and Bartha, 1981).
A general adaptive feature of many microbial populations is
the ability to turn on and off particular metabolic pathways
as needed. This mechanism allows for conservation of energy
and carbon flow and is an adaptive feature in ecosystems
with limited available substrates.
The importance of adaptation as a survival strategy in
the natural environment has been widely demonstrated with
respect to organisms of public health significance such as
Escherichia coli, Klebsiella pneumoniae. Vibrio cholerae and
Salmonella enteritidis (Anderson et al., 1979; Chai, 1983;
Colwell et al., 1985; Gauthier et al., 1987; 1989; Lopez-
Torres et al., 1988; Munro et al., 1987; Palmer et al.,
1984; Roszak et al., 1984; Tamplin and Colwell, 1986; Walsh
and Bissonnette, 1989). Viable but nonculturable bacteria
have been shown to exhibit structural and physiological


32
throughout the experiment, and the introduced strains did
not adversely affect the functioning of the unit. The GEM
persisted in the ASU, in the presence of the sludge
microflora containing predatory protozoa, for more than
eight weeks, although the population size did decline
gradually. Plasmid pDIO was stably maintained in the host
bacterium in the presence or absence of 3-chlorobenzoate,
but did not enhance the degradation of 3-chlorobenzoate in
the ASU.
All these survival studies indicate a significant
decrease in cell numbers of GEMs after introduction into
nonsterile aquatic samples as compared to filtered or
sterile samples. This apparently is a general phenomenon
which has been described for a variety of bacteria in
aquatic environments (Liang et al., 1982, Sinclair and
Alexander, 1984). The decrease may be attributed to biotic
factors such as predation, competition and antagonism by the
indigenous population.
Part V: Genetic Transfer bv Genetically Engineered
Organisms in the Natural Environment
Introduction
Among bacteria, genetic information can be naturally
transferred from one species to another by several known
mechanisms (conjugation, transduction, transformation and
possibly by vesicles, a yet unexplained mechanism). These
mechanisms can also be expected to apply- to GEMs that are
released into the environment. The potential for gene


124
Figure 5-7. Survival of genetically engineered Escherichia
coli in the presence of rhizospheric microorganisms (RM) ,
without corn roots present. ( ), total microflora
(E. coli + RM) ; ( ) ILs. coli in the absence of RM;
(O), E*. coli in the presence of RM.


161
(Graham and Istock, 1978) and marine sediments (Aardema et
al., 1983). The rate of transformation under natural
conditions tends to be less when compared to in vitro
experiments. This is due in part to the presence of
deoxyribonucleases (DNAses), DNA-hydrolyzing enzymes, and to
the sorption of DNA onto soil or sediment particles, thereby
reducing its availability for transformation (Gillett et
al., 1985; Lorenz and Wackernagel, 1987).
The purpose of this study was to examine the
persistence of eDNA in various aquatic systems and to
determine the effect of biotic and abiotic factors on
degradation rates. In addition, the kinetics of eDNA
degradation were determined.
Materials and Methods
Aquatic Samples
Samples were chosen to represent a wide range of
aquatic environments. Raw sewage was obtained from the
wastewater treatment plant (University of Florida,
Gainesville, FI.) that treats domestic wastewater from the
campus. The sample was collected at the point of entry into
the waste treatment facility. Lake water was collected from
a hypereutrophic lake (Lake Alice, Gainesville, FI.) at a
depth of 10-15 cm. Ground water was obtained from the
municipal water treatment plant in Gainesville, Florida. The
ground water sample was collected from a composite deep well
that tapped the Floridan Aquifer. Tap water was obtained


CHAPTER 5
FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN THE CORN RHIZOSPHERE
Introduction
Since the introduction of the concept of the
rhizosphere by Hiltner (1904), numerous studies have been
carried out to shed more light on the interactions between
plant roots and associated microorganisms (Lynch, 1982).
Roots release to the surrounding soil approximately 20% of
the dry matter produced photosynthetically by plants (Barber
and Martin, 1976). Root exudates are essential sources of
carbon and energy to heterotrophic microorganisms in the
rhizosphere. The types of microorganisms present in the
rhizosphere are influenced by the chemical composition of
the root exudates (Kraffczyk et al., 1984). In return,
rhizospheric microorganisms stimulate ion uptake by roots,
provide growth-regulating substances to the plant, and some
actively participate in nutrient cycling (Alexander, 1977;
Barber and Martin, 1976).
Recent advances in recombinant DNA (rDNA) technology
provide promising ways to improve and expand the useful
properties of plant-associated bacteria for enhanced
agricultural purposes. Inoculation of soils and seeds with
110


31
substrates enhanced survival of both GEMs. The recombinant
plasmids were extremely stable under all test conditions.
Trevors et al. (1989) investigated the survival of and
plasmid stability in Pseudomonas and Klebsiella spp.
introduced into agricultural drainage water. Microcosms
consisted of 30-ml glass bottles containing 9-ml of sterile
or non-sterile drainage water. Experiments were conducted
under aerobic and anaerobic conditions and the presence or
absence of added nutrients. The two strains of Pseudomonas
survived well in sterile drainage water incubated
aerobically, with or without added nutrients. However, the
Klebsiella strain only survived in the presence of added
nutrients. The GEMs did not survive well under anaerobic
conditions without nutrients, but showed good survival in
the presence of nutrients. Maintenance of the three
plasmids was found to be host-, plasmid- and environment-
dependent. Plasmid pBR322 was not stably maintained in
Klebsiella under all conditions tested, and pRK2501 was
readily lost from P¡. putida CYM318. Maintenance of RP4 by P.
fluorescens R2f was markedly influenced by nutrients, which
caused a loss of plasmids from cells.
A more complex microcosm was utilized by McClure et al.
(1989) to study the survival of Pseudomonas putida UWC1 in a
laboratory scale activated sludge unit (ASU). The engineered
strain harbored a cloned non-self-transmissible plasmid,
pDIO, that encoded the breakdown of 3-chlorobenzoate. The
ASU maintained a healthy, diverse protozoan population


12
agriculture include the enhancement of nitrogen fixation,
biological control of insects, and expression of toxin genes
of pathogens in food grain plants. Animals and humans
consuming such food will develop protection against the
potential pathogen, a concept that is similar to
vaccinations.
Pollution control. During the last 80-90 years, the
environment has become overloaded with human, animal, plant
and industrial wastes. Attempts to control or reverse these
adverse events have focused on developing improved waste
treatment systems and technologies that can control the
source of pollution. However, these systems are incapable of
effectively dealing with persistent toxic chemicals that
enter the environment at point sources such as dump sites,
plumes, agricultural runoffs and chemical spills.
It is this problem that has been the target of
biotechnology research whose object is either a) the
enhancement of growth and activity of indigenous organisms
at a pollution site, b) the addition of non-indigenous
'active-degraders' to a pollution site, or c) to enhance and
improve the degradative capabilities of organisms that can
degrade hazardous pollutants to innocuous products
efficiently and economically (Gillett et al., 1985).
Genetic engineering shows much promise for future use
in pollution abatement. Certain organisms, in particular the
genus Pseudomonas. have the capacity to degrade a variety of
hydrocarbons and are therefore potentially useful for


68
intrinsically affected by the presence of such toxicants,
with subsequent direct or indirect impacts on the rest of
the ecosystem. With the increasing use of chemicals in the
environment, it is important to understand how GEMs would
respond in such adverse conditions and whether they behave
differently (structurally or physiologically) from their
non-recombinant parent strains.
Materials and Methods
Bacterial Strains
Escherichia coli (strain HB101) and Pseudomonas putida
(strain 50014) were used as wild-type reference strains.
Genetically engineered E. coli (strain 50008) was obtained
by inserting an EcoRl DNA fragment from pRCIO into a
derivative of pBR322. The EcoRl fragment contained the gene
for mercury resistance and the genes involved in 2,4-D
degradation (Chaudhry and Huang, 1988). Detailed
construction of the clone was described in Chapter 3.
Pseudomonas putida (strain 50058) was used as the
genetically engineered strain and harbored R68.45 which
conferred resistance to carbenicillin, kanamycin and
tetracycline (Haas and Holloway, 1976).
E. coli and P. putida were grown in LB at 35C and
28C, respectively. For the genetically engineered E. coli
and P. putida, LB was supplemented with HgCl2 (40 ug ml-1),
and tetracycline (15 ug ml-1) and kanamycin (50 ug ml-1),
respectively. All cultures were incubated until late


LOG CFU/mi
76
DAYS
Figure 4-1. Survival of genetically engineered (GEM)
and wild-type strains of Escherichia coli and
Pseudomonas putida at 15C in lake water.
Wild-type and GEM strains recovered on non-selective
medium; ( ) and ( ), respectively. GEM strains
recovered on selective medium ( o).


22
flora, and the maintenance of the engineered genes or
plasmid vectors within the host cell. It is conceivable that
a precisely designed recombinant organism might carry
transcription signals or coding seguences that would have
unpredictable effects on an indigenous host organism (Omenn,
1986). Furthermore, the stability of the recombinant DNA is
important if bacterial cells containing recombinant plasmids
are to be used in environmental biotechnology applications.
At present, routine selective plating and/or DNA
probing has been used to assess the stability of marker DNA
sequences in plasmids and chromosomal sites, and to check
for gene transfer (McClure et at., 1989; Richaume et al.,
1989; Saye et al., 1990; Zeph and Stotzky, 1989). A
combination of well designed microcosms and effective marker
attributes can serve as reliable and powerful tools for
investigating genetic stability.
Part IV: Survival of Genetically Engineered Organisms
and Plasmid Stability of Recombinant Plasmids
in the Natural Environment
Introduction
The survival and growth of recombinant organisms in the
natural environment will depend primarily on 1) the nature
of the bacterial host and the cloning vector, 2) the
selective advantages or disadvantages conferred on the host
by the presence of the foreign DNA and 3) the ecological
niches occupied by the recombinant hosts (Curtiss et al.,
1977) .


21
monitoring and study of plasmid stability in soils
(Fredrickson et al., 1988; Van Elsas et al., 1989); 4)
observing genetic transfer in soils (Bleakley and Crawford,
1989; Zeph and Stotzky, 1989) and waste water (Mancini et
al., 1987); and 5) detection of deletions in an engineered
DNA sequence in soil systems (Jansson et al, 1989). These
studies demonstrate the usefulness of DNA probe technology,
especially when used in conjunction with conventional
selective plating procedures.
In-vitro amplification of rDNA by the polymerase chain
reaction (PRC) can greatly enhance detection of recombinant
target DNA in environmental samples. Using lake water and
raw sewage, Chaudhry et al. (1989) demonstrated the
usefulness of PCR over conventional plating methods for
monitoring GEMs in the environment. The PCR method detected
the presence of the GEMs when selective plating did not.
Similar results have been reported by Steffan and Atlas
(1988) .
While it is evident that a wide variety of
methodologies and approaches are available for monitoring
and detecting GEMs in the environment, further development
and refinement is necessary. At present, a combination of
protocols might serve as the best strategy for assessing the
fate of GEMs and rDNA in the environment.
Methods for Assessing Genetic Stability
Genetic stability studies address the concerns of
genetic transfer from engineered organisms to the natural


10
industrial chemicals (Gillett et al., 1985). At present, an
estimated 10,000 laboratories are currently conducting
biotechnological research in public and private corporation,
universities, and governmental agencies world-wide, and more
than 200 companies are marketing biotechnology products
(Jain et al., 1988).
The successful use of rDNA technology in the
pharmaceutical and chemical industries has dispelled early
fears and concerns about the practice and safety of genetic
engineering in the laboratory. As a result, the potential
for application has now broadened to include agriculture,
mining, pest control, pollution control and a host of other
environmental uses (Gillett et al., 1985; Keeler, 1988;
Lindow, 1985).
Potential Environmental Uses
The proposed environmental uses of GEOs can range from
transgenic animals and modified crop plants to bacteria
designed for specific tasks such as biodegradation of
pollutants. Some potential uses of GEOs are listed in Table
2-1.
Agriculture. The application of rDNA technology to
agriculture is directed to develop crop plants that will
provide more completely balanced nutrition, tolerate
environmental stresses, photosynthesize more efficiently,
exhibit enhanced food storage and express resistance to
pests and pathogens (Gillett et al., 1985; Keeler, 1988).
Other desired applications of genetic engineering in


185
treatment was significantly lower than that of untreated
lake water, suggesting that cell-associated degradation
played a more important role in DNA degradation than
extracellular nucleases. Although the autoclaved treatment
indicated a slow degradation rate C^x/2= 20*63 days), it
still suggests the presence of non-biological elements
involved in DNA degradation in lake water. Lake Alice is a
hypereutophic system that receives treated wastewater from
the university campus and therefore can potentially contain
a variety of chemical substances at any given time. This may
account for the degradation shown in the autoclaved
treatment.
The results of the groundwater treatments revealed an
interesting trend in the degradation kinetics of eDNA. Both
chromosomal and plasmid DNAs remained relatively stable in
ground water during the first 24 hours of incubation.
However, after 48 hours, gel electrophoresis showed no
indication of chromosomal or plasmid DNA. Optical density
measurements indicated some decay products still present at
48 hours, but none at 109 hours. This sudden and rapid
degradation of extracellular DNA suggests increased
biological activity. Similar results were noted by Maeda and
Taga (1974) for seawater samples. They reported no
significant DNA hydrolysis until after 3 days, at which time
a rapid increase in degradation was noted. The lack of
degradation in the filter-sterilized and autoclaved
treatments (Figure 7-7) also suggests that degradation was


189
chromosomal DNA is much more readily taken up by
microorganisms than plasmid DNA (Smith et al., 1981; Stewart
and Carlson, 1986). Plasmid DNA has been shown to be taken
up at a tenfold lower rate than chromosomal DNA (Smith et
al., 1981; Stewart and Carlson, 1986). There are, however,
some exceptions to the typically low efficiency of
transformation with plasmid DNA. Multimeric plasmids have
been shown to transform at a high efficiency (Smith et al.,
1981). Linear plasmids also transform well if a region on
each end is homologous to the chromosomal DNA or any
resident plasmid (Smith et al., 1981; Stwart and Carlson,
1986). The results of this study show that whereas
chromosomal DNA is rapidly degraded, plasmid DNA is more
stable and undergoes structural changes to the linear form.
This would suggest that plasmid DNA may still have the
potential for transformation since the linear form tends to
be the most stable and resistant form of DNA. In a recent
study, Paul et al. (1988) observed no transformation of
plasmid DNA in estuarine planktonic environments. This study
however does not preclude the possibility of transformation
occurring in natural systems. It is evident from the lack of
data that more research is needed on the fate of
extracellular DNA and its implication for natural
transformation. Such knowledge is important in view of the
fact that GEMs are now being seriously considered for
release into the environment.


59
CFU/ml (Millions)
Figure 3-9. Comparison of enumeration techniques
by the spread plate and drop plate methods for
genetically engineered Escherichia coli (50008).


98
days. This can be attributed to the initial growth and
decline of the wild-type strain and therefore does not
necessarily indicate that the engineered strain survives
better than the wild-type strain. On the contrary, the
survival patterns of the two strains (Figure 4-13) suggests
that the wildtype strain survives better or at least as well
as the engineered strain.
Plasmid stability. Results of the study indicate that
the engineered genes of both £. coli and P. putida were
relatively stable, with no loss occurring up to 21 days of
incubation in ground water (Figures 4-12 and 4-13,
respectively). No significant differences were noted between
viable counts or rates of decline for strains grown on
either selective or nonselective media.
Discussion
The microcosm model described here is a simple but
useful way to study the fate and survival of genetically
engineered microorganisms in aquatic environments. In this
study the model was applied to study the effect of selected
environmental factors on the survival of two genetically
engineered strains and their respective parent strains. It
has been suggested that the presence of the "foreign DNA in
recombinant microbial strains may affect or alter the host
bacteria in subtle ways that may affect its ecology or
population dynamics (Beringer and Bale, 1988; Curtiss, 1976;
Devanas and Stotzky, 1986). This study indicates that such


102
al. (1989) also reported poor survival of a recombinant
Pseudomonas strain in agricultural drainage water that was
directly influenced by low nutrient conditions. The rapid
decline of the recombinant £. coli strain in this study is
probably due to a temperature effect. The higher temperature
may have provided an additional burden to the GEM, thereby
affecting its growth and survival.
Studies of GEMs in model activated sludge/wastewater
units suggest that GEMs can survive and persist in the
system for long periods of time (Mancini et al., 1987;
McClure et al., 1989). Similar observations were made in
this study. With the one exception, the engineered strains
of £. coli and P. putida persisted in the activated sludge
effluent for up to 3 weeks. The richness of the effluent
apparently serves as a nutrient source for the organisms and
the presence of additional DNA did not affect their
survival. These factors have strong implications for genetic
transfers within wastewater facilities. Plasmid transfer
between GEMs and indigenous wastewater microflora has been
demonstrated in activated sludge and wastewater effluents
(Mancini et al., 1987; McClure et al., 1989).
In lake water, the addition of recombinant DNA appears
to have altered and improved the host response to
temperature. Interestingly, this response is not consistent
at all three temperatures. The recombinant DNA may also
afford these organisms a selective advantage over their
parent stains in terms of increased resistance to extraneous


143
Z SURVIVAL
120 ti
0 13 6 9
TIME (DAYS)
Figure 6-2. Percent survival of wild-type (HB101) and
genetically engineered (50008) strains of Escherichia coli
after exposure to lake water at 25C,
using viable plate counts.


87
Log CFU/ml
Time (hours)
Figure 4-7. Survival of genetically engineered (GEM)
and wild-type strain of Pseudomonas putida at 15C
in activated sludge effluent. Wild-type and GEM strains
recovered on non-selective medium; (-#-) and (-*-),
respectively. GEM strains recovered on
selective medium (-*) .


177
TIME (HOURS)
Figure 7-6. Degradation kinetics of spiked eDNA
in Lake Water. ( ) Untreated Sample; ( o ) Autoclaved
Sample; ( a ) Filter-sterilized Sample.


129
(Amy and Hiatt, 1989; Chaudhry et al., 1989; McClure et al.,
1989; Morgan et al., 1989; Steffan et al., 1989; Trevors et
al., 1989). The survival of GEMs in soils and soil extracts
has also been investigated (Bentjen et al., 1989; Devanas
and Stotzky, 1986; Fredrickson et al., 1988; 1989; Van Elsas
et al., 1989; Walter et al., 1987; 1989; Wang et al., 1989).
Since plant growth is primarily the result of soil-root
interactions, the root environment is considered as the
first candidate for efficient inoculation with useful
microorganisms. However, information regarding the ability
of GEMs to survive in the rhizosphere is still rather
scarce.
In this study, the complexity of the soil environment
was avoided by simulating the rhizosphere of growing corn
plants under hydroponic and sterile conditions. Only a few
factors were involved, allowing for a better understanding
of the observed phenomena. Moreover, it was also possible
to introduce known amounts of indigenous rhizospheric
microorganisms into the system to better simulate
rhizospheric conditions.
Devanas et al., (1986), using LB growth medium to
simulate the supply of nutrients in their system, observed
that microbial survival was primarily a function of the
nutritional status of the soil. In this study, root exudates
were the sole source of carbon for microorganisms added to
the rhizosphere. Root exudates are chemically diverse and
include simple low molecular weight compounds such as


Microcosm Design 44
Static renewal system 45
Flow-through system 45
Test Protocol for Survival Studies 47
Bacterial strains 47
Bacterial growth studies 49
Comparison of plating techniques 50
Comparison of selective media types 50
Comparison of Microcosms for Survival Studies... 51
Results and Discussion 52
Bacterial Growth Studies 52
Comparison of Plating Techniques 52
Comparison of Media types 57
Comparison of Microcosms 62
4 SURVIVAL OF AND PLASMID STABILITY IN GENETICALLY
ENGINEERED AND WILDTYPE STRAINS OF ESCHERICHIA
COLI AND PSEUDOMONAS PUTIDA IN AQUATIC
ENVIRONMENTS 65
Introduction 65
Materials and Methods 68
Bacterial Strains 68
Aquatic Samples 69
Survival Experiments 69
Microcosm design 69
Experimental procedures 70
Effect of temperature 70
Sterile vs non-sterile conditions 71
Toxicant effect 71
Plasmid stability 72
Statistical analysis 72
Results 73
Survival and Plasmid Stability in Lake Water.... 73
Temperature dependent studies 73
Sterile vs non-sterile conditions 79
Survival in the presence of a herbicide 81
Plasmid stability 83
Survival and Plasmid Stability in Activated
Sludge Effluent 83
Temperature dependent studies 83
Plasmid stability 93
Survival and Plasmid Stability in Ground Water.. 94
Survival studies 94
Plasmid stability 98
Discussion 98
5 FATE OF GENETICALLY ENGINEERED MICROORGANISMS
IN THE CORN RHIZOSPHERE 110
Introduction 110
vii


8
conjugation, transformation and transduction. Conjugation
involves the transfer of DNA from a donor to a recipient
bacterium and reguires cell-to-cell contact. The transfer
(tra) genes required for conjugation are encoded by
conjugative plasmids. In transduction, DNA transfer is
mediated by a temperate bacteriophage. These 'transducing
phage' may introduce novel DNA from donor cells to recipient
cells. Transformation is the process by which microorganisms
take up extracellular or 'naked' DNA and subsequently
acquire an altered genotype (Smith et al., 1981; Stewart and
Carlson, 1986). In all three processes, chromosomal and
plasmid DNA can be transferred from the host to the
recipient bacterium.
Protoplast fusion. Induced protoplast fusion has been
reported in both prokaryotic and eukaryotic microorganisms
(Hopwood and Wright, 1978; Peberdy, 1979). In this process,
bacteria lose their cell walls, their protoplasts fuse and
the cell walls are regenerated. After fusion, genetic
rearrangements can occur to give rise to new gene
combinations.
Mutagenesis. Mutations are heritable changes in genetic
material that can occur spontaneously or can be induced.
Errors in DNA replication and misrepair of DNA damage can
influence the occurrence of spontaneous mutations (Saunders
and Saunders, 1987). Induced mutations can also be achieved
by various chemical and physical agents termed mutagens.


42
Materials and Methods
Survival Chambers
Survival studies were conducted using modified membrane
diffusion chambers as described by McFeters and Stuart
(1972). The survival chambers were constructed from
6.5-mm Plexiglass acrylic sheeting (Commercial Plastics,
Jacksonville, Fla.) as depicted in Figure 3-1. The internal
liamen of the chamber was 6-cm in diameter and 6.5-mm wide,
and accommodated a 20-ml sample when assembled and filled.
The total surface area of the membranes within the chambers
was 56.8-cm and the surface area to volume ratio was 2.84.
Durapore membrane filters (0.2-um pore size, GVWP-293-
25, Millipore Corp., Bedford, Mass.) were cut into circular
pieces with a diameter of approximately 7.5-cm. Durapore
filters were used instead of cellulosic membrane filters
since the former are stronger and more resistant to
biodegradation. During assembly, the membrane filters were
inserted on either side of the central spacer and held in
place by the two outer retainers (Figure 3-1). A thin
coating of Lubriseal (Arthur A. Thomas Co., Philadelphia)
was applied between the membrane filter and chamber walls to
ensure a watertight seal.
Two 22-gauge hypodermic needles (Becto Dickerson Co.,
Rutherford, NJ) were fitted into the top of the central
spacer to allow filling and withdrawal of samples. To ensure
a secure and watertight seal, both needles were fixed into
position by applying a seal of silicone (Dow Corning Co.,


3
concerning the fate of GEOs in the natural environment.
However, research is already underway in many areas, and the
last several years has produced some useful and relevant
information that can be applied towards better understanding
the fate of GEOs in the environment.
The scientific, legal and policy issues associated with
environmental applications of biotechnology have been
addressed by several federal agencies. At present, four
regulatory agencies (USDA, EPA, FDA and OSHA) share
responsibility for controlling GEOs now covered by existing
laws. Three other agencies (USDA, NIH and NSF) are involved
in overseeing research activities (Fiksel and Covello,
1986). Current guidelines recommend research activities with
recombinant organisms and their products be confined to
contained settings within laboratories. Such guidelines have
made empirical methods such as microcosm testing,
indispensable tools for purposes of risk and fate
assessments.
This dissertation assesses the fate and survival of
GEMs and their recombinant DNA in natural environments. The
research focuses primarily on selected model GEMs developed
by transferring antibiotic or heavy-metal resistance genes
into Escherichia coli and Pseudomonas putida. two commonly
used host systems for recombinant DNA work. The overall
objective of the dissertation was accomplished by developing
and utilizing a microcosm approach to investigate specific
hypotheses.