bacteria in natural benthic aquatic commu-
nities, to identify response variables most
useful for detecting effects on introduced
organisms, and to evaluate the utility of
mesocosms for assessing the fate and effects
of GEMs in natural systems.

Comparison of xylE Gene Activity in
Different Molecular Constructs, H.M.
Abebe!, RJ. King!, S.E. Lindow’, KA. Short’,
and RJ. Seidler’ (‘ManTech Environmental
Technology, Inc., "Dept. of Plant Pathology,
University of California, ?U.S. EPA, Gulf
Breeze, FL)

The stability and level of expression of
plasmid-borne and chromosomally inserted
xylE marker genes, and assay systems for

these marked bacteria were assessed.

B. Environmental Effects
Studies

Genetic and Molecular Analysis of Sur-
vival Mechanisms of Pseudomonads, A./.
Anderson, R. Buell, J. Katsuwon, C. Heck, and
R Zaor, Biology Dept., Utah State University
Basic processes involved in colonization of
plant roots by pseudomonads were studied.

Route Specificity of the Toxicity of the
Kilodalton Protein, CY. Kawanishi and
M.E. Mayes, U.S. EPA, Research Triangle
Park, NC

Studies were conducted to ascertain the
effects of different routes of challenge with
the solubilized Bti parasporal crystal 28 kDa
polypeptide in rats. Changes in serum
parameters after intraperitoneal challenge

were monitored.

The Effect of a Genetically Altered
Bacterium on Nitrogen Transformation
Rates, Indigenous Microbiota, and.

Microbial Biomass in a Xeric Soil, L.K.
Gander', E.R. Inghan?’, J.D. Doyle’, and
C.W. Hendrick? ('ManTech Environmental
Technology, Inc., "Dept. of Botany and Plant
Pathology, Oregon State University, US.
EPA Environmental Research Laboratory,
Corvallis, OR)

This study examined the dynamics of
indigenous populations of bacteria,
protozoa, and nematodes in untreated soil,
and in soil treated with 2,4-dichlorophen-
oxyacetate (2,4-D), and amended with a
Pseudomonas strain engineered to degrade
this herbicide.

Effect of Organochlorine Compounds on
the Bioactivation of 2,6-Dinitrotoluene
in Fisher-344 Rats, S.E. George, R.W.
Chadwick, M.J. Kohan, and J.C. Allison,
U.S. EPA, Research Triangle Park, NC

‘The effect of organochlorine compounds on
metabolism and on GI tract flora were
evaluated.

Clearance of Environmental
Pseudomonads from CD-1 Mice
Following Intranasal Exposure, S.E.
George, M.J. Kohan, D.A. Whitehouse, and
L.D. Claxton, U.S. EPA, Research Triangle
Park, NC

The potential health effects associated with
intranasal exposure to engineered organisms
were assessed by monitoring of morbidity
and mortality of the host, and survival of
microorganisms in the lungs, nasal washing,
and intestinal tract.

Fate of Bacillus sphaericus Microbial Pest
Control Agent In the Environment, A.A.
Yousten and E.F. Benfield, Biology Dept.
Virginia Polytechnic Institute and State
University, Blacksburg

This research examines the fate of bacterial
spores during their interaction with the
aquatic environment as well as the fate of

ll


In order to ensure appropriate regulatory oversight of the biotechnology indus-
uy’, without interfering with the safe development and commercial use of biotech-
nology products, the U.S. Environmental Protection Agency (EPA) is developing
procedures to assess the potential risks that may be associated with the manufacture,
distribution, and use of microbial products. The EPA Office of Research and
Development (ORD) administers a research program structured to provide the
scientific tools for this risk assessment process; tools that allow sound evaluations of
the safety of various uses of bacteria, fungi, and viruses, including certain genetically
modified microorganisms.

Duluth, MN (5.4%)
Fate and Effects in Fresh
Water Environments

Corvallis, OR (37.3%)
Fate, Effects, and Transport
in Terrestrial Ecosystems

Gulf Breeze, FL (37.4%)
Marine and Estuarine
Ecosystems

Cincinnati, OH (1.5%)

Containment and Las Vegas, NV (4.8%)

Decontamination _ Research Triangle Aerosol Dispersal of
Park, NC (13.6%) Biological Agents
Human Health Effects

Relative participation of various EPA/ORD Laboratories in the Biotechnology Research Program based on FY
1992 funding levels.

5The U.S. Environmental Protection Agency functions under a number of statutes to carry
out its mission of protecting human health and the environment. While these statutes were not
written specifically to regulate biotechnology, there are two which have been interpreted as
investing EPA with the authority to do so: the Toxic Substances Control Act (TSCA) and the
Federal Insecticide, Fungicide,and Rodenticide Act (FIFRA). Under the Coordinated Framework
for Regulation of Biotechnology, EPA regulates many uses of microorganisms (see p. 8); the U.S.
Department of Agriculture (USDA) regulates agricultural uses of microbes, plants, and animals;
and the Food and Drug Administration (FDA) regulates foods, drugs, cosmetics, and biologics.

2


C. Control Strategies: focuses on assuring containment of biotechnology

products in field situations and production facilities

Field Releases

Methods of limiting the movement of microorgan-
isms and of monitoring them in the environment are
being developed. Experiments include the actual
application of microorganisms (including genetically
modified microbes) to specially designed and approved
experimental release sites representing different envi-
ronmental habitats. Controlled conditions allow for
practical evaluation of experimental techniques and
assure proper containment during field releases.

Mitigation

A variety of mitigation and risk reduction strategies
are being evaluated. These include novel biological
controls to limit the survival and gene exchange of
modified microorganisms, and physical and chemical

Studies are being conducted on the persistence of microorganisms
sprayed on plants, and on methods of reducing populations of these
microorganisms in soil.

decontamination methods for field sites. Since
environmental factors affect the effectiveness of such
procedures, each is being evaluated under a variety of
environmental conditions,

o 0 2 9 40
Days after inoculation

Regression lines of survival of
Erwinia herbicola in tilled plots
(From Donegan, K , R. Seidler, and
C Maytac. 1991. Can. J.
Microbiol, 37 708-712)


THE BIOTECHNOLOGY RESEARCH PROGRAM

The Research Program's activities are divided into three categories that reflect those areas of
research considered crucial to EPA's regulatory needs. These categories are: A. Environmental
Exposure Studies, B. Environmental Effects Studies, and C. Control Strategies.

A. Environmental Exposure Studies: aimed at understanding how
microorganisms survive, move, and interact in various environments

Detection and Enumeration

This work focuses on improving methods of
identifying and measuring populations of microorgan-
isms. Innovative detection and analytical methods are
being developed and assessed for their applicability and
potential usefulness in the risk assessment process.

Pigment production triggered by . . . . + :
catechol represents a quick and Microbial, serological, biochemical, and genetic

convenient means to enumerate methods are being tested and refined under laboratory
or verify the identity of certain conditions to maximize their sensitivity, and specific-

microorganisms in natural

ity, for microbes in soil, water, air, sewage, and animal
environments

and human digestive tracts.

Location of various samplers in a plot containing
strawberries that was sprayed with ice minus

Pseudomonas syringae
15m, 20m, 18m A
ay Ol
5) ~ s3 = |o gesmmeuaie
og 8 10 Towor
. ef a Bacteriologicel
Elk col egy foo. ‘Sampling Power
8) © Plot [2 *% | a sampling Platform
Ss ° ° 5 © Gravity plates
ag Multiple Sampii
Fl ee Andersen Samplers and all-glass
impingers have been used to monitor

survival and spread of microorganisms
following aerosol releases

Surface of an agar plate recovered from a Reynier sampler used

DAYO PLOTC. SITE 78 fg ‘Monitor the time and concentration kinetics of Pseudomonas
syringae applied in aerosols



STAFF

T.. EPA scientific staff combines expertise in
biotechnology and risk assessment procedures to bring
a unique scientific focus to the research program. They
are building the information base and developing
appropriate methods and protocols for risk assessment.
The staff scientists also foster interactive information
exchange with scientists in the fields of genetics,
biochemistry, ecology, and microbiology. They share
responsibility for a complimentary extramural program
administered through cooperative agreements,
interagency agreements, and contracts. Regular,
independent reviews of the research program serve to
guide the focus toward the needs of the regulatory
offices of EPA while maintaining a high standard of
scientific quality.

Preparation of an agarose gel for
analysis/recovery of DNA
restriction fragments.

‘Sampling zooplankton from a
‘temporary poo! site prior to

Researchers performing application of the mosquito
DNA sequence analysis to contral microorganism, Bacillus
track bacteria in the thuringiensis vat israelensis:
environment


B. Environmental Effects Studies: aimed at evaluationg the effects
of biotechnology products on organisms that share their environment

Ecological Processes

Attor IRfoduchon of Gutworms Fad Sprayed Losv
, s Fed Sprayed Leaves Environmental conditions can be
Be simulated on a small scale in “micro-
3 . cosms” in the laboratory. This allows
6 : scientists to test experimental
g 2 procedures. Microcosms also help to
1 identify and describe the effects of
‘oo 1 2 2 .
Days After Transfer of Larvae changes in the environ-
Pond veaeatng ment on natural
ridroma, a leaf-eating plant Fy . oy
pest, can be readily cultured in miei communities
the laboratory, and the larvae and the organisms
used for conducting fate and linked to them by the
genetic stability experiments with food web. Such effects
microbial pest control agents can also be estimated
(MPCAs) Microcosm studies
show that microorganisms can be through the use of
transported by insects from computer programs
sprayed plants to the soil, where designed to model the
they may multiply. behavior of organisms in
the environment.

Higher Organisms

There is a wide variety of microorganisms that may
be used as biotechnology products. For many, there is
very limited information regarding their effects on
other organisms. The focus of the current research is
on determining the means (genetic and molecular) by
which a products might have toxicologi-
cal or pathogenic effects, and
on identifying the host
organisms likely to be
affected. Results will be used

Tiny flourescent latex
particles applied in

enclosed chambers are to develop protocols for

used to simulate exposure ing the eff £ mi

of birds to microbial tesung U ee! ects ©: micto-
pesticides Such studies are organisms on varlous
important for determining vertebrates and invertebrates.
whether these pesticides

pose risks to wildlife.


United States Office of EPA 600/K-93/007
Environmental Protection Research and Development January 1994

Agency Washington DC 20460

Biotechnology

Research Program
Overview

U.S. Environment-!
Region 5, Lib

77 West Jacksaz s.r)
Chicago, IL 60604. 3590" 12th Floor

Protection Agency
19 )



RESEARCH SUMMARIES

The following summaries are abstracted from Review of Progress in the Biotechnology-
‘crobial P: 4

nt Pr

ram, EPA/GOO/R-92-147, and reflect

the status of EPA in-house research and research funded through cooperative agreements.
Requests for proposals for extramural research are issued annually. Most cooperative
agreements are funded for 2 or 3 years, and ORD initiated 32 agreements in the first 8

years of the program.

A. Environmental Exposure
Studies

Perspectives on Plasmid Stability: A
Study of the EPA Benchmark Plasmids,
T.T. Nguyen and RE. Lenski, Dept. of
Ecology and Evolutionary Biology, University
of California

This study focuses on the problem of
plasmid loss from a population of geneti-
cally engineered microorganisms.

Studies on Conjugal Transfer of Plasmids
From GEMs to Indigenous Aquatic Bac-
teria, 7: Barkay, U.S. EPA, Gulf Breeze, FL
The utility of catabolic gene assembly as a
tool for detection of conjugal transfer of
recombinant plasmids to indigenous
microorganisms was demonstrated under
optimal conditions. This experimental
approach will allow determinations of the
effect of environmental parameters on
transfer of conjugal plasmids.

Environmentally Induced Genetic Insta-
bility in Microorganisms, T.A. Kokjohn',
and RV. Miller (‘Argonne National
Laboratory, IL, *Dept. of Microbiology,
Oklahoma State University)

Pseudomonas aeruginosa was used as a model
system to study stress-induced genetic
alterations in bacterial cells.

Effects of Environmental Factors on
Bacterial Conjugation, M.A. Gealt, T.A.

Khalil, and S. Selvaratnam, Dept. of Bio-
science and Biotechnology, Drexel University
The effect of different factors on transcrip-
tion of tra and mob as might occur in
wastewater were studied to elucidate potent-
ial genetic interactions between released
bacteria and indigenous populations.

The Fate, Stability & Movement of
Foreign DNA in Filamentous Fungi: An
Environmental Study, M.B. Dickman! and
JF. Leslie ('Dept. of Plant Pathology,
University, Dept. of Plant Pathology, "Kansas
State University)

The stability of forcign DNA sequences in
fungi was evaluated to enhance the under-
standing of potential ecosystem effects of
DNA introduced via an eukaryotic vector.

Modeling Transduction in Aquatic
Environments, RV. Miller’, T.A. Kokjobn’,
and G.S. Sayler’ (Dept. of Microbiology,
Oklahoma State University, "Argonne
National Laboratory, IL, *Center for
Environmental Biotechnology, University of
Tennessee)

Pseudomonas aeruginosa is being used as a
model to study viral-mediated gene transfer
(transduction) in freshwater microbial
populations.

Stability of Foreign DNA in the Fungus
Colletotrichum gloeosporivides, J.L.
Armstrong! and D.L. Harris ('U.S. EPA,
Corvallis, OR, ManTech Environmental
Technology, Inc.)


Process Containment

This work focuses on identifying the causes of accidental emissions of biotechnol-
ogy products from production facilities, and on options for controlling emissions
and reducing exposure. EPA is developing engineering and cost models for assessing
risks associated with biotechnology processes in large-scale fermentation facilities.
Process equipment design, decontamination technology, worker exposure and
protection, and loss prevention techniques are integral components.

Number of Different

Microbial Species Reported
Use In the Literature 1980 -1986
Agriculture 296
Conversion of Biomass Wt
Industrial Chemical Production 1,058
Energy 356
Mining/Metal Recovery 167
Polymer/Macromolecule Production 1,213
Enhanced Oil Recovery 48
Waste/Pollutant Degradation 1,926
Other 133

Source: RIB Function/Organism Matrix - Fall 1986

Biotechnology Ap ons Under EPA Purview

Pollutant Degradation Nitrogen Fixation

*Enhanced OilRecovery = * Fuel Desulfurization

+ Wastewater Treatment —_* Biomass Conversion

* Lignin Degradation Pest Control

* Bioleaching of Ores Ice Nucleation (Snow making)



Transport

An understanding of the movement of microorgan-
isms through the environment is fundamental to evaluat-
ing the safety of biotechnology products. The mechanisms
and dynamics of transport between and within various
environmental components (e.g., air, soil, ground
water, water, plants, insects, animals, and humans) are
being studied. Where appropriate, this research may
extend to the development of mathematical modeling
frameworks and other decision support systems for
predicting transport and consequent exposure.

Survival and Colonization

A vital question preceding commercial development
and distribution of biotechnology products is, “What
is the potential for microorganisms to survive and
colonize particular habitats and under a variety of
conditions?” The pertinent cellular properties of the
microorganisms and relevant environmental factors
that influence survival and colonization are being
identified and described experimentally. This research
necessarily builds upon detection/enumeration
technologies as well as transport information. Here
again, much of the effort is planned to facilitate the
development of mathematical modeling frameworks
and other decision support systems.

Gene Exchange

This research includes analyses of genetic exchange
between microorganisms released to the environment
and their indigenous counterparts. Environmental,
genetic, organismal, and taxonomic factors which
affect and control gene stability and the rates of gene
exchange in the environment are being studied in a
variety of different environmental situations.

Pyramid-shaped mesh cage for
collection of emerging insects to
determine uptake and “fly-away”
of a microbial pest control agent
from the site.

Temporary woodland pools are
used to study the distribution,
survival, and effects of inverte-
brate populations of the
registered microbial pest control
agent, Bacillus thuringiensis.


EPA'S ROLE IN BIOTECHNOLOGY

T.. use of living organisms (or parts of organisms)
to make or modify products, to improve plants or
animals, or to develop microorganisms for specific
uses forms the basis of biotechnology.' The tremen-
dous potential of this technology encompasses the
culturing of microorganisms for toxic chemical
degradation and production of foods and pharma-
ceuticals; the creation of rapid, sensitive diagnositic
medical tools; and safe, effective control of agricul-
tural pests.

The promise offered by such developments is
evidenced by the growth of the biotechnology
industry. The industry is said to be “still inventing
itself”, as it has grown from under 100 companies
in 1970 to over 1,000 companies today.

Goats of the Risk Assessment Research Program

To provide the Agency with the scientific tools to:

= Allow beneficial uses of the technologies to move
forward in a safe manner.

= Accurately assess the risk of biotechnology products.

= Successfully control the use of products where
warranted,

SEE
“Given the growing
societal focus on
environmental issues
and increased aware-
ness of environmental
threats, the potential
contributions of envi-
ronmental biotechnot-
ogy may be far greater
than were earlier
anticipated.”

~from the Ernst & Young
Changing Environment’

*U.S. Congress, Office of Technology Assessment, Oct., 1991. Biotechnology in a Global
Economy, OTA-BA-494, U.S. Government Printing Office, Washington, D.C.

*Burrill, S.G., and K.B. Lee, Jr. 1990. Biotech 91: A Changing Environment. Ernst &

Young, San Franscico, CA., pages 1 & 4.


spores ingested by certain nontarget
invertebrates.

In Vitro Survival and Competition of
Environmental Psuedomonads Within a
Human Fecal Flora Culture, G.M.
Nekon', L.D. Claxton’, and S.E. George’
(Environmental Health Research and Testing,
Inc, ?U.S. EPA, Research Triangle Park, NC)
“This study is a continuation of research into the
potential adverse health effects due to the envi-
ronmental release of microorganisms.

Laboratory Observations of the
Inhibition of Soil Fungi by MPCA’s of
the Genus Pseudomonas, H.M. Abebe',
VP. Fieland', and RJ. Seidler (‘ManTech
Environmental Technology, 7U.S. EPA,
Corvallis, OR)

‘This work, an assessment of interactions
between bacteria and soil fungi, isa continua-
tion ofa study which demonstrated reduction
of soil fungal populations due to an accumula-
tion of a metabolic intermediate (2,4-dichlo-
rophenol) of 2,4-D degradation.

Interactions of Nontarget Invertebrates
with Bacillus thuringiensis var israelensis
in Natural Ponds, RL. Anderson', E Mead’,
LJ. Shannon’, and D. Janssen? ('U.S. EPA,
Duluth, MN, *American Scientific Interna-
tional, *Biology Dept., University of
Minnesota)

‘The objective of this project was to
determine, in temporary pools treated with
control mosquitoes, whether or not non-
target animals accumulated and retained the
spores of Bti.

Current Investigations on Micro-
sporidian Test Systems, W.S. Fisher, JW.
Fournie, CL. McKenney, Jr., and D.P.
Middaugh, U.S. EPA, Gulf Breeze, FL

A totally enclosed aquarium has been used

12

asa test system to assess the effect of a
variety of microbial pest control agents
(MPCA’s) on marine and freshwater non-
target species. Current focus is on micro-
sporidian (unicellular, obligate intracellular
parasites) MPCA’s.

Use of Ribosomal rRNA Sequences to
Characterize Diversity and Stability of
Microbial Populations, R Devereux,
Technical Resources, Inc., Gulf Breeze, FL
This research utilizes 165 rRNA-targeted
hybridization probes to define populations
of sulfate-reducing bacteria and assess
population stability in relation to ecological
Processes.

An Overview of Protocol Development
for Avian Pathogenicity Tests, A.
Fairbrother' and P. Buccholz? (U.S. EPA,
Corvallis, OR, *ManTech Environmental
Technology, Inc.)

‘This report is a review of the development
of standardized protocols that can be used to
determine the pathogenicity of microorgan-
isms in nontarget avian species. Emphasis is
placed on recent developments in respira-
tory protocols.

Test Procedures for Assessing Hazards of
Microbial Pest Control Agents to
Freshwater Fish, Virginia M. Snarski, U.S.
EPA, Duluth, MN

The interactions between fish and the
registered MPCA, Bacillus thuringiensis
subsp. israelensis (Bti), under laboratory
conditions, were investigated.

Field Calibration of Soil-Core
Microcosms for Evaluating Fate and
Effects of Genetically Engineered
Microorganisms in Terrestrial
Ecosystems, H. Bolton, Jr. and J.K.
Frederickson, Battelle Northwest Pacific
Laboratory, Richland, WA


Mechanisms of Effects of Recombinant
Streptomyces on the Carbon Cyd in
Soil, D.L. Crawford, Z. Wang, and B.H.
Bleakley, Dept. of Bacteriology and
Biochemistry, University of Idaho, Moscow
The genetic and chemical bases for the
effects of recombinant S. lividans strains
expressing lignin peroxidase on carbon
cycling were examined.

C. Control Strategies

Comparison of Survival on the
Phylloplane of Bacteria Released in
Greenhouse and Field Experiments, K:
Donegan’, J. Armstrong’, C. Matyac', and R.
Seidler? (‘ManTech Environmental
Technology, Inc.,?U.S. EPA, Corvallis, OR)
The use of greenhouses for predicting the
results of field releases, and the influence of
bacterial species, plant species, and environ-
mental conditions were investigated.

Potpourri of Bioaerosol Research at
ERL-Corvallis in 1990, B. Lighthart!, B.T.
Shaffer, and B. Marth? ('U.S. EPA,
Corvallis, OR, ?ManTech Environmental
Technology, Inc.)

This report summarizes progress in 1990 in
(1) increasing bioaerosol sampling effi-
ciency, (2) development of better tools
(Auto-DAT and EBARC) to evaluate
bioaerosol generation potential, and (3)
development of predictive simulation
models of bigaerosol dispersal and deposi-

tion.

Measuring Entrainment of Bacteria
From the Phyllosphere, M. Walter’, V.
Fieland', L. Ganio', and R. Seidler?
(ManTech Environmental Technology, Inc.,
U.S. EPA, Corvallis, OR)

A method of artificially inducing bacterial
entrainment from the phyllosphere was
developed, and various sampling methods

14

for detection of entrained bacteria were
compared. The effect of species of host
plant and the effect of entrainment on
bacterial viability were assessed.

Environmentally Released Micro-
organisms and the Enteric Microflora, W.
Dobrogosz', Y. Lin', M. Fiuzat', E. George’,
and L. Claston? ('Dept. of Microbiology,
North Carolina State University, ?U.S. EPA,
Research Triangle Park, NC)

Methodologies for growing enteric
anaerobes were compared, and various
bacterial species from mouse intestines were
enumerated.

Biotechnology Engineering Risk
Management, /. Burckle, U.S. EPA Risk
Reduction Engineering Laboratory

A number of reports have been written
addressing concerns associated with large-
scale production of biotechnology products.
‘These concerns include worker exposure,
release of GEMs from production sites, and
the efficacy of containment and destruction
techniques.

Biotechnology Quality Assurance, A.
Smiecinski and L. Stetzenbach, Environ-
mental Research Center, University of
Nevada, Las Vegas

A document intended to assist scientists in
understanding and implementing QA
principles as they apply to biotechnological
projects has been produced.

The Use of Lethal Bacterial Genes to
Limit the Survival of Intentionally
Microorganisms, W-H. Jeffrey, SM.
Cuskey, and R.B. Coffir? (' Technical
Resources, Inc.,?U.S. EPA, Gulf Breeze, FL)
Effects on growth, lethality, and mechanism
of action of the RK2 gene kilA were
examined.


OLS

Contents

EPA’s Biotechnology Research Program, begun in 1984, focuses on
predictive assessment of microbial biotechnology products. This
booklet describes the various types of research, and how they
contribute information needed for assessment and regulation of
these products.

EPA’s Role in Biotechnology ..........sessscssesssessesseesesstesnesseeenes 1
Stall oo eeesssesecsssesccssseeccsseeesssscessnsesssnnsesssnsecansneeessneceennsenssnseses 3
The Biotechnology Research Program ......ssssssssesssssesessseseesee 4
Research Summaries ....ssssccssesssssscessssscesssseccesseessssersssnneesssnees 9

BB Printed on Recycled Paper


Disclaimer
This document has undergone the United States Environmental
Protection Agency's peer and administrative review process and has
been approved for publication as an EPA document. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.

Comments or questions regarding this report should be directed to:

Dr. Richard Coffin
Environmental Research Laboratory
US. Environmental Protection Agency
1 Sabine Island Drive
Gulf Breeze, FL 32561-5299

Cover Photo: Micro autoradiogram of acridine
orange stained Pseudomonas aeruginosa PAOI
viewed under blue light excitation. Many cells
exhibit a metabolically active, but non-
reproducing filamentous morphology as a
result of incorporation of E. coli lethal genes.



The fungus C. gloeosporioides var.
aeschynemone (CGA) was selected as a model
organism to study the factors that determine
the stability of transformed marker genes
under simulated environmental conditions.

Genomic Plasticity and Catabolic
Potential of Pseudomonas cepacia, T.G.
Lessie, A. Ferrante, A. Byrne, M.S. Wood, and
HLP. Cheng, Dept of Microbiology, University
of Massachusetts

The construction of a physical map of the
chromosome of P. cepacia has been under-
taken to provide insight into the basis for
this bacterium’s novel catabolic functions
and extraordinary nutritional versatility.

Application of DNA Hybridization to
Compare Bulk DNA’s from Different
Soils, L.A. Porteous and ].L. Armstrong, U.S.
EPA, Corvallis, OR

A rapid, small-scale method of extracting
DNA pure enough for use in hybridization
experiments was developed. When coupled
with dot-blot techniques, DNA extracts can
be compared and quantified.

Update on the Assessment of Bacterial
Dispersal From Foliage by Rain, HA.
McCartney and J. Butterworth, AF.R.C.
Institute of Arable Crops Research, Rothamsted
Experimental Station, Harpenden, Herts,
England

The bacterial content of run-off from leaves
and of water droplets dispersed by splash
were assessed.

Field Release of a Genetically-Altered
Baculovirus with a Limited Survival
Capacity, HA. Wood and P.R. Hughes,
Boyce Thompson Institute, Ithaca, NY

This project is being conducted to evaluate
the spread and the survival potential ofa
genetically altered virus within the wild-type
population. The release was conducted with

10

an altered Autographa californica nuclear
polyhedrosis virus (AcCMNPV) which has a
deleted polyhedron gene, but no foreign
gene inserted.

Modeling the Fate of Bacteria in Surface
Waters, J.P. Connolly, RB. Coffir?, and
RE, Landeck (‘Environmental Engineering
& Science Program, Manhattan College, ?U.S.
EPA, Gulf Breeze, FL)

‘The overall objective of this project is the
step-wise development of the components
of a framework for modeling the movement
and growth of bacteria that have been
introduced into a surface water system.

Epiphytic Fitness Genes and Phenotypic
Adaptation, Steven E. Lindow, Dept. of
Plant Pathology, University of California
Studies have been initiated with Pseudomo-
nas syringae to determine the range of
differences in epiphytic fitness exhibited by
a given genotype of bacterium that is
exposed to different environments prior to
inoculation onto plant surfaces, and to
determine novel fitness determinants.

Molecular Studies of Microbial
Ecosystem Perturbations, D.A. Stahl,
University of Winois

‘Techniques have been developed using
comparative rRNA sequencing and
hybridization for use in the characterization
of microbial populations in natural
communities, thus avoiding limitations
associated with pure culture isolation.

Habitat Specific Differences in
Persistence and Effects of Introduced
Cellulolytic Bacteria Used as Surrogates
for GEMs, T. Bott and L.Kaplan, Stroud
Water Research Center, Academy of Natural
Sciences, Avondale, PA

‘The primary objectives of this research were
to determine the persistence of introduced


This work focused on calibration of soil-core
microcosms with their field counterparts for
microbial fate and ecosystem structural and
functional properties, and the resultant effect
of introduced microorganisms on these
properties.

Development of Test Methods to Assess
Fate of Microbial Pest Control Agents
and Their Effects on Nontarget Aquatic
Organisms, FJ. Genthner, G.M. Cripe, and
D.P. Middaugh, U.S. EPA, Gulf Breeze, FL
A fully contained single species test system
was developed to determine whether exposure
of a nontarget aquatic invertebrate to a
microbial pest control agent (MPCA) will
result in infectivity, toxicity, or pathogenicity.

Protocol Development for Testing the
Effects of Bacterial Pesticides on Bene-
ficial Beetles, R. James! and B. Lighthart®
(‘Mantech Environmental Technology, Inc.,
?U.S. EPA, Corvallis, OR)

In order to develop a standard assay for
testing the effects of bacterial pesticides on
beneficial insects, the effect of temperature,
dietary stress, and larval instar on the
susceptibility of Hippodamia convergens (the
convergent lady beetle) to a weak bacterial
pathogen was tested.

Use of 16S rRNA Probes to Correlate
Sulfate Reducer Community Structure
with Mercury Methylation, MR. Winfrey
and J. Winfrey, Dept. of Biology and
Microbiology, University of Wisconsin

Sulfate reducing bacteria (SRB) are known
to be active methylators of mercury, and
were found to comprise a larger portion of
the microbial population in low pH lakes in
northern Wisconsin. The use of SRB-
specific probes provides previously unattain-
able information on the community
structure of these microorganisms which

play a key role in nutrient cycling in
anaerobic environments.

Assessing Host Specificity of Fungal
MPCA’s to the Beneficial Wasp
Trichogramma pretiosm, D.K. Sewell! and
B. Lightharé (‘ManTech Environmental
Technology, ?U.S. EPA, Corvallis, OR)

This study was undertaken to develop
bioassay procedures to evaluate pathogenic-
ity of entomogenous fungi, compare specific
and nonspecific fungi, and evaluate factors
that may affect the expression of fungal
virulence to T. pretiosum or the susceptibility
of T. pretiosum to fungal pathogens.

Field Validation of Laboratory
Microcosms Using Bacillus thuringiensis
var. israelensis, L.]. Shannon, D.M. Janssen,
and R.L. Anderson

Both mixed flask culture and core micro-
cosms were evaluated to determine whether
they accurately and sensitively reflect Bri
survival, effects of Bti on target and non-
target species, and influence of physical,
chemical, and biological conditions in
natural systems on monitoring procedures.

Effects of a Lignin-Degrading
Recombinant Streptomyces on Microbial
Activity and Nutrient Cycling in Soil,
DLL. Crauford', Z, Wang}, J.D. Doyle’, H.
Bolton, Jr}, J.K. Frederickson’, S.A. Bensjen’,
and CW. Hendricks! (’ Dept. of Bacteriology
and Biochemistry, University of Idaho,
Moscow, ?ManTech Environmental
Technology, Inc., *Battele Pacific Northwest
Laboratory, Richland, WA, ‘Environmental
Research Laboratory, Corvallis, OR)

The effects of release of recombinant S.
lividans on bacterial and fungal activity, and
on carbon and nitrogen cycling in soil were
evaluated.