Methods for Studying Bacterial Gene Transfer in
Soil by Conjugation and Transduction
(U.S.) Corvallis Environmental Research Lab., OR
Uft Q«perttn*ffl ol Gommarce
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PB89-1950U4
EPA/600/3-89/042
June 1989
METHODS FOR STUDYING BACTERIAL GENE TRANSFER IN SOIL
BY CONJUGATION AND TRANSDUCTION
G. Stotzky, Monica A. Devanas1, and Lawrence R. Zeph2
Laboratory of Microbial Ecology, Department of Biology,
New York University, New York, NY 10003
Present addresses: * Department of Biological Sciences, Rutgers University, New
t
Brunswick, NJ 08903; 2U. S. Environmental Protection Agency, Office of Toxic
Substances, 401 M St., SW, Washington, D. C. 20460
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
C0RVALLIS, OR 97333
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TECHNICAL REPORT DATA-
(Ftent n»d lauruenau om iMt rtrmt btfort compkttnt)
«. asport no. 3.
EPA/600/3-89/042
>. RSCIPISNT* ACCSSSIOWNO.
PB89 1 QXOd A /M
4, TITlt ANO lUiTITLI
Methods for Studying Bacterial Gene Transfer
in Soil by Conjugation and Transduction
S. REPORT DATS
June 1989
ft. PSRPORHING ORGANIZATION COOt
7. AUTMOA(S)
G. Stotzky, M. Devanas, and L. Zeph
1. PSRPORMINO ORGANIZATION RCPORT NO.
». PIRPORMING ORGANIZATION NAM! AND AOORSSS
New York University, NY, NY.
10. PROGRAM CLCMSNT NO.
11. MNTIUCT^IUfclT WO."
13. SPONSORING AGtNCY NAM! ANO AOORCSS
US Environmental Protection Agency
Environi-nental Research Laboratory
200 SW 35th Street
Corvallis, OR 97333
13.TYPI OP RIPORT ANO PCRIOD COVIRSO
Puhllshprt Rppnrt
14. SPONSORING AOiNCY COM
EPA/600/2
11. SUPPLEMENTARY NOTKS
1989. A literature review and series of protocols on carrying out gene
transfar experiments in soil.
14, ASSTRACT
•f'li ¦ '
'The purpose of this document is to provide a series of protocols by
waich a trained technician can conduct studies on the transfer of
genetic information by conjugation or transduction in soil, with
emphasis on bacteria containing recombinant DNA. The level of the
document is geared to technicians with some background and experience
in standard laboratory methods used in microbiology but who have limited
knowledge of, and experience in, bacterial genetics, molecular biology,
and microbial ecology, especially in soil. Many of the specific
techniques described in £Vtis-document for studying gene transfer in soil
were developed by the authors during the past few years. The primary
motivation for these studies was the need for information on the
survival of, and gene transfer by, genetically engineered bacteria that
could be used in risk assessment of the release of genetically
engineered microorganisms to the environment.
17. KSY WORM ANO DOCUMINT ANALYSIS
i. MSCRtrroRS
b. IDS NT IP If RS/OPEN INOtOTIRMS
C. COS ATI Field/Group
1». OISTRJSUTION STATtfMfNT
- Release to Public —
It.IICyRlTY. CWAfS (ThuKtport)
unclassified
ai.no. or packs
170
*0 ttCURITY CLASS (Thuptt*)
Unclassified
33. PRICI
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The research described in this report has been funded by the U.S. Environmental
Protection Agency's Environmental Research Laboratory in Corval.lis, Oregon,
through the Terrestrial Microbial Ecology/Biotechnology.Program. This document
has been prepared for ERL-Corvallis through Purchase Order 8B1044NTTA. It has
been subjected to the Agency's peer and administrative review and approved for
publication. Mention of trade names or commercial producis does not constitute
endorsement or recommendation for use.
For further information, contact Ramon J. Seidler, Project Officer, phone: 503-
757-4661 or FTS 420-4661.
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EXECUTIVE SUMMARY
The purpose of this document is to provide a series of protocols by' which a trained
technician can conduct studies on the transfer of genetic information by conjugation or
transduction in soil, with emphasis on bacteria containing, recombinant ON A, The level
of the document is geared to technicians with some background and experience in
standard laboratory methods used in microbiology but who have limited knowledge of and
experience in bacterial genetics, molecular biology, and microbial ecology, especially in
soil. Consequently, Section I discusses soil as a habitat for microorganisms and stresses
the importance of the physicochemical and biological characteristics of soil in the
activity, ecology, and population dynamics of microorganisms in this habitat, which, in
turn, influence the transfer of genetic information., Section n briefly discusses the
molecular aspects of gene transfer by conjugation and transduction and reviews the
relevant literature on such .transfer in soil. Section III introduces the concepts of
microcosms as surrogates for the field and emphasizes that microcosms differ in
complexity of design; this Section is not intended to be an exhaustive review of
microcosms, as detailed protocol documents on microcosms are available and are
referenced. Section ^ provides general guidelines on the selection, preparation,
maintenance, and storage of soils. More specific guidelines on these aspects are
presented in Sections V and VI. Sections I through IV are intended to bring the novice
rapidly to a level of understanding that will enable the intelligent and thoughtful conduct
of "hands-on" studies of gene transfer. Section V provides details on how to study the
conjugal transfer of chomosomal- and plasmid-borne genes. Section VI provides details
on how to study gene transfer via transduction by bacteriophages. Both Sections V and VI
provide details on how to conduct these studies in vitro, which is necessary not only to
verify the bacterial strains, novel genes, and phages used, but also to learn the
.techniques, toestlmate the frequencies of gene transfer, and to establish base-line data
for comparison with in situ results. Once the techniques have been mastered in vitro, it
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will be possible to conduct studies on conjugation and transduction in soil, employing the
techniques developed specifically for use in soil. Section VII provides details on how to
identify, characterize, and confirm any presumptive recombinant bacteria that are
obtained, using the techniques of both classical microbiology, such as selective and
differential media, and molecular biology, such&s DNA "fingerprinting", plasmid profiles,
DNA-DNA hybridization with DNA probes, and serology. Section VIII provides guidelines
for obtaining the types of information that are necessary for quality assurance and
quality control. An extensive bibliography, with approximately 200 references, an
Appendix containing recipes for media and antimicrobial agents commonly used in studies
of conjugation and transduction, and examples of results of studies on gene transfer in
soil are included.
Many of the specific techniques described in this document for studying gene
transfer in soil were developed by the authors during the past few years, in part with
support from the U.S. Environmental Protection Agency, especially in the form of
cooperative agreements (CR812484, CR813431, CR813650) with the Corvallis
Environmental Research Laboratory (CERL). The primary motivation for these studies
was the need for information on the survival of, and gene transfer by, genetically
t i
engineered bacteria that could be used in risk assessment of the release of genetically
engineered microorganisms to the environment. The support, encouragement, and
suggestions provided by Dr. R. J. Seidler, J. L. Armstrong, and other members of CERL
are gratefully acknowledged.
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Methods for Studying Bacterial Gene Transfer in Soil
by Conjugation and Transduction
Table of Contents
L. Introduction
Microhabitats in soil 1-1
Gene transfer in soil 1-3
Biological factors 1-5
Effects of biological and physicochemical environmental factors
on gene transfer in soil 1-9
Microbial competition 1-10
Energy sources 1-11
Temperature 1-11
pH 1-13
Water content 1-14
Oxygen and Eh 1-15
Ionic composition l—15
Electromagnetic radiation 1-16
Surfaces ]-16
Interactions between factors 1-17
Conclusions 1-18
0. Literature Review of Conjugation and Transduction in Soil
CONJUGATION II-l
Introduction IM
Transfer of chromosomal genes II-2
Conjugative plasm ids IT-3
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Nonconjugative plasm ids 11-3
Mobilization 0-4
Cointegrate formation U-4
Mobilizing elements 0-5
Spectrum of conjugative bacteria 11-5
Survival of introduced genes in situ 0-6
Conjugation in situ D-12
Transfer of chromosomal genes 0-12
Transfer of plasmid genes 0-13
Conclusions 0-22
TRANSDUCTION 11-26
Introduction n-26
Transduction in situ fl-28
Conclusions 0-39
Terrestrial Microcosms •
Introduction 01-1
Simple microcosms 1D-1
Master jar studies 01-2
Soil replica plating 111-4
Complex microcosms 01-5
Selection, Preparation, Maintenance, and Storage of Soils
Introduction IV-1
Selection of soils IV-l
Preparation of soils 1V-2
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Maintenance of soils IV-3
Sampling of soils IV-4
Storage of soils IV-5
V. Methods for Studying Conjugation
IN VITRO V-l
Chromosomal transfer V-l
Media V-l
Inoculum preparation V-l
Mating V-3
Incubation V-7
Recovery of donors, recipients, and transconjugants V-8
Syntrophy V-9
Frequency of recombination (FOR) V-10
Plasm id transfer V-ll
Media V-ll'
Inoculum preparation V-13
Mating V-13
Calculation of frequency of conjugation V-l4
IN SOIL V-l 4
Chromosomal transfer V-l 4
Inoculation V-l 5
Incubation V-l 5
Sampling V-l 5
Recovery of donors;, recipients, and transconjugants V-16
Calculation of conjugation frequency V-17
Plasm id transfer V-17
Inoculation V-17
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Recovery of donors, recipients, and exconjugants V-18
Transfer to indigenous bacteria V-18
Limits of detection of gene transfer V-21
Stepwise Summary of Procedures for Studying Conjugation V-22
in Soil
VL Transduction
Transduction in vitro Vl-l
Preparation of phage lysates Vl-l
Preparation of bacterial cultures VI—2
Transduction procedure Vl-2
Transduction in soil VI-3
Inoculation and amendment of soil Vl-3
Incubction and sampling VM
Enumeration Vl-5
Detection of transductants in nonsterile soil VHi
Recovery of phages from soil VJ-7
Suppression of fungal growth on selective media Vl-8
Transduct ion frequency V1-9
Stepwise Summary of Procedures for Studying Transduction Vl-10
In SoU
VIL ldentificattoa, Characterization, and Confirmation of Recombinants
Introduction . VII-1
Selective media Vll-i
"Breakthrough" of indigenous microbes VII-3
"Viable but nonculturable" bacteria VIM
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Gene transfer on recovery media vs. jn situ VD-5
DNA "fingerprinting" and plasmid profiles VII-5
DNA probes VD-6
Introduction VU-6
Types o^DNA probes VU-8
Preparation of DNA probes VD-8
Hybridization techniques VD-10
Southern hybridization VII-10
' Slot/dot blot hybridization V1I-11
Colony hybridization V11-11
Sensitivity and specificity of DNA probes Vfl-12
Serological techniques VD-15
Heat induction of prophages VD-15-
Quality Assurance/Quality Control
Sample representativeness and custody VUJ-1
Sampling procedures VI11-1
Comparability VI1I-1
Calibration procedures and frequency VOI-l
Instrumental VIII-2
Analytical Vm-2
Microbiological VUI-2
Analytical procedures VIO-3
Experimental design and statistical analyses VUI-3
Data analysis and reporting VID-4
_lnternal_quality control checks -VIII-4-
Preventive maintenance VIO-4
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Corrective action
Literature Cited
Appendix
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L Introduction
Microhabitats in soil
Soil is unique among microbial habitats, as it is a structured environment with a
high solid: Water ratio (Stotzky, 1974, 1986). Inasmuch as all microbes are aquatic
creatures, their metabolism in soil is restricted to microhabitats wherein there is a
continual supply of available water. Hence, their distribution is essentially restricted to
microhabitats that contain clay minerals, as sand and silt do not retain water long
against gravitational pull. Clay minerals, because of their surface activity, retain water
against this pull, as the water adjacent to these active surfaces and coordinated with
charge-compensating ions on theclays becomes sufficiently ordered to form a quasi-
crystalline structure (i.e., the strong attraction of water molecules to the negatively and
positively charged surfaces of clay minerals'and their charge-compensating ions enhances
the hydrogen bonding of adjacent water molecules). The'ordering of this clay-associated
1 i 1
water decreases with distance from the clay surface until a distance is reached at which
the water is no longer under the attraction of the clay and is susceptible to gravity.
Clay minerals do not exist free in soil but are present as coatings, or cutans, on
larger sand and silt particles or as oriented clusters, or domains, between these 1
particles. The clay-coated particles and domains cluster, primarily as the result of
electrostatic attraction between the net negatively charged faces and the net positively
charged edges of clays, into microaggregates, that, in turn, cluster to form aggregates
that can range from 0.5 to 5 mm in diameter and are stabilized by organic matter and
precipitated inorganic materials. These aggregates retain water, the thickness and
permanence of which depend on the type and amount of clay and organic matter within
the aggregates, and this water may form bridges with the wster of closely adjacent
aggregates. These aggregates or clusters of aggregates, with their adjacent water,
comprise the microhabitats in soil wherein microbes function. The space between the
microhabitats constitutes the pore space, which is filled with air and other gases and
1-1
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volatiles (see front and back cover pages).
As the result of the discreteness of microhabitats in soil, the probability of
genetic exchange is' markedly less than the probability in ecosystems wherein water is
continuous. Except for periods when soil is saturated with water (e.g., after a heavy
rain, snow melt, or when irrigated), individual microhabitats are isolated by the
surrounding pore space, and movement of bacteria, transducing bacteriophages, and
transforming DMA among the microhabitats is limited to areas where water bridges
between microhabitats may occur. Even when the pore space is saturated, movement
between microhabitats may be restricted, as the surface tension of the ordered water
around aggregates may be too great to allow passive movement of bacteria or even
active movement by flagellated cells. (There is no convincing evidence that bacteria are
flagellated in soil, even though they, may have the genetic capability to produce flagella
when isolated from soil and cultured in liquid media or on agar.) However, filamentous
fungi appear to be able to bridge pore spaces between microhabitats, even when the pore
spaces are not filled with water, as these fungi grow apically from mycelia that have a
food and water base in a microhabitat and, therefore, are independent of the nutrient and
water conditions surrounding the growing mycelia. Moreover, the extending mycelia
probably have a surrounding water film in which bacteria or bacteriophages may be
transported from one microhabitat to another.
These conditions in soil differ markedly from those in sediments of aquatic
systems. Although clay minerals in sediments also occur as domains and as cutans on
larger particles and in aggregates, water-dependent microhabitats do not exist as they do
in soil, as the water in sediments is essentially continuous from one aggregate to the
next. Moreover, microbes in sediments appear to colonize primarily sand and silt
particles, rather than clays, as water surrounds these particles, and the need to overcome
the electrokinetic repulsion between net negatively charged clays and microbes is
eliminated. Hence, care must be exercised in extrapolating observations on gene
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transfer in sediments to soil and vice versa.
Gene transfer in soQ
The majority of studies on the transfer of genetic information in soil, especially
with genetically engineered microorganisms (GEMs; i.e., microorganisms whose genetic
information has beei} deliberately altered), have been conducted with bacteria, wherein
DNA can be transferred in situ by conjugation (cell-to-cell contact), transduction (via a
bacteriophage), and transformation (uptake of "naked" DNA by an intact cell). Although
these phenomena have been demonstrated in a wide spectrum of gram-positive and gram-
negative bacteria in the laboratory (i.e., in pure culture), there is sp£"se information on
their occurrence in soil and other natural habitats (Levy and Miller, 1989; Stotzky, 1989;
Stotzky and Babich, 1986; Trevors et al., 1987).
The first studies on the transfer of genetic information in soil were Conducted
only in sterile soil (Graham and Istock, 1978; Weinberg and Stotzky, 1972), as methods for
conducting such studies in nonsterile soil had not yet been developed. Studies conducted'
in sterile soil have little relevance to what occurs in nonsterile soil. Hence,
extrapolations from results obtained in sterile soil to what presumably occurs in
nonsterile soil should be viewed with skepticism, as Should studies conducted in soil
extracts, wherein microhabitats have been disrupted and physicochemical characteristics
altered. Nevertheless, studies in sterile soil can be valuable, when coupled with parallel
studies in nonsterile soil, as techniques for subsequent use in nonsterile soil can be
developed, and the effects of the indigenous microbiota and of surfaces and some other
physicochemical characteristics on survival, establishment, growth, and gene transfer
can be estimated.
Many of the early studies on gene transfer in soil were conducted with Escherichia
coli, which is not an autochthonous member of the micrcbiota of soil. There were
numerous plausible reasons why£. coli was used as the model bacterium in these
studies; e.g., 1) the genetics of coli were better defined than those of other bacteria;
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2) numerous strains with a spectrum of chromosomal alterations or containing different
plasmids were readily available; 3) there had been extensive genetic engineering of E.
coli strains for use in a variety of industrial applications; 4) because of the successful
experience with genetic engineering for industrial applications, it appeared reasonable'to
assume that strains of E. coli engineered to perform specific functions would be used
initially for releases to the environment; 5) E. coli has been shown to transfer plasmid-
borae genetic information to over 40 genera of gram-negative bacteria and even to some
gram-positive, bacteria; and 6) although primarily an inhabitant of the gastrointestinal1
tract of many, but not all, animals, £. coli is increasingly found in fresh and estuarine
waters and in soils in urban and agricultural areas, probably as the result, in large part,
of the presence of human beings. The tendency now is to conduct studies on.survival,
growth, establishment, and gene transfer with bacterial species that are autochthonous in
soil, especially as the genetics of these species'become more defined^
A major impetus for these studies has been the concern about the release of GEMs
to the environment, especially to soil, for agricultural
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conferred on the original or subsequent hosts by the DNA. These phenomena are
affected by the physicochemical and biological characteristics of soil and other natural
habitats (Table 1).
The relative importance of individual environmental characteristics varies with
i . t
the specific habitat (e.g., electromagnetic radiation is probably relatively unimportant in
soil, whereas it is extremely important in aquatic habitats), and their effects are usually
greater on introduced than on indigenous microbes. Moreover, none of these
characteristics exerts its influence individually but, rather, in concert with other
characteristics. Although the influence of one or a few characteristics may predominate
in a specific habitat, these influences have indirect, but cascading, effects on other
characteristics. Consequently, an alteration in one environmental factor may result in
simultaneous or subsequent changes in other characteristics and, ultimately, in the
habitat, and therefore, in.the ability of both introduced and indigenous microbes to
survive, establish, grow, and transfer genetic information (Stotzky, 1974, 1986; Stotzky
and Krasovsky, 1981). Because the possible permutations of interactions between these
environmental characteristics are vast, the relative success of microbes containing new
genetic information to transfer this information in soil cannot be easily predicted.
Biological factors
For detectable gene transfer to occur in soil or other natural environments, the
population densities of the donors and recipients of the gene(s) must be sufficiently
high. This is especially true in soil, where the spatial separation of microhabitats
reduces the probability of transfer. Consequently, for a bacterium containing a novel
gene to transfer this genetic information at a detectable frequency, it must be able to
establish (i.e., colonize) and grow to sufficiently high densities within the microhabitat,
especially if it is a recombinant that has resulted naturally from transfer in situ. If the
recombinant bacterium is introduced into soil, even at high densities, it will have
difficulty colonizing and growing, as the microhabitats are already filled with organisms
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Table 1, Factors ^ffecting the activity, ecology, arid population dynamics of
microorganisms in natural habitats.
Carbon and energy, sources
Mineral nutrients
Growth factors
Ionic composition
Available water
Temperature
Pressure
Atmospheric composition
Electromagnetic radiation
PH
Oxidation-reduction potential
Surfaces
Spatial relations .
Genetics of the microorganisms
Interactions between microorganisms
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that are highly adapted to these microhabitats after eons of selection. Even in the
gastrointestinal tract, an environment that is probably less complex than soil, especially
in terms of the number of spepies present, the introduction of billions of cells of ji. coli
K12 containing small, nonconjugative plasm ids, such as pBR322 and pBR325, did not
result in their colonization of the tract unless the indigenous microbiota, of which _E. coli
is a major component, was suppressed by antibiotics or starvation or the tract was
essentially devoid of indigenous microbes, as in gnotobiotic or neonatal animals (see
Stotzky and Babich, 1986, for references).
It must be emphasized that "gene transfer" in soil and other natural environments
implies not only the transfer of genes, but also their expression, as most methods used
initially to detect transfer rely on expression of the genes (i.e., phenotypic
characteristics). Hence, the transfer of genetic information in soil may be more
frequent than is actually detected.
The apparent lack of high frequencies of detectable gene transfer in soil may be
the result not only of insufficient population densities of appropriate donors and
recipients, but also of a reduction in the fitness and competitiveness of the recombinants.
in some microhabitats and of barriers that reduce the successful transfer and expression
of,genes. In pure.culture, the yield of plasmid-containing transconjugants is apparently
decreased by approximately the square of the parental densities below 10° cells/ml; e.g.,
*
the number of transconjugants is reduced by approximately 4 orders of magnitude if the
parental densities are only 10® cells/ml (Curtiss, 1976). However, this dependence on
parental densities was not observed in sterile freshwater (O'Morchoe et al., 1988).
Comparable studies have not been conducted in soil, but plasm id transfer is greatly
reduced when the survival of exogenously added parentals, especially of the donors,
decreases to levels below approximately 10* cells/g soil (Devanas and Stotzky, 1988b).
Moreover, if the foreign DNA does not confer some selective advantage to the
recombinant, the energy and precursors necessary for the replication of the additional
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DNA and for the synthesis of the products specified by this DNA, whether functional or
* ^ 1 . »
not, may reduce the competitiveness of the recombinant (Curtiss, 1976), especially in soil
microhabitats, which are usually oligotrophia.
There are numerous cellular barriers that can prevent or reduce the transfer and
expression of geiietic information. Among these are: 1) restriction/modification
systems, in which restriction endonucleases of the recipiient cleave incoming exogenous
DNA; 2) incompatibility of the exogenous DNA with replicons in the recipient, including
insufficient sequence homology for. recombination; 3) inability of the recipient to provide
the proteins, including the AecA protein, necessary for the establishment and replication
of nonself-sufficient exogenous DNA; 4) absence in the recipient of factors necessary for
transcription of the exogenous DNA; 5) absence in the recipient of appropriate enzymes
for post-translational modification of the gene product into a functional entity; and 6)
fertility inhibition, in whiph the exogenous DNA prevents the expression of transfer
genes, especially those that code for the production of pili (Miller, 1988). Despite these
numerous barriers, which usually do not occur simultaneously in a recipient, none is
probably sufficiently absolute to prevent gene transfer and expression in soil if the
number of donors containing the novel DNA is high enough (which it probably wQl be in
the case of planned releases) and the residence time of the novel DNA is long enough
(which it may be if the DNA survives). Morever, many of these barriers can be
surmounted by alternate strategies of DNA transfer and recombination and if the
recipients are stressed (Miller, 1984), which they undoubtedly wQl be in soil.
It would seem, therefore, that the combination of physical separation of donors
and recipients, poor survival (which will reduce the population levels of donors and
recipients), cellular barriers to gene transfer and expression, and decreased fitness and
competitiveness of the recombinant would reduce greatly the probability of successful
transfer and expression of genetic informatiorUn-bacteria-in soil,-However,-even an—
extremely low probability of transfer would eventually result, and probably has, in the
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successful establishment of recombinant bacteria In soil in sit'i. Bacteria have existed in
soil for millions of years, and some transferred DNA undoubtedly conferred some
selective advantage to and, thereby, increased the fitness and competitiveness of the
recipient. Moreover,, periodic fluctuations in the microhabitats, e.g., introduction of
nutrients, changes in pH, Eh. and other physicochemical characteristics, may reduce the
levels of the adapted indigenous microbiota sufficiently to allow a recombinant
bacterium to colonize and increase its population density before the microhabitat returns
to "normal" and again enables the autochthonous microbiota to proliferate. Such periodic
alterations in the environmental conditions within microhabitats appear to be involved in
the establishment in soil of fungal pathogens of plants and humans introduced naturally
into soils in which they were originally absent (see Stotzky, 1974, 1986). Furthermore, the
presence in soil of an antimicrobial agent to which a recombinant bacterium has gained
resistance (e.g., antibiotics, heavy metals) or of a compound that only it can metabolize
(e.g., a recalcitrant pesticide) will increase its survival, establishment, and growth and,
hence, the probability of gene transfer. The balance between the factors that mitigate
against and those, that encourage the transfer and expression of genetic information in
soil will not be known until extensive studies are conducted with a spectrum of
recombinant bacteria introduced into a wide variety of soils in different geographic
locations.
Effects of biological and physicochemical environmental factors on gene transfer In son
The fate of recombinant DNA in soil is ultimately dependent on the survival,
establishment, and growth in soil of the microbial hosts that house the genetic material.
The survival, establishment, and growth of the hosts are, in turn, dependent on their
genetic constitution and on the biological and physicochemical characteristics of the
recipient soil (Table 1). Detectable transfer of genetic information, regardless of the
mechanism of transfer, usually requires sufficiently high populations of donors (whether a
bacterium, a transducing bacteriophage, or transforming DNA) and recipients. Moreover,
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the ability to predict the fate of introduced GEMs and the potential transfer of their
novel genetic information to indigenous microorganisms in various types of soil would be
enhanced if the knowledge of the effects of different biological and physicochemical
characteristics could be related to the actual characteristics of the recipient soil.
Unfortunately, insufficient studies have been conducted on the effects of biological and
physicochemical factors on the activity, ecology, and population dynamics of microbes
and on their ability to transfer genetic information in soil and other natural habitats (see
Curtiss, 1976; Freter, 1984; Stotzky, 1974, 1986, 1989; Stotzky and Babich, 1986; Stotzky
and Krasovsky, 1981).,
Microbial competition. On the bastt of numerous studies conducted in vitro, in
vivo, and in situ, it is apparent that the survival, establishment, and growth of introduced
microorganisms, whether containing recombinant DNA or not, are usually reduced when
other species of microorganism^ are present. This is especially true in natural habitats,
such as soil, wherein the indigenous microbial populations are usually not only better
adapted to the habitat, but exert competitive, amensalistic, parasitic, and predatory
pressures on the introduced organisms. For example, the survival and growth of bacteria
that werfe not genetically engineered were significantly reduced when they were
inoculated into nonsterile environments (e.g., soil, water, sewage) in which they were not
natural residents, whereas they survived, and even grew, in the same environments when
*hsse were sterilized (e.g., the numbers of Salmonella typhlmurium, Agrobacterium
tumefaciens, and Klebsiella pnejmonlae increased by 1 to 2 orders of magnitude in sterile
soil, whereas there was a reduction in numbers when inoculated into nonsterile soil)
(Liang etal., 1982). There are numerous other examples in the literature of the apparent
lower survival and growth of introduced microbes in nonsterile than in sterile
environments (see Stotzky and Babich, 1986), and this relationship appears intuitively to
be valid. However, most of these studies did not consider the possibility of the "viable
but nonculturable" phenomenon (see below). Both genetically engineered and
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nonengineered bacteria.introduced into soil and other natural habitats could become so
debilitated or otherwise altered that they would not be recovered from these habitats,
especially on selective media, even though they could actually be surviving ancf growing
in their new environment. Because of the implications of such alteration to monitoring
the fate of GEMs. introduced into soil and other natural habitats, it must be clearly
established! that the apparent lack of survival and growth of an introduced GEM was not
an artifact of the experimental procedures.
Biergy sources. The presence of available energy sources appears to enhance the
transfer of genetic information, as these sources apparently increase the population
densities of the donor and recipient and the subsequent growth of the recombinant
bacterium. This, effect is considerably less pronounced in nonsterile than in sterile soil,
as the indigenous microbiota quickly mineralizes added carbonaceous substrates. If the
1 t '
donor or recombinant organism contains a gene that enables the metabolism of a
carbonaceous compound that can not be metabolized by other members of the indigenous
microbiota (e.g., a recalcitrant pesticide), the organism may have a temporary selective
advantage, as the result of its ability to utilize the compound (e.g., Chatterjee et al.,
1981). Nevertheless, this advantage is probably lost when the specific compound is
mineralized or transformed to an unavailable form. Moreover, the concentration of such
recalcitrant compounds in soil is usually low and less than the concentration of natural
organic materials that are available to essentially all the indigenous microbes.
Nevertheless, the additie>. to soil of a compound that only an introduced GEM can utilize
should enhance the survival of that GEM in soil, and no further addition of the compound
after it has been degraded should reduce the level of the GEM. This may be an efficient
method with which to restrict and control the population densities of some GEMs in soil.
Temperature. Temperatures near the optimum growth temperature appear to be
necessary for the.efficient in vitro transfer of genetic information in laboratory strains
of bacteria, although this is not always the case (see Stotzky and Babich, 1986). For
l-ll
-------
example, maximal conjugal transfer of the R-plasmid, Rldrd-19, in strains of E. coli
decreased progressively as the temperature was decreased from 37 to 17 C, and no
transfer was detected at 15 C (Singleton and Anson, 1981). Transfer of pRDl, derived
from a clinical isolate of Pseudomonas and maintained in the laboratory, from an E. coli
K12 donor to an E. coli K12 recipient decreased progressively as the t imperative was
reduced from 37 to IS C; when Erwinia herbicola was the recipient, transfer occurred
even at 12.5 C (Kelly and Reanney, 1984). Plasm ids pWKl and pWK2, that conferred
resistance to antibiotics and mercury, were transferred optimally in vitro from a species
of Citrobacter and a species of Enterobacter isolated from soil to E. coli at 28 C, and
frequencies were greatly reduced at 15 and 37 C. In contrast, the transfer of a plasm id
conferring resistance to kanamycin from a strain of Proteus vulgaris, isolated from the
human urinary tract, to JE. coli was about 5 orders of magnitude higher at 2S than at 37 C
(Terawaki et alM 1967). The in vitro transfer frequency of a plasm id conferring
resistance to streptomycin and tetracycline from an E. coli isolated from sewage to an £.
coli isolated from creek water was highest at 25 C and lowest at 35 C, with frequencies
at 15, 20, and 30 C being intermediate. The frequency of transfer in raw sewage in situ
was also higher at 22.5 than at 29.5 C (Altherr and K as week, 1982)..
Plasmids present in enterobacteria isolated from human beings, fecally-polluted
rivers, and sewage treatment plants were differentiated on the basis of their
thermosensitivity: "thermotolerant" plasmids were transferred equally well at 22 to 37
C, whereas "thermosensitive" plasmids were transferred at high frequencies at 22 or 28 C
but at low frequencies at'37 C. Only 3.1% of 775 conjugative antibiotic-resistance
plasmids evaluated were thermosensitive (Smith et al., 1978).
The maintenance of some plasmids by their host cells in vitro is also dependent on
temperature, and many plasmids are lost above the optimum growth temperature (see
_Siotzky„and Babich, 1986)- For exarnpie, the.Ti plasmid of A. tumefaciens (Watson et al.,
1975) and the nodulation plasmid, pW22, of Rhlzoblum trifolil (Zurkowski and Lorkiewicz,
1-12
-------
1979) were lost when the host cells were grown at 37 C. In contrast, the survival of _E.
eoli J3(RP4) and JC5465(pRDl) was reduced to a greater extent in soil maintained at 20
than at 4 C (Schilf and Klingmuller, 1983).
There have been few controlled studies on the effects of temperature on the
transfer of genetic information in soil. It appears, however, that conjugal, transfer of
both plasmid- and chromosomal-borne genes occurs in both sterile and nonsterile soil at
temperatures lower than those necessary for optimal transfer in vitro; e.g., transfer in E.
coli in soil occurred at temperatures between 15 and 27 C, considerably below the
requisite temperatures in vitro (e.g., Krasovsky and Stotzky, 1987; Trevors, 1987b;
Weinberg and Stotzky, 1972), and in Bacillus subtilis at 15 as well as at 27 C (van Elsas e£
ah, 1987). Transduction of E. coli occurred in soil at 25 to 27 C (Germida and
Khachatourians, 1988; Zeph eta)-. 1988). Transformation of £. subtilis in sterile soil
occurred at 37 C (Graham an4 lstock, 1978), in a simulated sterile marine system
Containing sea sand et 23 C (Aardema et al., 1983; Lorenz e^al., 1988), and in suspensions
of montmorillonite at 33 C (Stotzky and Golard, unpublished). Unfortunately, no studies
have been conducted to determine the effects of temperature on transduction and
transformation in soil.
Temperature in soil can not be conveniently controlled in situ. However, the
range In fluctuations in soil temperature may be an important consideration when
constructing a GEM for introduction into soil.
pH. The hydrogen ion concentration is an important physieochemical
characteristic of soil that is amenable to control in situ but whose effect on gene
transfer in soil has been insufficiently studied. Conjugal,transfer of plasmids in E. coli
was restricted in vitro to pH 8 to 8.5 (Curtiss, 1976). In soil, both intra- and Interspecific
plasmid transfer was not detected until the bulk pH was adjusted to 6.8 with CaCOj
(Devanas and Stotzky ,-unpublished)f andconjugaltransferofchromosomal genes was also
detected only at pH values near neutrality (Krasovsky and Stotzky, 1987; Weinberg and
-------
Stotzky, 1972) (pH values above neutrality were not evaluated in these studies).
Transduction of E. coli by phage PI was higher in a soil with a pH ol 7.9 than in a soil
with a pH of 6.8 (Germida and Khachatourians, 1988). However, as these soil* also
differed in texture, organic matter content, and other physicochemical characteristics,
differences in the transduction frequencies cannot be attributed solely to these
differences in pH. The effect of pH on transformation in soil has apparently not been
studied.
The pH of soil can affect gene transfer both directly (e.g., survival and growth of
the parentals) and indirectly (e;g., growth of the competitive and amensalistio indigenous
microbiota; adsorption of transducing phages, transforming DN A, and DNase on soil
particles). For example, the adsorption of bacteriophages on clay is pH-dep* dent
(Lipson and,Stotzky, 1987; Stotzky et alM 1981), and the adsorption of DNA on sea sand
increased as the pH was increased from, 5 to 9 (Lorenz and Wackernagel, 1987). The pH
determines the sign of the net surface charge of amphoteric materials (e.g., enzymes,
bacteria, bacteriophages) and the negative charge of Jonizable nonamphoter.': materials
(e.g., DNA). Therefore, the adsorption of these materials on soil particles, especially on
most types of.claiy minerals, which have a pH-independent neg&tlv? charge, will be
influenced by the pH of soil, which, in turn, will influence the persistence and activity of
these materials and the transfer of genetic information (see Stotzky, 1986).
Consequently, controlled studies on the effects of pH on genetic transfer in soil should be
conducted.
Watef cootent. The few data that are available on the effects of water content
on the transfer of genetic information in soil indicate that transfer frequencies are
higher when the soil water tension is near or at the optimum for microbial growth (i.e.,
the -33 kPa tension). For example, plasmlcJ transfer and survival of the donors (E. coll
j75(RP4) and.JC5466(pRDl)Mnd the exconjugants (Enterobacteriaceae strain KpRDl) and
Pseudomonas fluorescens (pRDl)) were higher in soil maintained at 16% water than when
1-14
n —ii* > r un
-------
allowed to dry to 4% (Schilf and Klingmuller, 1983). Similarly, plasmid transfer in sterile
soil was greater between strains of B. subtilis at a water content of 20 to 22%
i 1
(equivalent to the -33 kPa water tension) than at a water content of 8% (van Elsas et a).,
1987); between strains of £. coU in sterile soil at 80 than at 20% of the water-holding
capacity (WHC) (Trevors, 1987a); and between E. coli and other ehterobacteria and
Pseudomonas aeruginosa in nonsterlle soils at their -33 kPa tension (24 to 26% water)
than at 16% water (Devanas and Stotzky, unpublished). No studies appear to have been
conducted on the effects of water content on transduction and transformation in soil.
Inasmuch as microbial growth in soil is optimal at the -33 kPa water tension, genetic
transfer, regardless of the mechanism of transfer, is probably maximal at this tension,.
which is easily controlled by irrigation.
Oxygen and E^. At soil water tensions above -3.3 kPa, oxygen will become
progressively more limiting, and the Eh will be reduced (see Stotzky, 1974). The effects
of oxygen tension and Eh on the transfer of genetic information in soil has apparently not
been studied, and results from the few studies conducted in vitro are contradictory: e.g.,
the conjugal transfer of chromosomal genes between E. coli was similar under aerobic
and anaerobic conditions (Stallions and Curtiss, 1972); the expression of antibiotic
resistance by 45 different plasmids in E. coll was the same under aerobic and anaerobic
conditions, although the formation of sex pili was reduced under anaerobic conditions'
(Burman, 1977); the frequency of conjugal transfer of R-plasmids by two donor strains of
E. coli isolated from hum?n feces was reduced 10- to 1000-fold under anaerobic conditions
(Moodie and Woods, 1973).
loclc composition. The effects of the ionic composition of the soil solution, which
affects the activity of water (aw), as well as surface interactions among and between soil
particles, especially clay minerals, and bacteria, bacteriophages, DNA, and proteins (see
Stotzky, 1986), on genetic transfer requires study. The transfer, in vitro, of plasmid
Rldrd-19 in E. coli was apparently stimulated by concentrations of NaCl that are present
1-15
-------
in estuaries (Singleton, 1983). The survival of antibiotic-sensitive fecal coliforms was
i
reduced in seawater, whereas that of E. coli strains containing R-plasmids was,
unaffected (Smith et ah, 1974).
, Electromagnetic radiation. Although light probably affects only microbes residing
on the surface of soils, this physicochemical factor can be important In arid and semi-
arid soils, where photosynthesis in algal crusts, both procaryotic and euearyotie, may be
the major source ->f primary productivity Sfcujins, 1984). Hence, the transfer of
genes.conferrinjg resistance to ultraviolet,radiation may enhance the survival of
microbial surface dwellers (Marsh and Smith, 1969). The importance of electromagnetic
radiation on gene transfer in soil has apparently not been studied.
Surfaces. The effects of surfaces, especially those of clay minerals, on transfer
of genetic information in soil appear to have been studied to a greater extent, albeit also
' J
insufficiently, than those of other physicochemical characteristics of soil.
Montmorillonite appears to enhance conjugal transfer of both plasmid- and chromosomal-
borne genes in both sterile and nonsterile soil, whereas kaolinite appears to have
essentially no effect (e.g., Devanas and Stotzky, 1988a; Krasovsky and Stotzky, 1987; van
Elsas et al., 1987; Weinberg and Stotzky, 1972). In contrast, colloidal montmorillonite
reduced the in vitro transfer of plasmid Rldrd-19 in _E. coli by several orders of magnitude
(Singleton, 1983). This apparent paradox between in vitro and in vivo effects of clay
minerals may be the result of free clay particles jn vitro blocking sites on the bacterial
surface necessary for gene transfer, whereas few free clay particles exist in soil, as they
are relatively stably maintained in cutans and domains.
Surfaces, especially those of clay minerals, can exert both direct and indirect
effects on microbial activities, including transfer of genetic information, in soil.
However, the mechanisms by which some clays enhance conjugal gene transfer in soil are
not clear. Montmorillonite has been shown to stimulate the growth of bacteria, in part
by maintaining a suitable pH for sustained growth in the microhabitats, and to reduce the
1-16
-------
grow.:i of fungi, in part by complexing siderophores necessary for iron transport (see
Stotzky, 1986). Van Elsas et a[. (1S87) suggested that the enhanced transfer of a plasmid
»
in ji. subtilis in soil amended with montmorillonite, was not the result of an effect of the
clay on pH but of "a modification of the physicochemical soil environment, possibly
modifying cellular physiology or promoting.cell-to-cell contact" or because the "clay
apparently protected the recipient population". Unfortunately, no details were presented
on the mechanisms of such modifications and protection. Moreover, it is also not clear
which physicochemical properties of clays (e.g., cation- and an ion-exchange capacity,
specific surface area, surface.charge density, nature of the charge-compiensating cations)
are responsible for their effects on conjugation and survival of parentals and
. exconjugants in soil.,
The frequency of transduction in soil was not affected by montmorillonite,
i
although the survival of the transducing bacteriophage, PI, was increased (Zeph et al.,
1988). Transduction by phage PI was greater in a sandy (8% clay) than in a silty clay loam
(21% clay) soil (Germida and Khachatourians, 1988), but as these soils also differed in pH,
organic matter content, and other characteristics, and the types of clay present were not
described, the differences in transduction frequencies cannot be attributed solely to
differences in clay content in these two soils. The effects of surfaces on transformation
in soil have not been studied. However, both sea sand (Aardema et al., 1983; Lorenz et
ah, 1988) and montmorillonite (Stotzky and Golard, unpublished) reduced the in vitro
frequency of transformation in B. subtilis.
Interactions between factors. The value of studies conducted on the effects on
gene transfer of one physicochemfcal factor of soil at a time may be limited, as a change
in one factor can result in changes in numerous other factors, and several factors can
interact to affect gene transfer. For example, the in vitro conjugal transfer of plasmid-
Rldrd-19 in E. coli was inhibited more by deviations in pH from the optimum of 8.9 when
the temperature was simultaneously decreased from 3? to 17 C (Singleton and Anson,
1*17
-------
1983). Studies on the effects^ of interactions between multiple physicochemical
characteristics of soil are needed not only with respect to the transfer of genetic
.information, but on all aspects of microbial ecology in soil. Nevertheless, even the few
studies that have been conducted on the effects of these characteristics individually on
gene transfer in vitro and in soil and other natural habitats indicate that In vitro studies
of conjugation, transduction, and transformation, which are usually conducted under
standardized and optimal growth conditions, are not always adequate predictors of gene
transfer in natural habitats, wherein these characteristics fluctuate cpntinually and in
concert.
Conclusions
There is insufficient information on the frequency of transfer, whether by
i '
conjugation, transduction, or transformation, and on the survival and activity of
recombinant bacteria in soil and other natural environments that contain high numbers of
other microorganisms not involved in the transfer. The relatively few studies that have
been conducted in soil - and it must be emphasized that even these studies have been'
conducted primarily in the laboratory in microcosms of varying degrees of complexity or
under greenhouse conditions, as few field releases have been authorize^ - indicate that
transfer can occur in soil. However, insufficient information Is available on how transfer
and survival are affected by the physicochemical and biological characteristics of soil; on
the numbers of donors and recipients necessary for transfer; on the numbers of
recombinant bacteria that can be accurately detected; on the probability that low levels
of recombinant bacteria, especially below the level of detection, can multiply
sufficiently to become a significant portion of the soil microbial population; and on
numerous related questions.
The major lack of knowledge, however, is in the area of the potential effects that
recombinant bacteria could have on the structure and function of soil and other natural
habitats; e.g., what kinds of genes need to be transferred and how many recombinant
1-18
-------
bacteria need to be functioning; per unit volume of soil to result in detectable changes in
the activity, ecology, and population dynamics of microorganisms in soil? Even if a GEM
introduced into soil survives and transfers its novel genetic information to indigenous
microbes, there should.be little cause for cqncera unless the novel genetic information,
either in the introduced GEM or in an autochthonous recipient to which it has been
transferred, results in some unexpected and untoward impacts. Unfortunately, it is
difficult with existing knowledge and methodology to study and predict the occurrence,,
extent, and severity of such impacts.
Preliminary studies on the effects of adding high concentrations (e.g., 10® cells/g
soil) of various strains of JE. coli, Enterobacter cloacae. Pseudomonas aeruginosa, and •
Pseudomonas putida, with and without plasmids carrying antibiotic-resistance genes, to
soil have not shown any consistent and lasting effect's.on the gross metabolic activity (as
measured by C02 evolution), transformations of fixed nitrogen, activity of soil enzymes
(phosphatases, arylsulfatases, dehydrogenases), and species diversity of the soil
microbiota (Doyle et al.t 1988; Jones et al., 1988). However, in other preliminary.studies,
the introduction into soil of a strain of Streptomyces lividans that contained a plasm id
carrying a lignin peroxidase gene from the chromosome of Streptomyces virldosporus
enhanced the rate of mineralization of $oil carbon (as measured by C02 evolution) during
the 30-day incubation, especially when the soil was amended with lignocellulose (Wang
and Crawford, 1988). More such studies are needed, especially with GEMs that have been
engineered to perform specific enzymatic functions in soil.
It is obvious, based on the few studies that have been conducted on the transfer of
genetic information in soil, that more research is necessary to determine the frequency,
. the location, and the effects of physicochemical and biological factors on gene transfer
jnsoiland other natural habitats. The attainment of this knowledge will not only be_oL
immense academic interest to microbial and macrobial ecology and evolutionary theory,
1-19
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but also to risk assessment and regulation of the release of GEMs to soil and other
natural habitats.
1-20
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0. Literature Review of Conjugation and Transduction in SoQ
CONJUGATION
Introduction
Conjugation is the transfer of DNA from one bacterium to another by direct cell-
to-cell contact and is mediated by transfer (tra) genes. For example, the self-
transmissible fertility (FWac'tor in the gram-negative bacterium, E.coIi. is a plasmid
i —,
that has 15 to 25 tra genes that encode its transfer, i.e., enzymes and the appropriate F-
pili. The F-factor may «xist autonomously in the cell, or it can be Integrated into the
bacterial chromosome, where it is replicated and segregated to progeny cells with the
other genes on the chromosome, similar to a prophage in lysogenic bacteria. In £. coli.
which has been more extensively studied than other gram-negative and gram-positive
bacteria, the DNA moves from the donor F+ cell to the recipient F" cell through a
special surface pilus called an F-pilus, the synthesis of whose structural proteins are
directed by the tra genes. The F-pilus of the donor attaches to the recipient cell at
specific surface receptors and retracts until the surfaces of the donor and recipient are
touching. At the point of contact on the recipient, a channel is made through its cell
envelope, and the DNA is transferred, presumably through the pilus, into the recipient
cell. A bacterial cell in which the F-factor is integrated into the chromosome is called a
"High frequency of recombination" (Hfr) cell. The integrated F-factor can mediate
, transfer of part of the F-ifactor and part of the donor chomosome (an Hfr conjugation)
(Freifelder, 1987; Lewin, 1977; Vandemark, 1987).
Gram-positive bacteria can also transfer DNA by conjugation, but no sex pili have
been demonstrated. The recipient cells synthesize specific diffusible proteins called
pheromones. When these pheromones contact a donor cell, it synthesizes surface
proteins with adhesive properties that cause the donor and recipient cells to form
aggregates. The sex pheromones then induce the transfer of the plasmid to the recipient,
11 -1
-------
but the exact mechanism of transfer is not; known (Clewell, 1981; Freifelder, 1987).
Transfer of chromosomal genes
Transfer of the bacterial chromosome from an Hfr cell to an F~ cell begins at the
3'-end of the origin of transfer (oriT) site within the integrated F-factor, where a tra-
encoded endonuclease nicks and opens one of the two DNA strands. The free 5'-end of
the open single-stranded DNA attaches to a site on the cytoplasmic membrane, and the
3'-end acts as a primer for chain elongation, using the closed, intact, circular strand as a
template. The open DNA strand, after its release from the cytoplasmic membrane,
serves as a template for the synthesis of its complementary DNA and moves, 5'-end first,
into the recipient through a conjugation bridge (presumably the F-pilus in E. coli). This is
an example of the unidirectional "rolling circle" mechanism of replication. The entire
single-stranded DNA from the donor chromosome and the newly synthesized
complementary DNA strand must be moved'to the recipient for complete transfer of all
the genes on the chromosome, including the distal end of the F-factor, and to produce a
complete functional integrated F-factor upon reannealing and ligation in the recipient.
This process is estimated to require about 100 min in E. coli. This is a relatively long
time for oacteria to stay physically attached, especially in soil and other natural
habitats, and interruption of conjugation usually occurs after only 10 to 20% of the
chromosome has been transferred (Freifelder, 1987; Lewin, 1977). Therefore, the entire
F-factor is rarely transferred from an Hfr cell, as the remaining F-factor genes are at
the opposite free 3'-end of the nicked chromosome. Genes that show a high frequency of
recombination are those nearest the oriT site, and the frequency of transfer, of genes
decreases proportionately with their distance from this site.
An F-factor that has been integrated into the chromosome may excise itself, by
expressing the appropriate enzymes coded on its DNA, by a reverse recombinational
event and return to an autonomous, extrachromosomal state. Precise excision, i.e.,
nicking at the exact sites that were ligated to the chromosomal DNA on integration, will
IX-2
-------
restore the original autonomous F-factor. Imprecise excision may result in an '
autonomous F-factor that includes chromosomal genes (possibly introduced recombinant
genes) from one or both sides of the integration site of the F-factor. This modified F»
factor, sometimes referred to as an F-plasmid, can transfer these genes to another cell.
Conjugative plaamids
Autonomous F-factors, F'-plasmids, and R-plasmids (conjugative plasmids similar
to an F-factor that carry genes coding, for antibiotic resistance) are self-transmissible.
Hence, they may direct their transfer to other bacteria by expressing their tra genes.
Transfer of conjugative plasm id DNA also begins at the oriT site within the tra genes, as
it does in Hfr strains. However, as the length of the DNA sequences (i.e., the number of
genes) in a plasmid is significantly smaller 0 to 10%) than the length of a bacterial
chromosome, the entire plasmid is usually transferred. As'in chromosomel transfer, the
plasmid DNA is replicated during transfer (i.e., a copy remains in the donor cell), and the
recipient cell then expresses the tra genes and becomes a donor cell. Consequently, in
the presence of appropriate selection pressure, a plasmid may spread rapidly through a
bacterial population, even though plasmid-containing c?lls may originally have been an
insignificant part of the population. This rapid conversion of plasmid-free recipient cells
to plasmid-containing donor cells appears to be responsible for the rapid increase in
antibiotic-resistant nosocomial infections.
Nooconjugative plasmids
Autonomous, extrachromosomal, and self-replicating plasmids that do not have tra
genes are nonconjugative. Consequently, these plasmids, which may code for a variety of
functions (e.g., resistance to antibiotics and other antimicrobial agents, catabolic
enzymes, toxin production), can not direct their own transfer. However, nonconjugative
plasmids may be dispersed to appropriate recipient cells by coinhabiting conjugative
plasmids that can "move" them (mobilization) to other cells, or there is recombination
and integration into a plasmid that is self-transmissible (cointegrate formation).
11-3
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Mobilization. If a plasmid is deficient in DNA sequences that code for mobility
(mob) (e.g., for endonucleases that nick the oriT region) or transfer (e.g., F-pilus
production) functions, the plasmid will not be transferred, even if all other functions are
, present, and it will persist as an autonomous, self-replicating element. However, such
nonconjugative plasm ids may be transferred by conjugation if the missing functions are.
supplied gratuitously (in trans.) by another plasmid present in the same cell. For
example, the nonconjugative plasmid, ColEl, can be mobilized and transferred when an F-
factor present in the same cell supplies the missing pilus and transfer apparatus
(Freifelder, 1987).
The mobilization of nonconjugative plasmids under environmental conditions has
been reported. A nonconjugative plasmid, pHSV106, coding for resistance to ampicillin
and thymidine kinase from herpes simplex virus, was mobilized by plasmids present in
both laboratory and indigenous wastewater strains of bacteria in a laboratory-scale
wastewater treatment facility (Mancini e^al., 1987). In.sterile soil, triparental matings
(i.e., mating of a cell carrying a nonconjugative plasmid with a cell carrying a
conjugative plasmid and then transfer of either the nonconjugative or both plasmids to a
third cell) mobilized nonconjugative plasmids between strains of Streptomyces (Rafll and
Crawford, 1988). Transfer of a 2.8-megadalton (Mda) plasmid, pFT30, from cere us to
B. subtilis occurred in sterile and nonsterile soil when a 29.5-Mda plasmid was present in
B. cereus (van Elsas et al., 1987).
Cointegrate formation. When two plasmids coexist in a cell and there is a region
of homology between them, e.g., a transposon or an insertion sequence, the two plbsmids
may fuse to form a single plasmid termed a cointegrate plasmid. If one of the plasmids
is conjugative, the cointegrate plasmid, containing genes from both plasmids, can be
transferred. Once in the recipient, the cointegrate plasmid may separate into the
original two plasmids. Components of an infectious R-plasmid (i.e., the RTF region,
containing transfer and replication genes, and the R-determinant, containing drug-
11-4
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resistance genes) have been shown to form cointegrates with themselves and other R-
plasmids and then to separate into new species of plasmids (segregants) of various sizes
in the recipient (Freifelder, 1987; Lew in, 1977).
Mobil iring elements
Transfer of DNA that is not self-transferable can also be facilitated by mobilizing
elements, e.g, insertion sequences (IS), a transposon (Tn). These transposable elements
are specific segments of DNA that have the ability to move autonomously as discrete
units to other sites in DNA. The IS are a special type of transposable element containing
800 to 1400 base pairs with inverted repeat sequences that are 16 to 41 base pairs long at
each end. The IS encode only those genes necessary for their own transposition, including
a gene for an enzyme called transposase, which mediates recombination at special sites
in the terminal inverted repeat sequences. Several distinct IS have been detected and
i
characterized in procaryotic and eucaryotic genomes.
The Tns are composite units with terminal IS or short inverted repeat sequences
that flank antibiotic-resistance or other genes, as well as the gene for transposase, and
do not require homologous recombination genes for1 movement. These mobilizing
elements serve as regions of homology for recombinational events that can: 1) produce
cointegrates of transferable and nontransferable plasmids; 2) transfer copies of genes
from sites in the chromosome to sites on other replicons, e.g., plasmids; 3) cause'
insertional mutations within genes; and 4) provide a detectable marker after
incorporation into the genome (Freifelder, 1987).
Spectrum of coojugative bacteria
Some genera of gram-negative bacteria that have been shown, to be capable of
participating in the transfer of chromosomal- and/or plasm id-borne genes by conjugation
are: Enterobacter, Escherichia, Salmonella. Shigella. Providencia, Vibrio. Proteus.
Klebsiella. Pseudomonas, Serratia, Rhizobium. Bradyrhizobium. Bordetella. Neisseria.
Rhodopseudomonas, Azotobacter, Caulobacter. Agrobacterium. Erwinia.
II-5
-------
Chromobacterium. Acinetobacter. RhodospiriUum, and Flavobacterium. The gram-
! ,
positive genera that are known to transfer genes by,conjugation include: Staphylococcus,
Clostridium. Bacillus. Streptococcus. Lactobacillus. Nocardia. Actinomyces, and
Streptomyces.
Conjugal transfer of plasmids occurs in both gram-negative and gram-positive
bacteria, and plasmid transfer from _E. coli to various gram-positive bacteria has been
demonstrated, in vitro (Trieu-Cuot et al.. 1987).
Survival of introduced genes in situ
When the heterologous DNA is present in a plasmid, the survival and stability of
the DNA is dependent not only on the survival of the bacterial host, but also on the
maintenance and faithful replication of the plasmid during the cell cycle and the
partitioning of the plasmid to the progeny. Low copy number plasmids (I to 2 per
chromosome) are usually stably maintained and stably inherited by a partitioning
mechanism, whereas high copy number plasmids (greater than 5 per chromosome) may be
inherited by a random distribution mechanism of stochastic segregation, although some
form of active partitioning may also occur with high copy number plasmids (Sherratt,
1982). For example, when sterile soil was inoculated with £. coli K12 strain *1668, the
number of colony-formirig units (CFU) increased rapidly to a maximum population level,.
the increase being greater when inoculated in Luria Broth (LB) than in saline. When the
cells of«1666 contained plasmid pES019, which is a 4-Mda construct of the multicopy,
tetracycline (Tc)-resistant plasmid, pBR328 (Covarrubias et al., 1981), engineered to
contain gentamycin (Gn)-, chloramphenicol (Cm)-, and streptomycin (Sm)- resistance
markers (W. Watkins, personal communication; see Levin, 1982), the numbers of CFU also
increased in sterile soil. However, segregation of the plasmid apparently occurred, as
the numbers of CFU recovered on the nonselective medium, MacConkey agar (MAC),
were significantly higher than the numbers recovered on the medium selective for pES019
(MAC + 25 jig/ml Cm). This difference in numbers between the plasmidless and the
II-6
-------
plasmid-containing populations was not the result of an artifact (e.g., a "viable.but
¦I I ¦
nonculturable" phenomenon), as the same difference occurred when the CFU recovered
on MAC were subsequently transferred to MAC + Cm. The segregation occurred shortly
after the addition of the host-vector system to soil, and the plasm id-free population then
outgrew the plasmid-containing population. Nevertheless, despite the segregation, the
population of*1666(pES019) increased by 1 and 3 orders of magnitude when inoculated in
saline or LB, respectively, and persisted near these levels throughout the 27-
-------
decline in nonsterile soil of hosts that contained small, multicopy, nonconjugative
plasmids (2.6 and 8.0 Mda) or large, low copy number, conjugative plasm ids that ranged in
size from 26 to 64 Mda, regardless whether they were added in LB or saline (Devanas et
al., 1986).
Moreover, the survival of E. coli strains*1666 and PRC487 was greater when they
contained plasmids (pES019 and pACYC175 (8 Mda), respectively) (Devanas et al., 1986).
Tfci survival of*1666(pBR322) in the human gastrointestinal tract was also greater than
that of the plasmidless parental (Levy et al., 1980), as was that of the highly debilitated
E. coli strain «1776 when it contained pBR322 (Levy and Marshall, 1979). furthermore,
the survival of plasm id-containing strains of Jl. coli was similar to that of homologous
plasmidless strains in freshwater (Grabow et al., 1975), conventional sewage treatment
systems'(Sturtevant and Feary, 1969), and seawater (Smith et al., 1974), even in the
absence of thfc selection pressure of the antibiotics to which the plasmids conferred
resistance. Although most in vitro studies, usually conducted in chemostats under
nutrient-limiting conditions, indicate that plasrn id-free cells have a more rapid growth
rate and quickly displace plasm id-containing cells of the same species, this is not always
the case, as these studies show. Even in chemostat studies, the growth of E. coli that
contained either the cos DNA fragment from phage lambda (Edlin et al., 1984), the
transposable element; 1S50 (Hartl et al., 1983), derivatives of pBR322 that expressed
resistance to Tc (Lee and Edlin, 1985), or were lysogenic for phage lambda (Edlin et al.,
i I "
1975) was faster than that of the homologous hosts not carrying these DNA sequences.
The reasons for these observations are npt known. Although it is.tempting, in the case of
the greater survival of plasmid-containing bacteria in soil, to attribute this to the
antibiotic-resistance genes carried by the plasmids, there is no convincing evidence that
antibiotics are produced in soil, at least not at detectable levels (see Stotzky 1974,
1986). Regardless of the reasons involved, these observations indicate that the presence
of plasmids, even if they confer no known advantage to their hosts, is not always
11 - 8
-------
detrimental to the competitiveness of the bacteria and that results obtained In vitro,
despite their apparent logic, can not always be extrapolated to soil in situ.
There appeared to be a more rapid decrease in the numbers of some plasmid-
contdining E. coli strains when recovered from nonsterile soil on selective media (e.g.,
' 5
MAC + 25^ig/ml Tc ~ 5 i HT* .M mercury (Hg) as HgC^) than on nonselective media
(e.g., MAC), suggesting a loss of the plasm ids. However, this was an artifact, and there
was no loss of the plasmids: when cells from colonies recovered on nonselective media
were transferred to selective media, the »• imbers of CFU that subsequently developed on
the selective media were the same as those recovered on the nonselective media
(Devanas et al., 1986). The cells of some host^plasmid systems were apparently
sufficiently debilitated in soil to prevent their recovery directly on selective media with
the added stress of an antibiotic and/or a heavy metal, and resuscitation on less stressful
nonselective media was necessary for subsequent growth oh selective media.
i ¦
This phenomenon of "viable but nonculturable" has also been reported in other
environments with bacteria not containing recombinant DNA: e.g., coli, Vibrio
cholerae (Xu et al., 1982), and Salmonella enterltidis (Roszak et al., 1984) in natural
waters and E. coll and Streptococcus faecalis in chlorinated water (Bissonnette et al.,
1975; Camper and McFeters, 1979; Zaske et al., 1980) (see Roszak and Colwell, 1987). The
mechanisms, responsible for this phenomenon have not been definitively established. It
has been suggested that the cell membrane may be damaged (Zaske et al., 1980) and,
hence, not be capable of using an electrochemical gradient to generate high-energy
intermediates for sustained survival (Sjogren and Gibson, 1981) or that the cells may be
restricted in obtaining and retaining energy sources for endogenous metabolism and
maintenance (Morita, 1982). Regardless of the mechanisms involved, the reduced
viability or recoverability of host-plasmid systems introduced into stressful
environments, such as soil, may result in erroneous data on the survival of GEMs in such
environments.
_M.O
-------
Even DNA foreign to bacteria appears to be maintained in soil when introduced
into bacteria on a plasm id. For example, studies on the survival and maintenance'of a
0.9-Mda cDNA fragment that codes for a yolk protein in Drosophila grimshawii and
inserted into plasmid t>BR322 showed that the presence of the chimeric plasmid (C3S7)
' i i 1 | i
had little effect on the survival of the host (E. coli rtBlOl) in sterile soil (Devanas and
Stotzky, 1986). When E. eoU HB10l(pBR322) and HB10KC357) were added in LB to
nonsterile soil, the presence of the insert of Drosophila cDNA reduced somewhat the
stability of the plasmid during the 27-day incubation. When the host-plasmid systems
were added in saline, the rate of decrease of the system containing the chimeric plasmid
was similar to that of the system containing only pBR322, and the decrease of both was
greater than when added in LB. The presence of foreign genetic material has been shown
to reduce the stability of plasmids when the host-vector systems were grown in a1
chemostat (Warnes and Stephertsbn, 1986), and other studies have indicated that pBR322
is unstable in nutrient-limited chemostat cultures (Jones et al., 1980} Jones and Melling,
1984). However, when £. coli HB10HC35?) was added in either LB or saline to nonsterile
soil, there was no selective loss of the chimeric plasmid, as determined by the isolation
of HB10HC357) on a selective medium and colony hybridization with a 32P-labeled DNA
probe specific for the cDNA (Devanas and Stotzky, 1986). Nevertheless, HB10KC357)
appeared to bfe less able to cope with conditions of starvation and competition in
nonsterile soil, as the addition of LB on day 16 to soil that received the inoculum in saline
1 1 ¦ 1
did not elicit growth similar to that of HB101(pBR322). Although £. coli HB101 is used
extensively for cloning, it is a poor choice for studies in soil, as it is auxotrophic and
lactose-negative. Hence, an important phenotypie marker (i.e., pigment production) is
not available, and it is difficult to distinguish E. coli HB101 from gram-negative
indigenous soil bacteria, which are usually also lactose-negative, on MAC.
The survival of P. fluorescens, resistant to kanamycin (Km) and Sm, and B.
subtilis. resistant to Tc, in two soils of different texture and planted with wheat was
11-10
-------
studied in situ (van Eisas et al., 1986). During 120 days, P. fluorescens decreased more
slowly in the silt loam soil (a reduction of approximately 2 orders of magnitude) than in
the coarser loamy sand (a reduction of approximately 5 orders of magnitude), whereas EJ.
subtilis declined rapidly in both soils within the first 20 days and then stabilized,
with the population being present primarily as spores.' These results were similar in both
rhizosphere and root-free soil. Although this is one of the few studies with engineered
bacteria that has been conducted in soil in situ, it should not be concluded that the
survival of engineered gram-negative bacteria is greater than that of gram-positive
bacteria in soil, as the bacteria were altered by two very different mechanisms (i.e., P.
fluorescens by Tn£ mutagenesis of the chromosome and B. subtilis by protoplast
transformation with plasmid pFT30).
In a current in situ study, the survival of root-colonizinfe Pseudomonas
aureofaciens 3732RNL11, that contains the lacZY genes from £. coli K12
(encoding ^-galactosidase' and /£-galactoside permease, respectively) and genes
conferring resistance to nalidixic acid (Nx) and rifampicin (Rf) on the chromosome
(Drahos et al., 1986), in the rhizosphere is being investigated (E.L. Kline, H. Skipper,
and EJ. Brandt, personal communication). The genetically engineered bacterium, in
aqueous .suspension, was applied to seeds of winter wheat planted at a depth of
approximately 2.4 to 3.8 cm, and its survival was monitored by periodically plating.
1 " '
dilutions of rhizosphere soil on a minimal medium containing lactose, Nx, and Rf,
checking for fluorescence under ultraviolet light, and enumerating colonies that
showed /^-galactosidase activity (i.e., they turned blue when they cleaved the X-gal
reagent, 5-bromo-4-chloro-3-indolyl-/£-D-galactoside). The level of detection is
approximately 1 to 10 CFU/g soil or root. The numbers of bacteria decreased (after
£ 4
colonization of the emerging roots) from approximately 10 to 10' CFU/g root during the
31-week growth period (seeds were planted in November, and the crop was harvested in
June). The survival kinetics of the host strain that did not contain the lacZY genes were
H-11
-------
similar to those of the recombinant bacterium. After harvest of the wheat crop, the
levels of the bacteria fluctuated between 10* CFU/g root,and undetectable.levels on
soybeans that were subsequently planted in the same site without tillage. Survival of the
bacteria was not detected in root-free soil. No apparent transfer of the lacZY genes to
indigenous fluorescent pseudomonads occurred, as determined by dot blot hybridization
of a "P-labeled DNA probe (a 502-base pair nucleotide sequence of the Tn^ used to
insert the genes) with DNA extracted from more than 5,000 fluorescent bacteria isolated
from the rhizosphere (third- wash of roots) and nonrhizosphere soil.
The survival of engineered genes, introduced in GEMs, in soil and other natural
habitats may be increased by their transfer to indigenous bact* Ma that are more fit for
survival in these habitats (Stot2ky, 1989; Stotzky and Babich, 1986).
Conjugation in situ
Transfer of chromosomal genes. Conjugal transfer of chromosomal genes from
prototrophic to auxotrophic strains of E. coli occurred in both sterile (Weinberg and
Stotzky, 1972) and nonsterile soils (Krasovsky and Stotzky, 1987; Stotzky and Krasovsky,
1981) inoculated with these strains. The, frequency of transfer was significantly higher in
sterile than in nonsterile soils, indicating that the presence of the indigenous microbiota
interfered with conjugal transfer.
The physico<:hemical characteristics of the soil affected the frequency of transfer
of chromosomal genes. For example, the addition of the clay mineral, montmorillonite,
with a high cation-exchange capacity and surface area, enhanced the frequency, whereas
the addition of the clay mineral, kaolinite, with a low cation-exchange capacity and
surface area, had no effect. The enhancement by montmorillonite was apparently the
result of the increased growth of the donors and recipients in the microhabitats, which
probably resulted in increased conjugation because of the larger number of donors and
recipients. Montmorillonite has been shown to increase the growth and metabolic
activity of bacteria,~prImarUy by maintaining the pH of microhabitats at levels
11-12
-------
conducive to sustained growth (see Stotzky, 1974, 1986). The effect of pH on the transfer
of chromosomal genes was further demonstrated in studies that showed that both the
survival of the donors and recipients and the frequency of gene transfer increased as the
pH of the soil was raised to neutrality (Krasovsky and Stotzky, 1987; Weinberg and
Stotzky, 1972).
Transfer of plasmid genes. Numerous studies have demonstrated the transfer of
plasmids from bacteria Isolated from soils and waters to recipient strains of the same
and different species in the laboratory (see Stotzky and Babich, 1986). However, few
studies have demonstrated such transfer in soil and other natural environments. For
example, when a strain of Enterobacter cloacae, isolated from the rhizosphere of
Festuca heterophylla and that did not fix nitrogen and was sensitive to antibiotics,'was
mated, in.vitro, with E. coli containing the plasmid, pRDl, which carried genes for
nitrogen fixation and resistance to three antibiotics, the exconjugants were able to fix
nitrogen and were resistant to the antibiotics. However, many of the exconjugants
contained plasmids that were smaller than pRDl or contained no plasmids, indicating that
all or part of the pRDl had become incorporated into the chrorposome of E. cloacae
(Kleeberger and Klingmulter, 1980). When _E. coli JC5466 containing the broad-host-range
plasmid, pRDl, was added to nonsterile soil, the frequency of transfer of the plasmid to
indigenous bacteria was approximately 1 x 10"9 exconjugants/recipient, whereas the jn
vitro frequency of transfer to mixed bacterial populations Isolated from soil ranged from
7.1 x 10"® to 4.S x 10"* (Schilf and Klingmuller, 1983). The survival of E. coli strains
J5(RP4) and JC5466(pRDl), Enterobacteriaceae strain KpRDl), Enterobacteriaceae strain
2(RP4), and P. fluorescens (pRDl) (the latter three being exconjugants of soil isolates
obtained by in vitro matings) was also reduced in nonsterile soil, at either 4 or 20 C with
a soil water content of 16% or at 20 C with a water content of 4%, presumably as the
result of either a loss of the.plasmidsjiraselec t i vejJec rease_inJhe host-plasmid systems
when they were exposed to nonselective conditions in soil. In contrast, the survival of a
11-13
-------
chromosomal transconjugant that resulted from conjugation in soil of E. coli strains*493
I 1 . . 1
(Hfr, prototrophic) and *696 (F~, auxotrophic) was greater in nonsterile soil, when
reinoculated after isolation from soil and growth on laboratory medium, than that of the
parental* (Krasovsky and Stotzky, 1987).
Pertsova et al. 0984) reported the transfer in nonsterile soil of plasm id-borne
genes that coded for the degradation of 3-chlorobenzoate from P. aeruginosa and P.
putida to indigenous strains of Pseudomonas that were taxonomically different from the
donors. Transfer of antibiotic-resistance plasm ids among strains of Klebsiella in a sandy
loam soil sown with radish seeds (the seeds were inoculated with plasmidless strains and
the soil with strains containing the resistance plasmids) was detected on about 24% of
the radish seedlings after one week, but not thereafter, at frequencies of 10~7 to 10~®,
whereas in an aqueous suspension of redwood sawdust, transfer was detected in about
30% of the donor-recipient combinations at frequencies of 10"® to 10"^, and in broth,
transfer was detected in about 60% of the combinations at frequencies of 10~® to 10"'
(Talbot et ah, 1980).
Plasmid transfer also appears to occur among gram-positive bacteria in soil,,
although this has not been studied as extensively as with gram-negative bacteria. Both
conjugative and nonconjugative plasmids were transferred (the latter by tripartntal
matings) between strains of Streptomyces in sterile soil, although less frequently than on
agar (Rafii and Crawford, 1988). The transfer of a 2.8-Mda plasmid, pFT30, carrying a
Tc-resistance gene, from B. cereus to B. subtilis occurred in sterile soil at a frequency of
7.x 10~8 (van Elsas e£ ah, 1987). Transfer was increased to a frequency of 1.6 x 10"® by
the addition of nutrients and bentonite (montmorillonite), but it was lower at 15 C and a
WHO of approximately 20% than at 27 C and a WHC of approximately 60%. Detectable
transfer of plasmid pFT30 (frequency of 9 x 10"®) occurred in nonsterile soil only when it
was amended with bentonite, and the clay also enhancedjhesurvivalofthereciplenfs'.
Although not stated, the transfer of this 2.8-Mda plasmid was probably facilitated by the
11-14
-------
presence of a 29.5-Mda plasm id in the donor. Bacilli isolated from soil samples collected
in hog and cattle feedlots throughout the United States contained plasmids that showed'a
high degree of homology wjth plasmids from clinical isolates of Staphylococcus aureus.
suggesting the occurrence of natural intergenerie plasm id transfer (Polak and Novick,
1982).
The transfer of nonconjugative plasmids by mobilization, either directly or by
cointegrate formation, by conjugativeplasmids has been studied extensively in pure
culture, but there have been few studies on the frequency of such transfer in soil and
other environments. Transfer of nonconjugative plasmids by triparental mating has been
demonstrated in coll in a laboratory simulation of a sewage treatment plant (Gealt et
al., 1985; Mancini et al., 1987; McPherson and Gealt, 1986) and in S. lividans in sterile soil
(Rafii and Crawford, 1988). Nonconjugative plasmids, as a result of their smaller size
and, hence, lower "burden" on the host, especially if present in low copy number, may be'
retained longer than larger conjugative plasmids in bacteria in soil, and GEMs that will
be released to soil will undoubtedly carry their heterologous DNA on nonconjugative
plasmids, if hot on the chromosome. Consequently, extensive studies should be
conducted on the mechanisms and frequency of transfer of nonconjugative plasmids in
soil.
In sterile soil, the conjugative plasmidis, pDU202 (64 Mda) and.pRR226 (26 Mda),
w^ich were derepressed for transfer, were transferred between strains of E. coll (Fig. 1)
, 1 t
only when nutrients (LB) were added, and the donors and recipients increased to high
numbers, and when the soil had an optimum water content (i.e., -33 kPa water tension; no
detectable transfer occurred and survival of the donors and recipients was reduced at
lower water tensions) (Devanas and Stotzky, 1988a). Similar results were obtained in
sterile soil with E. coli containing a 60-Mda plasmid (Trevors, 1987a; Trevors and Oddie,
1986). However, in nonsterile soil at the optimum water tension, no transfer of plasmids
pDU202 and pRR226 was detected between the same strains of £. coli, even when
11-15
-------
¦ ?>••••
• • Q..
o.
......
#.
*' •
•
i
i
•
%
m
\
•
*
\
MCMT f
•
.
10
15
day*
21
31
Figure 1. Survival and transfer of plasmids pDU202 and pRR226 from Escherichia coll
DU1040 to_E. coU PRC487 in sterile soil. Cells were inoculated in Luria Broth (LB).
Total numbers of bacteria were enumerated on MacConkey Ajar (MAC): strain PRC487
was lactose-positive strain and strain DU1040 was lactose-negative. Cells containing
plasmid pDU202 were enumerated on MAC containing 25>ug/ml chloramphenicol (Cm)
and those containing plasmid pRR226 on MAC containing 25x^/^1 tetracycline (Tc)
(Devanas and Stotzky, 1988a).
11-16
-------
nutrients were added initially and during the incubation, which resulted in the growth of
, _ I
the donors and recipients (Fig. 2) (Devanas and Stotzky, 1988a). The soil microbiota
apparently interfered in some manner (probably biochemically and not physically) with
the transfer of the plasmids. In contrast, Trevors (1987b) reported that a 60-Mda plasm id
was transferred between strains of E. coli in nonsterile soil at 22 C and at 20 or 80% of
the WHC, albeit at low frequencies, when the donor to recipient ratios were 1:1
(frequencies of 5 x 10"9 and 3.2 x 10"® at 20 and 80% of the WHC, respectively) or 1:10 (S
x 10"9 at 80% of the WHC), but not when they were 1:100 (at 80% of the WHC).
However, the broad host-range plasmid, RP4, was transferred from £. coll to not
only another strain of E. coli and, related Enterobacteriaceae (e.g., Enterobacter
aerogenes. Klebsiella pneumoniae, both autochthonous soil bacteria, and Proteus vulgaris
(Fig. 3)), but also to P. aeruginosa in both sterile and nonsterile soils (Figs. 4 and 5)
(Devanas and Stotzky; 1988a, 1988b). In sterile soil, there was growth of the donor and
recipients during the first few days after inoculation, especially when inoculated in LB,
and then the numbers remained essentially constant. Exconjugants were usually detected
2 h after inoculation (the first time measured) and throughout the incubation at levels
ranging from 10* to 10® CFU/g soil (oven-dry basis).
In nonsterile soil, the numbers of some recipients increased during the first few
days after inoculation and then either remained relatively constant or decreased by 1 to
« .
2 orders of magnitude to 10s to 107 CFU/g soil, depending on the recipient (Figs. 3 and
5). In contrast, the numbers of the donor decreased, sometimes after an initial Increase,
to either undetectable (less than 102 CFU/g soil) or low (e.g., 10* CFU/g soil) levels. The
numbers of exconjugants detected generally depended on whether the donor and
recipients were inoculated in saline or with nutrients (LB), on the type and concentration
of clay minerals with which the soil was amended, and, in part, on the species of the
recipient and the survival of the donor. For example, the numbers of exconjugants of the
Enterobacteriaceae remained at about 10* CFU/g soil throughout a 21- to 28-day
11-17
-------
5
9
N
i
i
Figure 2. Survival and transfer of plasmids pDU202 and pRR226 from Escherichia coli
DU1040 to E.coli PRC487 in nonsterile soil. Cells were inoculated in LB or saline. Total
bacteria were enumerated on MAC, bacteria containing pL)U202 on MAC containing Cm,
and bacterircontaining pRR226 on MAC containing Tc (see Figure 1 for details) (Devanas
and Stotzky, 1988a).
11-18
-------
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Figure 3. Survival and transfer of plasmid RP4 from Escherichia colt J53 to
Enterobacter aerogenes. E. coli DU1040, Proteus vulgaris, and Klebsiella pneumoniae in
nonsterile soil. Cells were inoculated in saline. Total bacteria were enumerated on
Luria Agar (LA): E. coli J53(RP4) on MacConkey Agar containing tetracycline (Tc);£.
aerogenes. E. coli DUI040, P. vulgaris, and K. pneumoniae on LA containing streptomycin
(Sm); and E. aerogenes, £. coli DU1040, P. vulgaris, and K. pneumoniae carrying RP4 on
LA containing Sm and Tc (Devanas and Stotzky, 1988a).
-------
1 « » IS . »• IT 1 4 7 1« ao »T
DAYS
Figure 4. Effect of clay minerals on survival and transfer of plasmid RP4 from
Escherichia coli J53 to Pseudomonas aeruginosa PAOl in sterile soil. Soil (Xitchawan, K)
'was amended with 3 or 12% (v/v) montmorillonite (M) or 12% (v/v) kaolinite (K). Cells
were inoculated in saline or Luria Broth (LB). Total bacteria were enumerated on Luria
Agar, J53(RP4) on MacConkey Agar containing tetracycline (Tc), PAOl on Pseudomonas
Isolation Agar (PlXT.lind PA01(RP4) on PIA containing To (Devanas and Stotzky, 1988a).
11-20
-------
DAYS
Figure 5. Effect of clay minerals on survival and transfer of plasmid RP4 from
Escherichia coli J53 to Pseudomonas aeruginosa PAOl in nonsterile soil (see Figure 4 for
details) (Devanas and Stotzky, 1988a).
Xlill
-------
incubation, even though the donor and recipients were inoculated in saline and the
numbers of the donor decreased by several orders of magnitude or, with E. aerogenes' and
P. vulgaris as the recipients, to undetectable levels (Fig. 3). In contrast, when P.
aeruginosa, as the recipient, was inoculated in saline, its numbers decreased slowly by
about 1 order of magnitude, but the numbers of the E« coli donor, also inoculated in
saline, decreased to an undetectable level after 20 to 27 days (Fig. S). The numbers of
exconjugants (P. aeruginosa(RP4)) detected differed with the type and amount of clay
present in the soil, but in all soils, the numbers of exconjugants decreased to
undetectable levels after 20 to 27 days, similar to the decrease in the donor. When the
donor and recipients were added in LB, the numbers of recipients remained essentially
constant, total bacteria increased Initially and then slowly decreased, the donor
increased by about 3 orders of magnitude during the first few days after inoculation and
then decreased rapidly by about 5 orders of magnitude, and the exconjugants, which were
initially detected 2 h after inoculation of the parentals, increased to approximately 5 x
10® CFU/g soil, followed by a rapid decrease to undetectable levels'(Fig. 5).
Conclusions
The results of these studies again emphasize the importance of the type of
bacteria and plasmids involved and of the physicochemical characteristics of the
recipient environment to the survival of, and genetic transfer by, GEMs. For example,
plasmids pDU202 and pRR226 were not transferred even between strains of _E. coll in
nonsterile soil, whereas the broad host-range plasmid, RP4, was transferred both intra-
and intergenerically fn nonsterile soil. P. aeruginosa survived better than Z. coll in
nonsterile but not in sterile soir, reflecting the relative competitiveness of these species,
the former being an autochthonous soil inhabitant. Similar survival data for these
species in soil (Klein and Casida, 1967; Zechman and Casida, 1982) and in soil extracts
(Walter et al., 1987) hf.ve been reported. The presence of nutrients generally enhanced
survival and gene transfer in nonsterile soil but had essentially no effect in sterile soil,
11-22
-------
and suboptimal water tensions inhibited transfer. Montmorillonite, especially at high
concentrations, apparently enhanced the frequency of transfer of both plasmid- and
chromosomal-borne genes*
it is not known whether the fluctuating survival patterns of the recombinant
bacteria reflected continuous conjugation or the growth and survival of recombinants
formed early in the incubation, as the recombinants were not inoculated but resulted
from gene transfer in soil. Consequently, the duration of gene transfer in soil may be
more apparent than real, as it is not possible to distinguish between continual gene
transfer and cell division of recombinant cells formed shortly after introduction of the
parentals. Nevertheless, the detection of exconjugants at levels ranging from 10"* to 10®
CFU/g soil for at least 20 days after inoculation of the parentals indicated that,the novel
genes survived. Therefore, there is a possibility that, depending on the host-plasmid
i
system involved, the introduction of GEMsinto soil and other natural environments could
alter the homeostasis of these habitats.
These results also indicate that more research is needed on the transfer of genetic
information by conjugation from GE.Ms and between indigenous bacteria iq soil and other
natural habitats in situ.. Furthermore, the differences in survival and genetic transfer
between the various host-vector systems studied indicate that the survival of, and
genetic transfer by, GEMs that may be released to soil or other natural environments
must, at this state of knowledge, be evaluated on a "case-by-case* basis.
• One aspect that is clear from these studies is that the frequency of transfer of
genetic material is higher in sterile than in nonsteril* soil, indicating that the presence
of the indigenous microbiota interferes with transfer. What is not clear is whether this
interference is the result of: 1) a reduction in the population densities of donors and
recipients caused by competition for nutrients, water, oxygen, space, etc. or by
amensalistic, parasitic, and predatory activities of the indigenous microbiota; 2) the
production of molecules by the indigenous microbiota that interfere with transfer (e.g..
11-23
-------
blocking or alteration of receptor sites on the recipients); or 3) other mechanisms. The
first possibility is probably not a major factor, as the frequency of transfer ir\ nonsterile
soil was reduced even when the population densities of the donors and recipients were
high enough to result in transfer in sterile soil (e.g., Figs. 1 and 4). The second possibility
is suggested by observations made in studies on the In vivo frequency of transfer of a
multiple drug-resistance plasmid from Salmonella typhosa to E. coli in the bladder of
healthy rabbits (Richter et ah, 1973; Stotzky and Krasovsky, 1981). The frequency was
reduced by greater than 1 order of magnitude when Proteus mirabilis and a
nonconjugative strain of E. coli (exogeins) were also introduced into the bladder. When
polystyrene latex particles of approximately the same size (0.8 ;im diameter) and at the
same concentration as the exogens were introduced into the bladder, the .frequency of
transfer of the plasmid from S. typhosa tc £. coli was not reduced, indicating that the
reduction caused by the exogens was not the result of a steric interference but of a
chemical interference in the conjugal process. Similar results were observed when these
studies were conducted in vitro in urine removed from the bladder or in a liquid mating
medium. Whether such interference occurs in soil is not known and requires study.
However, the apparent inability of narrow host-range plasmids (e.g., pDL'202 and
pRR226) to be transferred in nonsterile soil while they were transferred in sterile soil,
whereas broad host-range plasmids (e.g., RP4) were trcnsferred.in both nonsterile and
sterile soil, suggests that the transfer of some plasmids may be more susceptible to
chemical interference than the transfer of others.
It Is also not known whether the enhancement of the transfer of genetic material
by the addition of nutrients (e.g., better transfer when the parentaLs were added in LB
than in saline) was related to this interference. Although the addition of nutrients
increased the total numbers of detectable donors and recipients, the numbers initially
added, either in LB or saline, were; sufficiently high for transfer to have occurred.
However, the nutrients may havs increased the numbers of parentals in those
11-24
-------
microhabitats where transfer was occurring, or the nutrients may have increased the
robustness (or reduced the debilitation) of the parentals, which facilitated transfer. This
is another aspect that requires study in soil.
The studies.on conjugal transfer of chromosomal genes in soil showed that
auxotrophic bacteria (in these studies, genetically deficient for the synthesis of some
amino acids) can coexist by cross-feeding (syntrophism) in soil and on agar media
inoculated directly from soil by the soil replica-plate technique (Stotzky, 1965b) rather
than by only genetic recombination (Krasovsky and Stotzky, 1987; Weinberg and Stotzky,
1972). Similarly, cometabolism or "shared detoxification" of inhibitors can contribute to
the survival in soil of toxin-sensitive microbes in the absence of genetic recombination
(e.g., Daughton and Hsieh, 1977; Senior et_ al., 1976; Slater and Godwin, 1980; Slater and
Somerville, 1979). Furthermore, the transfer of genetic information in soil may be more
apparent than real, as the transfer may occur not in soil but on the agar used for the
subsequent isolation and enumeration of the donors, recipients, and putative
recombinants (Walter et al., 19J8a). These aspects, in addition to the "viable but
nonculturable" phenomenon, must be considered and controlled in studies on transfer of
genetic information in soil.
The apparent higher frequency of conjugal transfer of chromosomal-borne
• '
(Krasovsky and Stotzky, 1987) than of plasmid-borne (Devanas and Stotzky, 1988a, 1988b)
genes in soil suggests that conjugation may not occur long enough in soil for the transfer
of an entire large, conjugative plasmid. This suggestion was supported by preliminary
observations that the frequency of transfer of marker chromosomal genes was reduced as
their location was more distant from the oriT site. Studies on how plasmid size and the
location of chromosomal genes affect the frequency of gene transfer in soil should be
conducted, especially as such studies may provide clues for the construction of
recombinant DMA that would have a low frequency of transfer.
1-25
-------
Although nonconjugative plasm ids are used extensively as vectors for the
introduction of heterologous DNA into bacteria, the introduced DMA can be moved to the
chromosome, e.g., by a Tn, and from there be transferred to another bacterium. Hence,
conjugal transfer of chromosomal genes could be an important mechanism for the
dispersal of geneti§ information, e.g., from an introduced GEM to indigenous soil
bacteria. However, appropriate genetic manipulation of a chromosome containing
heterologous DNA can presumably reduce the probability of transfer in soil (Watrud et
alM 1986).
TRANSDUCTION
Introduction
Transduction is another method by which novel genes can be transferred to
indigenous soil bacteria after the release of GEMs to the environment. Moreover,
bacteriophages may serve as reservoirs of bacterial DNA in soil and other natural
habitats, as the packaging of genetic material in a transducing bacteriophage probably
represents an evolutionary survival strategy for bacterial genes (Stotzky, 1989; Stotzky
and Babich, 1986; Zeph et al., 1938). The survival of a transducing phage that contains
the novel DNA could be longer than that of the introduced GEM itself, and these novel
genes could reappear long after the GEM can no longer be detected if the phage infects
I t
susceptible indigenous bacteria. The probability of the occurrence of this scenario is
difficult to predict, as the persistence of, and transduction by, phages in soil and other
natural ecosystems has been insufficiently studied.
Two mechanisms of transduction, designated generalized and specialized
transduction, have been described in which virions that contain bacterial DNA can be
formed after infection of a bacterium by a phage. In generalized transduction, phages,
whose~DNA is replicated shortly after infection of the bacterial cell (lytic cycle), can
package DNA from the bacterium, whose chromosome has been cleaved into small
11-26
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segments by nucleases, in place of phage DN A during incorporation of DNA into the
i ,
phage capsid (Lin e£ alM 1984), Usually, less than X% of the bacterial genome' is
packaged into the phage virions. Any single or group of bacterial genes may be
incorporated, depending on the size of the phage capsid, and, thus, the designation,
generalized transduction. The transducing phages that carry bacterial DNA are capable
of adsorbing on and injecting their DNA into new bacterial host cells, but the DNA will
be defective for replication of the phages.
In specialized transduction, the bacteriophage genome is integrated into the
bacterial chromosome, usually at a specific site, and replicates faithfully with the
bacterial chromosome. The integrated phage genome is termed prophage DNA, and the
bacteria'that contain it are termed lysogenic (Lin et al., 1984). Upon induction of the
lytic cycle of phage replication, imprecise excision of the phage genome can result in the
packaging of adjoining bacterial genes on the chromosome into the virion with the
prophage DNA. The most extensively described specialized transducing phage is phage
lambda, which can transduce the genes fot galactose catabolism and biotin synthesis that
are located on either side of the integration site of this prophage in E. coll. The
imprecise excision event occurs at a low frequency, e.g., in about one bacterial cell per
e 7
10° to 10 cells. The ability to transfer only genes that are close to the site of phage
integration is termed specialized transduction. A specialized transducing phage may be
t 1
defective for some of its genes and may require the activity of "helper* phages to
replicate in the new host bacterium.
Two types of transducing phages, temperate and lytic, that differ in their mode of
replication in the bacterial host, have been described. Temperate bacteriophages are
capable of maintaining single or multiple copies of their genome in the host bacterium in
a prophage state; the phage DNA is either inserted into the bacterial chromosome
through a recombinational event (e.g., coliphage lambda or Mu-1) or remains as an
independent and autonomous replicon (e.g., coliphage PI or phage F116 of P. aeruginosa).
11-27
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similar to a bacterial plasmid. A temperate phage can initiate a lytic cycle of
replication and virion formation (induction), although this occurs at a low frequency
under normal conditions (e.gM one in 10^ to 10® DNA replication cycles for phage PI).
Exposure of lysogenic bacteria to temperatures above the optimum for growth or to
mutagenic agents can result in the induction of lytic reproduction at a higher
frequency. Only between 1 to 10% of the infective virions produced after induction jn
vitro will usually establish lysogeny after infection of new host cells, as most infections
result in lysis of the cells.
Lytic transducing phages undergo the normal lytic cycle of infection and
formation of new virions in the bacterial host, and they are not capable of establishing
lysogeny. During replication of the phage DNA, genetic material from the host
bacterium, that has escaped degradation by phage-encoded nucleases, can be randomly
packaged in to,intact virions at a low frequency. Hence, lytic transducing phages are all
generalized transducing phages. In a typical lysate, about 1% of the virions will contain
only bacterial DNA (Stent and Calender, 1978). Consequently, a phage lysate of
sufficient titer (i.e., at least 10s phages/ml) can be expected to contain at least one
virion carrying a copy of each gene on the bacterial chromosome.
Transduction In situ
Gene transfer in situ by transduction has been studied to only a limited extent.
There have been few studies on transduction in marine or freshwater ecosystems (Baross
et al., 1974; Morrison et al., 1978; Saye et al., 1987) and in terrestrial ecosystems
(Germida and Khachatourians, 1988; Zeph et al., 1988; Zeph and Stotzky, 1989), even
though transducing phages for a variety of bacteria have been isolated from soil. Zeph et
al. (1988) studied the transduction in soil of bacterial genes that confer resistance to
antimicrobial agents by ooliphage PI Cm £ts, which carries a resistance gene for Cm,
and a variant of this phage, PI Cm ets::Tn5Ql» which also carries a Hg-resistance gene on
the transposon, TnSOl. A temperature-sensitive mutation
-------
inactivation, at temperatures above the optimum for growth (e.g., at 42 C), of a
repressor protein coded for by phage PI that maintains lysogeny.
Lysates of phage PI (105 to 10® plaque-forming units (PFU)/g soil (oven-dry basis))
were added to soil with J|. coli (10^ to 10® CFU/g soil), and soil dilutions were
periodically plated on selective media to quantitate the numbers of E. coll. phage PI, E.
coti transductants (Cm- or Hg-resistant cells lysogenized by phage PI), and total soil
bacteria. Significantly highermaximum numbers of coli transductants were observed
in sterile (approximately 10* transductants/g soil) than in nonsterile (approximately 10®
transductants/g soil) soil (Fig. 6). Amendment of nonsterile soil with nutrients (LB) on
day 0 did not significantly affect the numbers or survival of E. coli transductants during
the 28-day experiment, even though the numbers of total E. coli increased by 1 order of
magnitude in *he nutrient-amended soil (Fig. 7). Weekly amendment of the soil with LB
also (lid not result in significant increases in numbers of E. coli, phage PI, or E. coli
transductants (Fig. 8) (Zeph and Stotzky, 1988). These results demonstrated that phage
PI was capable of infecting and transducing E. coli under the relatively low nutrient
conditions that presumably occur in natural soil. Although the survival of phage PI was
slightly greater in soil amended with the clay mineral, montmorillonite, than with the
clay mineral, kaolinite, this improved survival did not result in significantly higher levels
of transduction (Fig. 9).
When phage PI was added to sterile soil in lysogenic E. coll J53(RP4XP1 Cm
cts::Tn501) together with nonlysogenic£. coli W3110 (both at approximately 10® CFU/g
soil) and nutrients (LB containing 2 mM CaC^ and 10 mM MgSOjJl^O; LCB), phage PI
was released from the lysogenic E. coll and infected E. coll W3110. which resulted in
approximately 1Q5 tr&rsductants/g soil (Fig. 10). In nonsterile soil, using E. coli
W311Q(R702) as the recipient and E. colt J53(PL Cmets) as thelysogen, the number of E.
coli W3110(R702)(P1 Cm cts) transductants was significantly lower, as 102
transductants/g soil were recovered on day 1 only. The multiplication and survival of the
a.
-------
I E.coll
g.«Sil
Tranaductanta
* Total Gram Nagativa
• E.coll
Figure 6. Comparison of transduction of Escherichia eoli W3U0(R702) by phage PI CM
cts in sterile (A) and nonsterile (B) soil. Bacteria and phage inocula were added in LCB
(Luria Broth containing 10 mM MgS04*7H20 and 2 mM CaCl2) on day 0. Means+ SEMs,
which are shown if larger than the dimensions of the symbols (Zeph et aL, 1988).
11-30
-------
Total Gram NoQetiv*
a --a
Figure 7. Comparison of the effect of inoculating Escherichia coli J53(RP4) and phage PI
Cmcts:;Tn501 in saline (A) or LCB (B) on transduction in nonsterile soil (see Figure 6 for
details) (Zeph et al., 1988).
11-31
-------
8
^ 6
O
(0
* •
u.
*• 4
v.
0
1 ;
o
Or 2
o
Total dramNagatlva
e. coii JSi(H*4)
PI PHaga
15
Days
23
Figure 8. Transduction of Escherichia coli J53(RP4) by PI Cm cts::Tn501 inoculated in
LCB into nonsterile soil. Sterile LCB was added on days 7,14, and 21, as indicated by the
arrows (see Figure 6 for details) (Zeph and Stotzky, 1988).
11-32
-------
0E.coM
Tranaductants
Figure 9. Transduction of Escherichia coli.J53(RP4) by phage PI Cm ct3:;Tn501 in
nonsterile soil amended with (A) 12% kaolinite (12K), (B) 6% montmorillonite (6M), and (C)
3% montmorillonite (3M). Bacteria and phage inocula were added In LCB (see Figure 6
for details) (Zeph et alM 1988).
II -33
-------
Days
Figure 10. Transduction in sterile (A) and nonsterile (B) soil inoculated with either
Escherichia coli J53(RP4) lysogenic for phage PI Cm cts::Tn501 and nonlysogenic E. coli
W3110 (A) or _E. coli J53 lysogenic for phage PI Cm cts and nonlysogenic E. coli
W3110(R702) (B). Bacterial inocula"wef^"added Tn ECB"on day"0 (see Figure-S for"details)
(Zeph et al., 1988).
11-34
-------
lysogenic donor and nonlysogenic recipient E. coli were also significantly lower than in.
}
sterile soil (Fig. 10). Despitr. "he lower survival and frequency of transduction in
nonsterile than in sterile soil, these studies showed that phage PI released from one
lysogenic E. coli can lysogenize susceptible bacterial cells under conditions similar to
those in natural soil.
E. coli transductants isolated from soil were verified by heat-induction of phage
PI and with a biotinylated DNA probe, which demonstrated the presence of phage PI
DNA in the isolates (Zeph and Stotzky, 1989). Approximately 300 isolates of indigenous
soil bacteria, isolated on MAC containing 75 >ig/ml Cm and inoculated with a 10"^
dilution of soil, were negative when subjected to both verification procedures (Table 2),
even though the theoretical minimum limit of detection was 10 transductants/g soil, and
' i • •
the specificity and sensitivity of the DNA.prob^ was satisfactory. Consequently, few, if
any, indigenous,bacteria in the soils studied were apparently capable of being transduced
by phage Pi. .
Germida and Khachatourians (1988) studied the generalized transduction by phage
Pi of amino, acid and Tc-resistance markers on the chromosome of E. coli in a sandy and
a silty clay loam soil. .The frequency of'transduction of these markers to added recipient
JJ
strains of E. coli was 10 . which was similar to the frequency obtained in vitro. Thus,
conditions in nonsterile soil were apparently not detrimental to transduction by phage PI.
A generalized transducing phage of P. aeruginosa. F116L, has been shown to
transfer plasmid RP4 to P. aeruginosa PAOl in soil (Zeph and Stotzky, 1988). When a
, i
lysate of phage F116L (107 PFU/g soil), raised in P. aeruginosa PAOl carrying plasmid
RP4, was added to sterile soil with nonlysogenic cells of P. aeruginosa PAOl (10® CFU/g
soil), presumed P. aeruginosa transductants resistant to Tc were detected at
approximately 10^ transductants/g soil (Fig. 11). These presumptive transductants were
confirmed by the demonstration that their antibiotic-resistant phenotype was the same
as the antibiotic-resistance markers present on plasmid RP4 (Km, Ap, and Neomycin
11-35
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Table 2. Verification by heat induction of lysis and w'.th a biotinylated DNA probe
of presumptive transductants of Escherichia coli and indigenous soil bacteria
isolated on MacConkey Agar containing either HgC^ (30 ;iM) or Cm (75^ig/ml).
Presumed Heat induction Hybridization
transductants Soil of phage Pla with PI DNA^
Indigenous Nonsterile 0/297 0/297
JE. coli. W3110(R702) Sterile 15/15 NDC
Nonsterile 8/8 8/8
E. coli J53(RP4) Sterile 12/13 ND
Nonsterile 25/25 7/7
a No. of isolates that'were positive/no. tested.
** No. of colonies or dot blots that were positive/no. tested.
c Not determined.
(Zeph and Stotzky, 1989)
11-36
-------
10
o
CO
0)
u.
ft.
c
o
s
u.
o
0)
o
8
4
Ph*0« F1161
Figure 11. Transduction of Pseudomonas aeruginosa PA01 by phage F116L in sterile soil.
The phage F116L inoculum was raised on I\ aeruginosa PAOl carrying the drug-resistance
plasmid, RP4. The bacteriunrand phage were inoculatediirLuriaBroth (LB) (Zeph and
Stotzky, 1988).
I 1-37
-------
(Mm), in addition to Tc) and by plasmid screening by gel electrophoresis, which showed
the presence of plasmid DNA of the same molecular mass as plasmid RP4 (Fig., 12). No
RP4 transductants of JP. aeruginosa PAT2 or of j\ aeruginosa PAT2 lysogenic for phage
F116L were detected when these recipient bacteria were added to sterile soil with phage
F116L carrying plasmid RP4.
Conclusions
Transduction as a mechanism of genetic transfer may be as, or more, important in
soil and. other natural habitats as conjugation and transformation. Bacteriophages are
capable of multiplying in natural soil, which emphasizes the potential for transducing
phages to transfer genes in situ. Furthermore, the survival of viruses, including
bacteriophages, in soils and waters is enhanced by their adsorption on clay minerals,
especially on montmorillonite, and probably on other particulates. Consequently, DNA in
an indigenous bacterium or introduced in a GEM could be incorporated into the genetic
material of a phage and could persist in soil longer than in the host bacterium itself.
Such persistence would be undetected (i.e., cryptic) in the absence of a bacterial host
susceptible to infection and reproduction of the phage. However, if an appropriate host
were subsequently infected, the recombinant DNA could be rapidly dis{>ersed among
susceptible bacteria; e.g;, a single nondefective phage that was able to accommodate the
extra DNA and with a burst size of 100 could transfer the recombinant DNA to
approximately 10s bacteria after only four lytic cycles. Hence, there is the potential for
a novel gene to persist undetected and then unexpectedly reappear in soil and other
environments if the transfer of the gene occurred by transduction.
Gene transfer to indigenous bacteria by conjugation can theoretically be
controlled by engineering the novel genes into the chromosome and through the use of
suicide plasmid vectors (Cuskey et ah, 1988). However, placing the novel genes in the
chromosome will not necessarily decrease the probability of their transfer by
transduction, especially as no techniques have been developed for limiting transduction in
11-38
-------
1 2 3 4 5
Figure 12. Agarose gel electrophoresis of plasmid and chromosomal DNA extracts of
three individual isolates of presumed transductants of Pseudomonas aeruginosa PAOl
Isolated from sterile soil that was inoculated with phage F116L and _P. aeruginosa PAOl
(see Figure 11). Lanes 1 to 3: P. aeruginosa PAOl isolates; Lane 4: P. aeruginosa
PA0KRP4); Lane 5: Escherichia coli J53(pDU202): Mr of pDL'202 = 64 Mdaj Mf of RP4
65 Mda (Zeph and Stotzky, unpublished). ' _
I 1- 39
-------
situ. Few studies on the frequency of lysogeny in bacteria exist, and it is likely that even
these studies underestimate the frequency, as it has been demonstrated that most strains
of _R. aeruginosa and several strains of P. fluorescens are lysogenic (Holloway, 1969).
To date, studies on transduction in situ have focused on two species of bacteria,
i.e., E. coli and £. aeruginosa, and on a limited number of genetic markers, i.e., genes O
encoding resistance to antimicrobial agents and amino acid auxotrophy. Studies on
transduction in situ are needed with more bacterial species and genetic markers,
particularly with GEMs that are being \ised or proposed for environmental releases, e.g.,
Rhi2obium meliloti containing recombinant nitrogen-fixation genes.
11-40
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m. Terrestrial Microcosms
Introduction
Terrestrial .microcosms have been defined as analogues of the field, as a field
within the laboratory (Pritchard, 1988), or as physical models of the natural environment
in which experimental conditions in the laboratory are intended to mimic a field setting
(Pritchard and Bourquin, 1984). The extent of such simulations can be as broad.as
isolating in the laboratory a "piece of the field" that acts ecologically similar to an
identical companion "piece" in the actual field (Hicks e£al., 1989) or as simple as adding
sterile soil to broth in test tubes (Walter etal., 1987). The spectrum of experimental
systems that have been called microcosms has resulted in numerous discussions on "what
is a njicrocosm?". Greenberg et al. (1988) stated that "the term microcosm seems to
mean all things to all men, ranging, for examplej from 'synthetic' to 'natural' systems".
The consensus of opinion, regardless of the style or complexity of the microcosm, was
that "it is essential that the questions to be asked with the micrbcosm, and the
experimental design, are carefully thought out" and "that those workers who use
microcosms should understand the uses, limitations and bias of the particular systems and
ensure that people using their results are also aware of these facts". Bull (1980) pointed
out that one benefit of the use of microcosms is their replicability in statistical trials;
however, they are not exact reproductions of the field but, rather, analytical tools that
can be used as the basis for environmental studies.
Simple microcosms
The simplest terrestrial microcosm consists of soil in a container. Sterile soil
provides a gnotobiotic environment in which to evaluate GE.Ms in the absence of the
indigenous microbiota. Soil may be amended with nutrients and clay minerals, adjusted
to optimum pH values and water contents, and added to test tubes, flasks, or other types
of containers. Such simple microcosms have been used to evaluate the survival, growth,
111 -1
-------
gene transfer, and effects of GEMs in soil in the laborator (Devanas et al., 1986; Doyle .et
al., 1988; Jones et al., 1988; Krasovsky and Stotzky, 1987; Stotzky, 1965a, 1989; Stotzky
and Babich, 1986; Weinberg and Stotzky, 1972) and changes in plasmid frequencies in
natural soil microbial populations (Wickham and Atlas, 1988).
Quantities of soil between 2 and 50 g are recommended for simple microcosms, as
tHis facilitates handling of soil samples in the laboratory, enables the initial dilution of
the entire soil sample in the same container, and ensures good mixing of the diluted soil
sample. Sufficient dilution and mixing of the soil are necessary to prevent aggregation
and settling of the soil in pipettes during dilution, which would introduce error and
reduce the reproducibility of replicate samples. The soil microcosm used as an example
in this document consists of a screw-cap test tube (18 mm x 150 mm), loosely capped and
maintained in a high humidity chamber, into which approximately 2 g of soil at the -33
• ¦' i '
kPa water tension (approximately equivalent to the water content at field capacity) is
placed. The container should be large enough to ensure that the surface: volume ratio of
the soil sample is sufficiently high to maximize gas exchange between the soil and the
head-space of.the container (e.g., for large samples of soil, an Erlenmeyer flask is better
than a test tube). The selection, preparation, maintenance, and storage of soils is
discussed in Section IV.
Master jar studies. The. over-all metabolic activity of microbes in soil can be
determined with respirometric techniques that monitor either COj evolution or 02
uptake. These methods, especially when COj evolution is used, probably provide the best
and most easily measured index of the gross metabolic activity of mixed microbial
populations In soil (see Stotzky, 1960, 1965a, 1974, end Anderson, 1982, for details of the
techniques). The "master Jar" technique (Stotzky, 1965a) (Fig 13) enables removing
subsamples of soil during an extended incubation for various analyses (e.g..
transformation of substrates, species diversity, enzyme activities, survival of introduced
bacteria, including GEMs and their genes), in addition to continuous measurement of COj
ni-2
-------
oducrd from
b«>l i»
-------
evolution, without disturbing the remainder of the soil and, thereby, eliminates
artifactual peaks in CO2 evolution that can result from the physical disturbance of the
soil. The soils are incubated under controlled temperatures and maintained at their -0.33
kPa water tension by continuous aeration with water-saturated, C02-free air. The
amount of CO2 trapped in NaOH collectors is determined, after precipitation of the COj,
with BaC^. by automatic potentiometric titration with HCL
The gross metabolic activity of the heterotrophic soil microbiota is measured by
the addition of a nonspecific substrate (e.g., glucose), and the activity of specific
components of the microbiota is evaluated by the addition of specific substrates whose
mineralization is dependent on the ability of these components to synthesize appropriate
enzymes (e.g., celluloses, starches, lipids, Hgnins,,aldehydes, proteins). At various times
after the start of the incubation, subsamples of soil, in their own containers, are removed
from the master jars and subjected to a variety of microbiological, chemical, and
enzymatic analyses. Species diversity is determined by inoculating decade serial
dilutions of the soil onto or into selective media, usually at least in triplicate. The soils
can also be amended with the specific substrate (e.g., toluene, xylenes, 2,4-D) on which
the gene product of a novel gene in an introduced GEM functions, to determine whethei"
this provides an ecological advantage to the GEM and how this affects both nonspecific
and specific metabolic activities, as well as other processes (Doyle et al., 1988; Jones et
al., 1988).
Sefl replica plating. The soil replica plating technique (Krasovsky and Stotzky,
1987; Stotzky, 1965b, 1974; Weinberg and Stotzky, 1972) can be used to determine the
growth rates of GEMs and their homologous hosts, the ability of the GEMs to compete
with representative soil microbes, and the transfer of genetic information. For example,
the GEMs or the hosts are inoculated into the center of petri dishes containing sterile
soil, and representatives of the indigenous soil microbiota (bacteria, including
actinomycetes, and fungi) are inoculated into equidistant sites around the GEMs. The
II1-4
-------
plates are incubated in a high-humidity chamber, and replication from the soil plates to
selective agars are made periodically with a replicator constructed with stainless steel
nails and acrylic, plastic and sterilized with alcohol and flaming. The design of the
replicator permits numerous replications from the same soil plate without significant
disturbance of the soil. The growth of all the inoculated org$$isms is recorded on maps
of the soil plates, and growth rates (in mm/day) are calculated. The technique can also
be used with nonsterile soil, but highly selective media must be used to prevent
overgrowth of the GEMs by the indigenous soil microbiota (see Section VII).
Complex microcosms
Undisturbed soil microcosms consist of soil cores of varying size that are brought
Into the laboratory with minimum disturbance of the structure and biotic composition of
the soil. The soil sample is removed intact.from the coring device, and thereby, the soil
system is relatively undisturbed. Disturbed microcosms are those in which the soil.,
ecosystem is reconstituted in the laboratory, e.g., the placement of excavated soil into
i
containers in which seeds or seedlings are planted.
Undisturbed soil tores have been utilized to study, for example, pesticide
degradation (Johnen and Drew, 1977; Wingfield et al., 1977), microbial community
responses to environmental perturbations (Elliot et al., 1986), soil denitrifier populations
under anaerobic and aerobic conditions (Martin et al., 1988),. the fate and ecological •
effects of GEMs (Bentjen et al., 1989), and the.effects and mobility of xenobiotics in soil
containing plant root systems (Hicks et al., 1989; see Fig. 14). In the latter study,
uniform and realistic distributions of the xenobiotics were attained with simulated rain
events, and the data on environmental perturbations were consistent with those obtained
in parallel field studies. Other studies with intact soil cores have also adequately
predicted events subsequently observed in field studies (see Pritchard and Bourquin,
1984). The soil cores can_be_sampled for_the,enumeration-of-microbial populations at
111 -5
-------
:.T
0fi»coc'O^
Hign 0«n»irv
^oivainviin*
lm»ci
Soil Cot*
Figure 14. Terrestrial soil-core microcosm test system (Hicks etal., 1989).
111-6
-------
different depths through sampling ports inserted in the walls of the soil core cylinder
(Ardakani e£al., 1973). This also enables the soil sample to be replaced with fresh soil.
Examples of various types of microcosms (Atlas and Bartha, 1981; Gillett, 1988; Johnson
and Curl, 1972) and guidelines for the use of soil core microcosms, with descriptions of
various soil core designs, sampling procedures, and statistical analyses, have been
published (Anon., 1987).
With disturbed soil microcosms, it is assumed that biotic and abiotic processes and
interactions representative of the in situ situation will be reestablished after suitable
incubation or treatment (Pritchard and Bourquin, 1984). Small nonsterile soil cores (5 cm.
x 2.5 cm deep) were used to study changes in microbial populations and respiration after
organic amendment (Elliot et al., 1986). Holben et al. (1988) used a soil-vermiculite
microcosm (700 g of this mixture in plastic pots) to study the survival of a model GEM.
A coring device was inserted through the side of the plastic pot, a sample of the mixture
was removed for enumeration of the GEM, and a test tube was inserted, into the hole
formed by the coring device. Survival iand plasmid transfer have been studied with GEMs
added to a rhizosphere microcosm that consists of a peat-vermiculite mixture containing
radish or bean plants. The pots or flats containing the simulated soil and plants were
maintained within large chambers that enable the control of relative humidity,
temperature, and light/dark cycles (Fig. 15) (Armstrong et al., 1987; Gile and Gillett,
1982; Knudsen et al., 1988).
The soil perfusion technique is a highly sensitjve, easy to sample, and continuous
system for measuring the kinetics of biochemical transformations in soil. Sieved soil is
added to a glass column and perfused with water at a cycling rate that achieves the
desired saturation and aeration of the soil column. Various amendments can be added to
the soil, and the perfusate is periodically analyzed for the biochemical transformations
of interests, e.g., nitrogen transformations. This technique has been used to study the
impact of GEMs and various environmental factors, e.g., sulfur dioxide, acid
111-7
-------
A.r .nifi
i
Figure 15. A microcosm used to study the survival, fate, and genetic stability of
i .'combinant bacteria in the rhizosphere and in herbivorous insects. Physical conditions
(e.g., temperature, humidity, light/dark cycles) can be controlled in this system (Gile and
Gillett, 1982)..
I I 1-8
-------
precipitation, and heavy metals, on ecologic processes in soil (Doyle et al., 1988; Jones et
al., 1988; see Stotzky, 1974, 1986). The enumeration of added GEMs and of the
indigenous microbiota and measurement of gene transfer can be accomplished by either
sampling or sacrificing individual soil perfusion columns at various times, diluting, and
plating the soil dilutions on selective media.
An apparatus for growing plants with the root systems maintained sterile has been
described for the collection of root exudates and for measuring root respiration and the
release of other volatiles from roots (Stotzky £t al., 1962a). The apparatus consists of a
gr.owth tube containing the planted seed or seedling, a root chamber containing a solid
growth medium (e:g., sand, vermiculite, soil), an irrigation chamber, an irrigation
reservoir, an aeration system, a root exudate collecting system, and inoculation ports.
The root system is separated from the above ground portion of the plant by a silicone
sealr which eliminates the need for enclosing the whole plant. This system could be
easily utilized for studying gene transfer in the rhj-osphere.
1II-9
-------
IV. Selection, Preparation, Maintenance, and Storage of Soils
Introduction
Knowledge about the structure, physicochemical composition, and microbial
events in microhabitats in soil is sparse and mostly conjectural. What can be assumed
with a high degree of certainty is that these microhabitats, and.even sites within a single
microhabitat, are heterogeneous and differ in the type and amount of clay minerals,
organic and inorganic substances, and other physicochemical characteristics and,
therefore, in microbial composition and activities, including the transfer of genetic
information (Stotzky, 1974, 1985, 1989).
Selection of soils.
The selection of soils for studies on gene transfer among bacteria should be based
pn the relevance of the soils to projected field,release sites; for example, recombinant
rhizobia should be studied in representative agricultural soils on which legumes are grown
commercially. A complete soil analysis should be obtained, to determine the relevance
and representativeness of the soil samples to the experimental purpose and design and to
correlate the survival of, and gene transfer by, the introduced GEM with the
physicochemical characteristics of the soils. The soil parameters determined should
include pH, organic matter content, cation-exchange capacity, water content at the -33
. t
kPa water tension (and, preferably, also the water-holding capacity (WHC) and the
permanent wilting point (PWP)), elemental analysis (including both plant nutrients and
toxic heavy metals), and clay mineralogy (Babich and Stotzky, 1977). This information
can most easily be obtained by sending soil samples to the soil testing laboratory of the
appropriate State Agricultural Experiment Station for analysis, or analyses can be made
using the methods described in Klute (1986) and Page et al. (1982).
The base-line microbiological characteristics of a soil sample that should be
obtained include numbers of total bacteria, actinomycetes, gram-negative and gram-
IV-1
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positive bacteria, spore-forming bacteria, nitrifiers, denltriflers, cellulose utilizers,
fungi, and protozoa. The numbers of soil bacteria resistant to specific antimicrobial
agents (e.g., antibiotjcs, heavy metals), especially to those agents to which the GEMs to
be studied are resistant, should be evaluated. Other physiological groups of bacteria and
the activity of selected enzymes (e.g., acid and alkaline phosphatases, arylsulfatases,
dehydrogenases) can also be measured to provide an index of over-all microbiological
activity. This information is often useful, as changes in any of these parameters may
provide indications of the persistence and ecological effects of the GEMs in soil.
Preparation of soils
The preparation of a disturbed soil sample for use in laboratory studies on gene
transfer and the ecology of GEMs begins with particle stae separation. The soil is sieved
(usually through a 2 mm sieve) to remove stones, gravel, roots, other debris, and large
organisms. Hence, the soil particles will not vary significantly in size distribution from
sample to sample, and the water content at the -33 kPa tension Will be relatively
constant. The sieved soil sample may then be amended: e.g., with CaCOj to raise the
pH; with mined clay minerals to adjust the concentration or type of clay; with water to
adjust the water tension; with an inoculum of a rricrobiologically-active soil (e.g., from
the field or a flower pot) to provide an active microbiota; with nutrients (e.g., a nutrient
broth or glucose plus mineral salts) to establish an active indigenous microbial
population; or, usually, with some combination of these. Conditioning the soil by
incubating it with a mixed nutrient amendment (e.g., glucose plus mineral salts), in
addition to sufficient water to bring the soil to its -33 kPa tension, before initiation of an
experiment may be necessary if the soil has dried excessively during storage, *hich could
result in some members of the microbial community declining in number or becoming
dormant.
To determine the effects of the indigenous soil microbiota_on_ihe_transfer_of
genetic information by the added GEMs, parallel studies should be conducted in sterile
1V-2
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soil. The length of time for soil sterilization and the number of sterilization cycles
varies with the size of the soil sample (Johnson and Curl, 1972). Sterilization with moist
heat is accomplished, by autoclaving the samples at IS psi and 121 C for 30 min. To
ensure sterilization, especially if the soil sample is larger than 50 g, a second autoclaving
sho&d be conducted 24 h later. This second autoclaving should kill bacterial spores,
which have been heat-shocked by the first autoclaving to germinate into vegetative cells,
and microorganisms protected inside soil aggregates and associated with organic
matter. A sterility check should be performed by adding a nutrient medium to the soil
and, after incubation for 24 to 48 h, the preparation of a streak plate with a It 10 soil:
water dilution on a nonselective medium to detect the presence of microorganisms.
Sterilization with moist heat results in the formation of toxic coiTi^inds in soil, such as
reduced manganese and various organic and inorganic volatiles. Therefore, the
autoclaved soil samples should be maintained, preferably at room temp*. 'ire and in a
high humidity chamber, for several days before inoculation of the GEMs (S ^iiVy, 1986).
Gamma-irradiation Is an attractive alternative technique for the sterilization of so?, as
it does not result in as many changes in soil (e.g., substantial release of nutrients or toxic
chemicals) as does autoclaving (McLaren et ah, 1962; Stotzky and Mortensen, 1959).
However, it is not as convenient as autoclaving, as it requires a source of gamma rays,
and large soil samples cannot be sterilized effectively, as the penetration of gamma rays
i
into soil is poor.
Maintenance of aoOs
The water content of the soil can be maintained at a constant -33 kPa tension
during the experiment by incubating the soil samples in a high humidity chamber or by
periodic addition of sterile water on a weight basis. Generally, the water content of the
soil sample at the beginning of the study is adjusted to the -33 kPa water tension, as this
~is the water content at field capacity (i.e., the water retained by soil 24 to 48 h after a
rainfall or irrigation) (Cassel and Nielsen, 1986). This water tension, as well as other
IV-3
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critical soil water contents (e.gM WHC, PHP), can be obtained from the State
Agricultural Experiment Station, as mentioned above.
Experiments.conducted with aerobic microorganisms require adequate aeration.
Hence, the soil containers should allow sufficient diffusion of air by placing closures
loosely on the container, and the container should be less than one-half full to provide an
. adequate heed space for air above the soil sample. Alternatively, the soil can be aerated
with a constant or intermittent stream of water-saturated air, which will provide both
sufficient oxygen and water to maintain the soil at its -33 ItPa water tension (Stotzky,
1965a).
The effects of nutrient amendment of the soil on survival and gene transfer can be
studied by periodic additions of either simple (e^.,. glucose) or complex (ejg., cellulose,
humic materials) organic compounds or w|th a specific substrate utilizable only by the
GEM, as with GE.Ms capable of degrading xenobiotics (e.g., toluene, 2,4-D). The effect
of nutrient amendments on available water content, pH, and other physicochemlcal
characteristics of the soil must be evaluated.
Sampling of soils
Sampling of soil inoculated with GEMs can Involve the sacrifice of individual
microcosms or of multiple samplings from the same microcosm. Individual microcosms
can consist of test tubes or flasks containing subsamples of the same soil that are
sacrificed at different times.(see Section III). Usually, triplicate or more replicates are
sacrificed at each sampling time. The soil sample Is commonly diluted with either sterile
tap water, saline (0.85% NaCl), or a phosphate buffer (pH 7) to a It 2 (e.g., 10 g soilt 10
ml diluent) or a It 10 (e.g., 2 g soil: 18 ml diluent) dilution (see Wollum, 1982, for other
diluents), and the subsequent appropriate serial decade dilutions are used to inoculate
either spread or pour plates containing selective media. Numerous techniques for
dispersal of the microbes in the diluent have been described, and generally, each"
procedure is selective for a specific portion of the soil bacterial population (Casida,
IV-4
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1968). The efficacy of recovery of inoculated GEMs is usually dependent on the type and
ionic strength of the diluent, the amount of mixing, and the temperature at which the
sample, is mixed (Jensen, 1968).
Methods for multiple samplings from undisturbed soil microcosms, e.g., soil cores,
for studying the survival of, and gene transfer by, GEMs are presented in Section 111.
Multiple samplings from the same microcosms should be performed so that the soil
structure of the microhabitats is not significantly disrupted, as this could affect
microbial events and subsequent samplings. Microbial counts may vary with the depth in
the soil core, and contamination of a soil sample from one depth with soil from other
depths must be avoided.
Storage of sbtta
Soil samples should be stored in bulk, so that repeat experiments can be conducted
with the same soil samples without resampling in the field, which could result in
variability in samples and microbiological responses. Bulk soil samples can be stored in
open plastic bags, usually in metal or plastic garbage cans, and the water lost as a result
of air-drying can be replaced, along with desired nutrients, clays, or other amendments;
to condition the soil before experimental use, as discussed above. However, the
remoistening of air-dried soil can result in increases in microbial populations caused by
the release of nutrients, and the soil should be allowed to incubate for at least one week
before the initiation of an experiment. Soil samples can also be stored in thin-walled
polyethylene bags that prevent the loss of moisture and allow the exchange! of oxygen,
carbon dioxide, and other gases. However, storage in even such bags can result in
significant changes in the composition of the soil microbial community (Stotzky et al.,
1962b). Storage at 4 C reduces the loss of water, but such storage probably affects the
microbial composition of soil, as this temperature may not be favorable for the survival
of some soil microbes and vice versa.
IV-5
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V. Methods for Studying Conjugation
IN VITRO
Conjugal gene transfer in bacteria can be demonstrated in various ways, depending
on the location of the genes (i.e., on the chromosome or on plasmids) that will be
transferred ICurtiss, 1981). The frequency of transfer (i.e., the frequency of
recombination, as this is what is actually measured) of the experimental strains should be
determined in standard laboratory conjugation studies, so that the potential for gene
transfer by conjugation in these strains, especially when introduced into soil, can be
assessed. Moreover, this will enable meaningful comparisons between the experimental
strains and well-studied and documented strains, both in vitro and in situ.
In all studies of gene transfer, both In vitro and in soil, appropriate controls must
be included to determine the frequencies of spontaneous mutation, as these'frequencies
may differ in vitro and in soil. In vitro mutation frequencies are most easily, determined
during verification of the phenotypes of the donors, recipients, and plasmids. Mutation
frequencies in soil are determined by inoculating the recipients and the donors, with and
without the plasmids of interest, individually into soil and, after various periods of
incubation, plating appropriate soil dilutions on all selective media used to enumerate
donors, recipients, and recombinants. For example, a lactose-positive, Cm-sensitive
recipient should be plated, on MAC containing Cm at the same concentration that will
allow the growth of a lactose-negative, Cm-resistant donor and of the resultant
recombinants. The frequency of gene transfer must be corrected for the frequency of
spontaneous mutation in theparentals.
Chromoaomal transfer
A model for the transfer of chromosomal genes in soil that can be used to develop
studies with other bacteria is the transfer of prototrophic markers from Hfr E. coli K12
donor strains to auxotrophic F" E. coli K12 recipient strains (Krasovsky and Stotzky,
V-l
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1987; Weinberg and Stotzky, 1972). The mating type and phenotype of the E. coli strains
used in these studies were: KS03, Hfr, prototrophic, streptomycin-sensitive (Sms);*493,
Hfr, prototrophic, Sms; and *696, F", auxotrophic for leucine, proline, and arginine (Leu"*
ProB", Arg"), and streptomycin-resistant (Smr) (Bachman and Low, 1980; Curtiss and
Renshaw, 1969).
Media. The type of medium, i.e, liquid or agar, used for the growth of cultures
will depend on the design of the experiment and the information desired. For rapid (e.g.,
overnight) production of a large number of cells, as for an inoculum, shaken broth
cultures are best. A solid medium should be used to distinguish .metabolic, traits of
individual colonies, which is necessary for the enumeration and phenotypic screening of
transconjugants. The preferred type of medium for various procedures is described
below. Commercial sources of standard media and formulations of specific media are
listed in the Appendix.
Cultures should be' maintained at 4 C on agar slants, e.g., on Bacto-Penassay Agar
(PA), and transferred monthly. Some of the phenotypic markers that are commonly and
most easily used to demonstrate gene transfer are unstable or revert and, therefore, can
be lost from the gene pool, unless there is selection pressure for. the markers.
Consequently, to ensure that the resistance markers are not spontaneously lost from the
cultures, the strains should be maintained on selective media (see below), or frozen
suspensions should be used as the source of cells for each experiment (Lewin, 1977).
The phenotypes of all strains should be verified immediately before use in an
experiment. For example, the phenotypes of the E. coli strains described above were
verified and maintained on the following media: the prototrophs on Minimal Agar (MA);
the autotrophs on MA ~ 200 >ig Sm, 20 yg L-leuclne, 30 )ig L-arginine, and 22 jig L-
proline/ml; and the recombinants on MA + Sm (Curtiss, 1965: Curtiss etal., 1968;
Krasovsky and Stotzky, 1987).
V-2
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Inoculum preparation. Parental strains (i.e., donors and recipients) are usually
grown overnight (ca. 18 h) at 37 C.in 125- or 250-ml Erlenmeyer flasks containing 25 or SO
ml of Bacto-Penassay Broth (PB). The period of growth for adequate production of cells
depends on the generation time of the strains. The donor and recipient strains are shaken
at ca. 120 rpm; however, the donor strain is grown in stationary culture for at least 30
min before mating to minimize the mechanical loss of pili. Both cultures are grown to a
density of ca. 109 cells/ml, which is determined most easily by spectrophotometry at 520
nm, after correlation and standardization of absorbance units with the colony-forming
units (CFU) of each strain. The cultures can be either washed, at least twice, with a
suitable mating medium (e.g., Minimal Mating Medium, 3M; LB) by centrifugation at
1,000 £ for 20 min at 4 C or just centrifuged to concentrate the cells. The cultures are
then resuspended in the required amount of mating medium to yield the desired
concentrations of donor and recipient cells.
Mating. An excess of recipient cells is preferable in both In vitro and in situ
matings (Curtiss, 1981). Different donor: recipient ratios (ranging from 1: 20 to 1:1) are
obtained by varying.the numbers of the recipient and donor cells added. The parental
cultures can be mated in liquid or on agar media (Atlas et al., 1988; Curtiss, 1981;
Krasovsky and Stotzky, 1987; Miller, 1972; Weinberg and Stotzky, 1972). Conjugation can
occur with either type of medium, provided that the medium contains adequate levels of
nutrients and energy to support the growth of the parentals and the transconjugants and
that no growth-inhibiting substances are present. Schema'for mating, for both .
chromosomal ind plasmid transfer, in liquid and on solid surfaces are available (e.g.,
Curtiss, 1981; Walter et al., 1987) (Figs. 16 and 17).
The selection of either a liquid or a solid medium for the evaluation of rates of
transfer of both chromosomal- and plasm id-borne genes requires some knowledge of the
type of pili produced by the donor. Rigid pili require a static solid environment, such as
that provided by agar, whereas flexible pili usually attach to conjugative partners on a
V-3
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COMBINED MATING
TECHNIQUE
BROTH MATINS
Situ
UtiM Cutvr*
St«K 30'C
24hf«.
100 ul
S«««cv« WUdiA
COLONY CROSS STREAK
¦ B«M«!
UM4
MEMBRANE FILTRATION MATING
Dooof
F**r >ncuMt»d 31**.
ton-M*aiv« U«j*
Madvt
F**»
Vcrt«* F<
1 mn iO
lOmMTr*. pH 7.S
COMBINED SPREAD PLATE MATING
Doner Ffeceant
100 W VMd
S«t«aiv» U«d«
Jigure 17. Combined mating techniques for studying the transfer of genes by conjugation
in vitro (Walter et al.. 1987). (LB = Luria Broth)
V-4
-------
' f ,
25 ml F3 (24 h) , 25 el PB (24 h)
centrifuge, wash twice with 3M
I
a,
1
1
add 3 al 3M
1 "ml 1 al
add 4 nl 3M
• 5 ml R
i
5 al
T
J
25 ml flaek
37 C (5 h)
Vortex .Genie (15 sec)
k 4
I
z
I
0.1 =1 D ~ R ~ T
HA
(48 h)
B '~ I
HA
1'
1
KA ~ So
^(4S hj
KA ~ sa ~ amino acids
(48 h)
R ~ T
~ ;
1
Figure 16. Flow diagram for studying the transfer of chromosomal genes by conjugation
in vitro (Krasovsky and Stotzky, 1987). (R = recipient; D = donor; T = transconjugant; PS
= Bacto-Penassay Broth; 3M = Minimal Mating Medium; MA = Minimal Agar; Sm =
streptomycin)
V-5
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solid medium and in liquid. The pili produced by the strains of E. coli used herein as
examples function adequately both in liquid and on solid media, and frequencies of
conjugal transfer are comparable with both types of media.
For broth ma tings, each parental culture should be mated in at least triplicate.
The environmental conditions for mating depend on the system being used, e.g., an F-
mediated chromosomal transfer between E. coli should be incubated at 37 C, without
shaking, in ca. 10 ml of a nutrient broth contained in a 250-ml Erlenmeyer flask (Curtlss,
1981). The incubation time for chromosomal gene transfer can vary, and sampling at
intervals for at least 100 min when usingE. coli is recommended. Nutrient content,
incubation conditions, and other experimental parameters should be optimized and
standardized in vitro before experiments on in situ gene transfer are designed with
specific strains.
For plate matings, parental cultures can be streaked separately and sequentially
on agar or they can be mixed before placement on a solid medium, to ensure that the
parental cells make contact and form conjugative pairs or aggregates (Fig. 17) (Curtiss,
1981; Walter et al., 1987). In the colony cross-streak method, the parental strains are
streaked individually at right angles to each other on the surface of the solid mating
medium: several streaks of one culture are streaked in one direction across the surface,
and the second culture is streaked perpendicular to and crosses the streaks of the first
culture. Hence, the parentals are mixed in the area where the two streaks cross. If the
medium is selective for the transconjugants, neither parental will grow, and only
recipient cells that conjugated and successfully received and incorporated the
prototrophic markers will produce colonies. The frequency of gene transfer by this
procedure is generally difficult to quantitate, as even if the numbers of each parental
streaked and of the transconjugant colonies that develop are known, there is no definitive
way to estimate the, numbetoLmating. pair.-; that actually occurred in the mix of the
cross-streak. If the medium is nonselective, both parentals will grow, and it will be
V-6
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difficult to quantitate donors, recipients, and transconjugants, as all the ceils must be
J
removed from the mating plate, diluted, and then each phenotype enumerated on
selective media. Consequently, the colony-streak procedure is not recommended when
quantitation of conjugation frequency is required. Conversely, it is.a simple method with
which to obtain transconjugants for further use In studies in soil. Moreover, it can be
useful in the rapid screening of different strains for their ability to conjugate on a solid
medium.
— In the combined spread-plate procedure, the parentals are mixed as uniformly as
possible bver the surface of an agar plate. This can be achieved by mixing aliquots of
each parental culture in a test tube or flask before spreading the mixture, or the
parentals car. be uniformly spread sequentially on the plate (Walter et al. *87). The
• i
same limitations in the interpretation of results described tbove for the colony cross-1
streak method apply to the combined spread-plate procedure.
In the membrane filter procedure* aliquots of the parental strains are mixed in a
test tube or flask, and the mixture is filtered, with vacuum, through a sterile filter
membrane. The parental strains are impinged on the surface of the membrane, which
increases,the probability of the mating pairs being in.close proximity. Membranes of
nitrocellulose (e.g., Millipore, Bedford, MA) or polycarbonate (e.g., Nucleopore,
Pleasanton, CA) with pore sizes smaller than the diameter of the test strains, e.g., 0.22
or 0.45 )im, are commonly used. The membrane is placed aseptically on a nonselective
agar medlun) on which the parentals will grow and conjugation can occur. • The membrane
•»
is then removed from the i^jar surface, the cells are resuspended^ diluted, plated on
selective media, and incubated, and the CFU of each phenotype are recorded (Curtiss,
1981). The limitations in quantitation are similar to those for the colony cross-streak
and combined spread-plate methods.
Incubation. The mating mixtures should be incubated at temperatures and for
durations that are optimum for conjugation of the strains. Depending on the bacteria
V-7
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involved, the mating mixture can be either shaken gently (30 to 120 rpm), to enhance
aeration, or incubated without shaking in a vessel with a large surface: volume ratio to
permit adequate diffusion of oxygen into the medium, e.g., 10 ml of mating mixture in a
250-ml Erlenmeyer flask (Curtiss, 1981; Miller, 1972). The temperature selected for the •
i ' » •
mating depends on the optimum temperature for growth of the parental strains. For
many laboratory strains, 37 C is optimum (Khalil and Gealt, 1987).
For example, for chromosomal transfer between the E. coll K12 strains*503 and
-------
and the lounterselecting agent can be used for isolating recipients (and transconjugants).
For example, in the mating between E. coli strains*503 and*696 described above, MA
was used for the recovery of donors and transconjugants, MA + Sm + amino acids for the
recovery of recipients and transconjugants, and MA + Sm for the recovery of
transconjugants. Aliquots of the mating mixture were also plated on MA + Sm amended
with various combinations of amino acids for which the recipient was auxotrophic, to
determine the numbers of partial and complete transconjugants. Other selectable
phenotypic characteristics can also be utilized, e.g., catabolic functions, heavy metal- or
i t
antibiotic-resistance, for quantifying the frequency of transfer of chromosomal genes
(Curtiss, 1981). The markers used for phenotype verification and strain maintenance can
usually also be used for the recovery of the parental strains,' but recovery of the
transconjugant will require specific and unique media to detect the transferred gene{s) of -
interest.
Syntrophy. A potential artifact that can confound the enumeration of
transconjugants in soil is syntrophy, or cross-feeding, a type of mutualism that enables
different auxotrophic strains to grow together, but not apart,, when the nutrients
necessary for the growth of each auxotroph are absent or their concentrations are very
low, which is not uncommon in.the soil environment. Syntrophy can enable auxotrophic
organisms to grow on minima] media selective for transconjugants, as the amino acids,
vitamins, and other growth factors required by one auxotrophic strain may be excreted
by the other strain, which can result in an exchange of nutrients under conditions that
would not support the growth of either auxotroph when cultured alone. Consequently,
any isolates that grow on selective minimal media must be verified to be
transconjugants, rather than auxotrophic parentals that are cross-feeding on the recovery
media, by streaking purified colonies of presumed transductants individually on selective
minimal media or by the colony cross-streak method_(Krasovsky and Stotzky, 1987;
Weinberg and Stotzky, 1972).
V-9
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Frequency of recombination (FOR). This measure of successful conjugal transfer
of chromosomal genes Is calculated from the ratio of the numbers of CFU recovered on
media selective for the transconjugants to, the total numbers of CFU recovered on mirdla
selective for the donor (on which the transconjugants will also grow):
CFU on TC-medium
FOR = ;—:——
CFU on D-medium - CFU on Tc-medium
where: TC-medium is the selective medium on which only transconjugants will grow; and
D-medium is the selective medium on which donors and recombinants will grow.
When an Hfr strain is mated with an F~ strain, the amount of the donor genome
and the specific genes transferred depend on the duration of mating and on the location
of the integration site of the F-factor (i.e., the oriT site), respectively. By sampling and
plating cultures of mating bacteria on selective media at different times, the order of
genes on the donor chromosome can be determined by the times at which they appear in
the recipient and by their order'and relative location (Curtiss, 1981; Miller, 1972). Genes
that are located near each other on the chromosome are transferred together with a high
frequency. Moreover, the frequency of chromosomal transfer of genes near the oriT site
may be greater than that of plasm id transfer. Bacteria in soil may not conjugate long
enough to transfer a large plasmid, but they probably conjugate long enough to transfer
several genes located close to the oriT site. Conversely, if the marker genes are located
too far upstream from the oriT site, the apparent FOR may be low, despite significant
conjugation between pareritals and the transfer of genes that were not detected. These
principles of chromosomal gene transfer must be considered when designing studies to
observe the potential for gene transfer by conjugation in the environment.
V-10
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Plasmid transfer
Many of the procedures used for the study of chromosomal transfer can also be used to
study plasmid transfer by conjugation. Host strains that will maintain the plasmid(s),
being studied, before and after conjugation, are required. The strains should not contain
other plasmids that may interfere with conjugation, either by surface exclusion (i.e.,
prevention of "superinfection" by plasmids of the same type) or by competing during cell
division for membrane-bound segregation systems that provide for maintenance and
inheritance (i.e., incompatability) (Freifelder, 1987; Lewin 1977). Ideally, the strains
should be devoid of plasmids other than the ones being studied, as shown by phenotypic
expression and by the inability to detect other plasmid DNA by agarose gel
electrophoresis after extraction of plasmid DNA by standard procedures (Curtiss, 1981;
Maniatis et al., 1982; Miller, 1972) (see Section VII). Selective enumeration of donors,
recipients, and exconjugants is facilitated if the chromosome of the donor and recipient
strains is "tagged", i.e., contains antibiotic-resistance marker(s) or some other easily
characterizable phenotype, e.g., the production of a.red color by lactose fermenters on
MAC (Atlas et al., 1988; Curtiss, 1981; Devanas et al., 1986; Miller, 1972) or a blue color
on X-gal agar by cells containing the ZT genes of the lactose operon (Drahos et al.,
1986). The lacZY genes can also be used as plasmid markers.
The choice of plasmids to use experimentally will depend on the type of system to
be studied and the type of information desired. The enumeration of donors and
exconjugants is facilitated if the plasmid encodes catabolic pathways, resistance markers
to antibiotics and/or heavy metals, or some other easily characterizable phenotype
(Maniatis et al., 1982; Miller, 1972).
Media. The media used for the maintenance of the hosts, with or without
plasmids, and for mating and recovery of the donors, recipients, and exconjugants depend
on the nutritional requirements of the bacterial strains. Most bacteria will grow
adequately on standard laboratory media, such as Nutrient Broth (NB), Tryptone Glucose
-------
Yeast Extract Broth (TGYB), and LB and on agarswith the same nutritional
composition. Environmental isolates, however, may require growth factors that are not
present in standard commercial media. Such fastidious strains can usually be maintained
on media supplemented with extracts of soil or with specific nutrients (see Appendix). In
o
contrast, many commercial media may be too nutritious for indigenous soil bacteria and,
possibly, also for introduced bacteria after they have adapted to the oligotrophia
conditions in soil.
Slower growth and, hence, fewer cells occur on agar than in liquid media, thereby
reducing the number of mutants that develop in the cultures and extending the intervals
between necessary transfers of the cultures to fresh medium. A solid medium is also
better if resistance markers must be maintained by a constant selection pressure, as the
destruction or alteration of the selective chemical (e.g., an antibiotic) is less than in
liquid media, as the result of the slower rates of diffusion of degradative extracellular
enzymes from a resistant colony on agar. Liquid culture is generally better for obtaining
large numbers of bacteria in a short period of time (e.g., in 19 h), which is important for
the preparation of inocula.
Bacteria will usually not maintain extrachromosomal genetic information that is
not used (i.6., that is not induced to transcribe). For example, plasmids that encode
resistance to antibiotics (R-plasmids) will not be replicated, segregated, or maintained
after several transfers if there is no selection pressure of the respective antibiotic(s) in
the medium. Bacterial strains containing R-plasmids are best maintained on a selective
agar medium that contains at least one of the antibiotics to which the plasmid encodes
resistance. If the plasmid is especially unstable and the loss of plasmid genes is likely,
several or all of the compounds (e.g., antibiotics, heavy metals, substrates for
degradation) for which the plasmid encodes resistance or utilization should be included in
the selective maintenance medium. For example, an E. coli strain that carries the
plasmid, RP4, is maintained on LA containing 23 ^g/ml Tc. When the host is a strain of
-------
Pseudomonas, which are generally more resistant to many antibiotics than most members,
of the Enterobacteriaceae, the basal resistance level to specific antibiotics of the
' i 1 1 '
plasmidless host' must be determined and the selective antibiotics (e.g., Tc) added at
higher concentrations for both maintenance of the plasmid and selection of
exconjugants. For example, P. aeruginosa. PAOl is resistant to Tc at concentrations
above 100 yg/ml, and the quantity of Tc necessary for maintenance and selection of
plasmid RP4 in this strain is 200^ig/m2.
Inoculum preparation. Strains can be grown in a nonselective broth to obtain large
numbers of cells. However, if the plasmid phenotype is unstable and a significant
proportion of the cells is likely to loose the plasmid when grown under nonselective
conditions, then selective pressures must be maintained. In either situation, the
phenotype of the donor and recipient strains must be verified before use. One method to
maintain selection for the plasmid markers is to grow the strain overnight on a selective
agar medium. For example, cells of E. coli containing plasmid RP4 were grown on MAC
+ 25^g/ml Tc, scraped, diluted with saline to 104 CFU/ml, of which 0.25 ml was added to
25 ml of LB to yield a density of 103 CFU/ml, and grown overnight at 37 C with shaking
(Devanas et al., 1986). This produced a. suspension of cells that contained the plasmid, as
all cells grew on the selective medium. Consequently, loss of plasmids, that could result
from their partitioning (segregation) ducing growth under nonselective conditions, can be
. minimized. If the donor strain readily looses.the plasmid under nonselective conditions,
suspensions of cells scraped directly from a selective medium can be used. The
concentration, washing, diluting, and inoculation procedures are as described above for
chromosomal transfer.
Mating. The conditions selected for mating will vary with the type of system to
be studied. For example, F-factors, their derivatives, and other plasmids derepressed for
transfer are transferred readily at high frequencies (ca. 100%); other conjugative
plasmids have lower frequencies (10~^ to 10"^) (Lewin, 1977). Various proportions of donor
-------
to recipient cell numbers should be evaluated, as the optimum donor: recipient ratio for
plasmid transfer depends on the species and strains used (Curtiss, 1981; Miller, 1972). This
ratio should be determined in preliminary studies; In the model systems of £. coli
J53(RP4) as the donor strain and,£. aeruginosa PAOl, £. aerogenes, K.,pneumoniae, and
P. vulgaris as the recipient strains, the overnight broth cultures (usually 10® CFU/ml)
were diluted with sterile saline to cell densities of approximately 10® CFU/ml and then
added to the mating medium (LB) in a donor: recipient ratio of 1: 5, which ensured that
each potential donpr was likely to encounter a suitable recipient (Devanas and Stotzky,
1988). Successful mating and transfer may occur within 1 to 4 h with plasmids depressed
for transfer. For other plasmids, longer periods of mating maybe necessary. The mating
.mixture is then diluted, and samples are plated on appropriate selective agar media for
recovery of donors, riecipients, and exconjugants, as described above for transfer of
chromosomal genes.
Calculation of the frequency of conjugation (FOC). The frequency of plasmid
transfer by conjugation is calculated by dividing the number of exconjugants by the
number of donors in the mating mixture (Curtiss, 1981):
CFU of exconjugants
FOC = :
CFU of donors
IN SOIL
Chromosomal transfer
The design of studies to examine the potential for conjugal transfer of
chromosomal genes in soil must consider all the variables that affect chromosomal
transfer in vitro, as well as the physicocherriical factors inherent in the soil
environment. The selection of donor and recipient strains and the methods used for their
maintenance, culture, and preparation as inocula are essentially the same as those
employed for in vitro studies. Consequently, only additional aspects unique to studies of
in situ chromosomal gene transfer are described.
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Inoculation. The use of donor and recipient cells that are in log phase appears to
enhance conjugal chromosomal transfer in soil (Weinberg and Stotzky, 1972). One method
for the preparation of log phase cells for inocula uses cells that were grown overnight,
washed by centrifugation (2 to 3 times at. 1,000 g), followed by resuspensioh of the cells
of each strain in a phosphate buffer, usually at pH 7.
The sequence of inoculation of the donor and recipient cells has been shown to
affect their survival and the frequency of conjugation (Krasovsky and Stotzky, 1987;
Weinberg and Stotzky, 1972). The survival of both donors and recipients was higher when
the recipient cells were inoculated before-thc donor cells than when the donors were
added before the recipients. When the donor and recipient cells were inoculated together
(i.e., mixed before addition to soil), their survival and the FOR were greater than when
they were inoculated sequentially (Krasovjsky and Stotzky, 1987). However, mixing of the
parentals before their addition to soil may result in mating pair formation outside of the
soil, thereby producing artifactually inflated frequencies in soil. Hence, mixing donors
and recipients before addition to soil is not recommended for studies designed to observe
the transfer of chromosomal genes in soil.
Incubation. Soils used for In situ mating experiments should be incubated under
conditions that maintain the moisture and temperature conditions defined for the
experiment. To maintain the optimum soil water tension (i.e., -33 kPa), high-humidity
chambers can be used (Stotzky, 1986). One to 2 inches of water are placed in the bottom
of large glass containers (e.g., chromatography tanks, desiccators) and covered with lids
that have a vent that is loosely plugged with nonabsorbent cotton. This arrangement
produces a water-saturated atmosphere that maintains the optimum soil water tension.
Temperature ranges of natural soils can be simulated by placing the high-humidity
chambers in incubators maintained at the desired temperatures.
Sampling. The choice of sampling regimes will depend on the type of microcosm
used and on the purpose of the experiment. Simple test tube microcosms, in which the
-------
entire soil sample is "sacrificed", reduce the sampling error associated with repeated
replicate samplings from larger batch volumes. However, sampling error can be
introduced at the level of preparation and inoculation of the individual tubes. Hence,
precise weighing of soil and uniform inoculation are necessary. Different typss of, soil
microcosms and sampling systems are discussed in Section III.
The samples of soil are subjected to serial decade dilution with standard
laboratory diluents, e.g., sterile distilled or tap water, saline, buffers, 0.1% peptone. The
range of dilutions suitable for donors, recipients, and recombinants will depend on the
cell densities added. Dilutions from 10~5 to 10~7 should be suitable for the enumeration
of the indigenous soil microbiota. The types and numbers of indigenous microbes present
in the soils being.studied should be evaluated before any experimental strains are
introduced, so that observations made after introduction of the strains, which may alter
the profile of indigenous microbes, can be compared with these baseline measurements.
Aliquots of the dilutions of sample are plated on appropriate selective media, incubated,
and the CFU recorded. A tared sample of the soil is dried for 24 h at 100 C, and the
CFU are expressed and compared on the basis of 1 gram of oven-dried soil.
Recovery of donors. recipients, and transeonjugants. The appropriate selective
media are based on the phenotypic markers of the donor, recipient, and transconjugant
strains, as in in vitro studies. In studies with nonsterile soil, the media used for the
isolation of honors, recipients, and transeonjugants may not be selective enough to
eliminate or reduce sufficiently the indigenous soil microbiota to enable isolation and
distinction of the introduced strains. To suppress the indigenous populations of fungi, SO
to 200 jjg/ml Cy should be added to the recovery media. The concentration of Cy needed
will depend on the type of soil and on the numbers and types of fungi present.. Indigenous
populations of bacteria may be counterselected by the antibiotics used to isolate the
parentals or by a combination of antibiotics to which most of the indigenous populations,
but not the parentals, are sensitive, e.g., a mixture of Sm, Ap, Nx, and Rf. Media that
-------
suppress indigenous gram-positive bacteria can be formulated based on the phenotype of
the experimental gram-negative strains, as in the case of E. colin503 and-x696, which
are tolerant of 400 yg/rnl eosin and 65 >ig/ml methylene blue, whereas gram-positive
bacteria are not (Krasovsky and Stotzky, 1987), The concentrations of the various
antimicrobial agents necessary to suppress the indigenous miorobiota will vary with
different soils and should be determined in preliminary studies.
To distinguish between transconjugants that resulted from chromosomal transfer
(as well as exconjugants that resulted from plasmid transfer, see below) in soil from
those that may have resulted from matings between donors and recipients on th^
recovery medium, Nx is incorporated into the selective, medium, which will inhibit gene
transfer by Nx-sensitive donors (Curtiss, 1981; Walter etai., 1988a) (see Section VU).
Calculation of frequency of reoombinatioa (FOR). The FOR is calculated by
dividing the number or transconjugants by the number of donors isolated from the soil
(see FOR above).
Plasmid transfer
Studies to measure conjugal transfer of plasmid genes in soil require' careful
design and selection of plasmids and of donor and recipient strains, as well as extensive
controls for both gene transfer and environmental variables. The nutrient content of the
soil, the method of inoculation of the parental strains into the soil,,the recovery of
donor, recipient, and exconjugants from the soil, and the transfer of the plasmid to
indigenous bacteria must also be considered.
Inoculation. The sequential addition of donor and recipient cells may affect the
survival of the parental strains and the potential for gene transfer, particularly if one
mating partner is not added for some time after the addition of the other partner. For
example, preincubation of the recipient, P. aeruginosa PAOI,-in soil did not appear to
have any significant effect on its survival, whereas the donor. E. coli J53(RP4), declined
rapidly after addition to soil. Gene transfer occurred immediately after log phase donor
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cells were added to preincubated recipients (Fig. 18), whereas exconjugants were
recovered only after 1 to 3 weeks of incubation of the soil when log phase recipients
were added to preincubated donors (Fig. 19) (Devanas and Stotzky, 1988b).
The presence of nutrients, whether introduced with the inoculaor as amendments
during the incubation, affect the survival and potential for gene transfer in soil (Devanas
et al., 1986). Studies on survival, recovery, and gene transfer in soil should be designed
to include both unamended soil as well as soil amended with nutrients.
Recovery of donors, recipients, and exconjugants. The coil is sampled the same as
for chromosomal transfer, and the procedures for the recovery of donors, recipients, and
exconjugants are the same as those for in vitro plasm,id transfer. However, the, recovery
of some strains from soil may sometimes be reduced, as the stress of being in soil may
sufficiently debilitate these strains and affect their subsequent recovery. This
phenomenon'of "viable but nonculturable" has been described for bacteria in various
aquatic systems (Roszak et al., 1984; Zaske et al., 1980). A similar phenomenon appears
also to occur in soil, as shown by the differences between the CFU of some strains
recovered on highly selective media (e.g., MAC+Tc) and on less stressful selective media
(e.g., MAC) (Devanas et al., 1986). A number (50 to 100) of the colonies recovered on the
less stressful media should be transferred to the more stringent selective media to
determine if the differences in the proportion of CFU recovered by direct isolation on
each medium are real or if the reduced numbers that developed on highly stressful
recovery media were the result of a "viable but ronculturable" condition (see Figs. 18 and
19). The stress of being in soils may also retard the rate of growth of cells even on
nonselective and nonstressful media. Consequently, repeated observation of the recovery
plates, marking the colonies as they appear, and reincubating the plates until no new
colonies appear may be necessary.
Transfer to indigenous bacteria. Gene transfer to indigenous bacteria is generally
difficult to demonstrate, as selection for gene transfer to these bacteria is based solely
It ^ A
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o
DAYS
Figure 18. Effect of preincubation of recipient Pseudomonas aeruginosa PAOl cells on the survival and transfer of plasmid RP4
from donor Escherichia coli J53(RP4) cells in nonsterile soil. All cells were inoculated in saline. Donor cells were added
immediately (i.e., on day 0) or after the soil had been incubated with recipient cells for 7 to 14 days. E. coli J53(RP4) was
enumerated on MacConkey Agar (MAC) containing 25jtg/mi tetracycline (Tc); P. aeruginosa PAOl on Pseudomonas Isolation Agar
(PlAh and PAOURP4) on PIA containing 200jig/ml Tc. When the colony-forming units (CFU) of J53(RP4) on MAC ~ Tc were
lignificantly lower than the CFU of E. coli JS3 on MAC, selected colonies from the MAC plates were transfered to MAC + Tc. All
transfers grew on MAC ~ Tc, indicating that the plasmid was not selectively lost from the J53(RP4) population but that the stress
n soil produced a "viable but nonculturable" population of J53(RP4) that did not grow on MAC ~ Tc when isolated directly from
oil (Devanas and Stotzky, 1988b).
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DAYS
Figure 19. Effect of preincubation of donor Escherichia coli J53(RP4) cells on the survival and transfer of plasmid RP4 to
recipient Pseudomonas aeruginosa PAOl cells in nonsterile soil. All cells were inoculated in saline. Recipient cells were added
immediately (i.e., on day 0) or after the soil had been incubated with donor cells for 7 to 14 days. E. coli J53(RP4) was enumerated
V .
on MacConkey Agar (MAC) containing 25^
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on the presence of the transferred marker genes. All colonies tfiat appear on media
.selective for exconjugants must be tested for the presence of the marker genes by,
phenotypic (e.g., morphology, gram stain, biochemical characteristics) and physical (e.g.,
DNA extraction, agarose gel electrophoresis, DNA fingerprinting, DNA probes) methods
(see Section VII). Srch analyses should clarify whether the exconjugant isolated is the
added recipient or an indigenous soil bacterium to which the introduced genes have been
transferred. The intrinsic resistance of the indigenous soil bacteria to the antimicrobial
agents used to evaluate the transfer of marker genes must be determined by plating
appropriate soil dilutions of a control (i.e., uninoculated) soil sample on the various
selective media used.
Limits of detection of gene transfer. There are always limits to the detection of
microbial populations in any natural environment. These populations may: 1) not grow
on any of the recovery media; 2) be present in such low numbers that unrealistically large
volumes must be sampled; 3) adhere tightly to surfaces and are not removed for recovery,
by the dilkients in serial dilution; and 4) be obscured or overgrown on the recovery media
by faster-growing strains. All these problems occur in soil and make the detection of
introduced GEMs and, especially, of any recombinants that may result from gene transfer
difficult. The lower limit of the numbers of CFU detectable in soil samples is a function
of the size of the soil sample, the ratio of the initial dilution of the soil sample (e.g., 1:
i ' 1 1 1
,10), and the volume of the dilution plated. For example, the recovery of 1 CFU when 0.1
ml of the 10"1 dilution of soil is plated represents a lower detection limit of 100 CFU/g
soiL The limit of detection of a recombinant bacterium in a sample of soil is generally in
the range of 1 to 20 CFU/g soil, and the level of confidence in this range is a function of
the number of replicate soil samples evaluated and the number of CFU of the
recombinants recovered (Devanas et al., 1986).
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Stepwise Summary of Procedures for Studying Conjugation In Soil.
_ Prepare soil (e.g., sieve; amend with clays, CaCO^etc.; condition;'add to appropriate
microcosm).
- Prepare suspensions of donor and recipient cells.
- Wash suspensions by dilution or centrifugation,
- Adjust concentration of donor and recipient cells for appropriate conjugation ratio (1:1
to 1: 23).
- Add donor and recipient cells sequentially to soil ,w;th sufficient water (or a nutrient
solution) to bring the soil to its -33 kPa water tension.
- Incubate soil, preferably in a high-humidity chamber.
Sample soil periodically (either by sacrificing multiple microcosms or from a batch
system).
- Prepare appropriate serial decade dilutions of the soil.
- Plate dilutions on selective media suitable for growth of donor, recipient, and
recombinant cells, as well as of indigenous soil bacteria.
- Incubate plates, and record numbers of colony-forming.units that develop on each
medium.
* Verify presumptive donor, recipient, and recombinant cells for phenotype and/or
genotype (see Section VII).
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VL Methods for Studying Transduction
Tr»n,--rtuctior in vitro
Preparation of bacteriophage lysates. Two methods for preparing phage lysates of
high, titer are commonly used, depending on the phage-host system that is being studied.
The first method involves inoculating a liquid culture of a susceptible host bacterium
with an appropriate phage or inducing (e.g., by heat, ultraviolet radiation, chemical
mutagens) a culture of a lysogenie bacterium. An example of the use of a lysbgenic
culture is described for the preparation of lysates of phage PI. Examples of other
methods for preparing phage lysates in liquid culture can be found in Adams (1959).
Phage PI Cm^ts contains a temperature-sensitive mutation that enables the
induction of the lytic cycle at temperatures above (e.g.,, 42 C) those optimal for growth
of the host bacterium, E. coli (e.g., 37 C). E. coli AB1137, lysogenie for phage PI and
stored on LA slants containing SO^ig/ml Cm to maintain the temperate phage PI DNA in
the bacteria, is grown at 30 C in LCB to ar| optical density of. 0.2 (at 600 nm) and shaken
at 42 C for at least 2 h until lysis is evident (30 C is used for growth, to reduce
spontaneous induction of lytic reproduction as the result of the temperature-sehsitive
mutation). Chloroform (0.5 ml/100 ml of culture) is added, with mixing, for 1 min to lyse
cells and release additional phage virions, and the remaining intact cells are removed by •
. filtration through a 0.45 )jm nitrocellulose filter membrane (Millipore). The phage
particies are stored iq LCB at 4 C. Phage PI titers between 10® to 10*® PFU/ml are
commonly obtained.
An alternative method, which is more suitable with certain phages for the
preparation of lysates of sufficient titer, is the agar-ov»rlay method. An example of this
technique, used with the generalized transducing phage, F1J6L, that infectsP.
aeruginosa, is described below (Miller and Ku, 1978). Although phage F116L does not
contain a temperature-sensitive mutation, many lysogenie bacteria can be induced to
lysis by growth at temperatures above their optimum, as the repressor protein that is
-------
involved in the maintenance of lysogeny will be impaired in activity or inactivated by
such higher temperatures. Phage F116L (105 PFU in 0.1 ml) and P. aeruginosa PAOl (10®
CFU in 0.1 ml) are'mixed in 2.5 ml of melted LA (top agar), to give a multiplicity of
infection (MOI; no. of phages: no. of host cells) of 0.1, and poured on the surface of LA
°plates (bottom agar). After incubation overnight (ca. 18 h) at 37 C, the top agar layer is
removed in 5!ml of LB, the suspension is vortexed for 1 min and centrifuged for 10 min at
5000 £ to pellet the bacteria and agar, and the supernatant is filtered through' a 0.45 )im
nitrocellulose membrane to remove remaining bacterial cells. The phage F116L stocks
(10® to 1012 PFU/rnl) are stored at 4 C.
The lysates can be purified of bacterial cell debris and soluble cytoplasmic
components by various techniques, e.g., differential centrituration on CsCl gradients,
polyethylene glycol (Miller, 1972). However, the removal of such contaminants is seldom
' "¦ _ - - f . -
necessary or recommended.for studies that are intended to simulate in situ situations.
Preparation of bacterial cultures. The bacterial cultures used as hosts for in vitro
transduction should be grown to log phase in liquid culture (e.g., in LCB), and the
optimum MOI for the specific host-phage system, determined in preliminary experiments,
should be used. For transduction to E. coli of resistance to Cm by phage PI Cm cts, an
MOI between 1 and 5 is typically used with lo' to 10® CFU/ml of E. coli.
Transduction procedure. Transduction experiments in vitro should be conducted in
liquid rather than on solid medium, to ensure adequate mixing of the phage and bacteria
during the adsorption period. Phage PI is allowed to adsorb on JE. coli for 30 min at 30 C
without snaking in LCB. The presence of Ca2+ and Mg2* in this medium promotes phage
PI adsorption on the bacteria. The adsorption period is restricted to 30 min to prevent
both the phage from completing a reproduction cycle and initiating a burst and the
multiplication of transduced£. coll cells. After the 30-min adsorption period, E. coli
transductants are enumerated by plating serial decade dilutions of the bacterium-phage
suspension on either LA or MAC containing 30^ig/ml Cm. The E. coli inoculum is also
-------
plated on this medium, to determine the number of spontaneous mutants to Cm
resistance.
Quantitation of the bacterial and phage inocula should be conducted to determine
the exact MOI in the transduction mixture. Dilutions of the E. coli suspension are plated
on LA, and the plates are incubated at 3(PC until colonies appear. Phage PI is titered on
LCA bottom and top agar at 42 C, and plaques are counted after 24 h.
The frequency of transduction is expressed as the number of transductants divided
by the number of bacteria originally present in the transduction mixture. The number of
phages added can be used as the denominator instead of the number of bacteria, to give a
transduction frequency related to the total number of phage particles.
Transduction in soQ
The design of transduction experiments'in soil must consider the complex nature
of soil and the fact that studies will be conducted over a period of several days or longer,
thereby making it difficult to calculate the frequency of transduction. The major factors
to consider are: the MOI; the use of either a phage lysat'e or a lysogenic bacterium as
the source of the transducing phage; whether the recipient bacteria should.to be added to
soil after washing (e.g., in saline) or with nutrients (e.g., LB); and the methods used to
enumerate selectively the added bacteria, transductants, arid phages.
Inoculation and amendment of soli. The optimum MOI in soil should be determined
before extensive experiments are desigrfed, as it may differ from that in vitro. The
number of phages added should be appropriately adjusted to the number of bacteria added
to the soil. In studies of transduction by lysates of phage PI in soil, 10s to 10® CFU/g
soil of recipient E. coli J53(RP4) or W3110(R?02) are typically added, to avoid adding
unrealistically high numbers of bacteria, and the MOI is approximately 3. E. coli and
phage PI are added individually in 0.1 ml of LCB or saline, so as to bring the final wet
weight-pf-the soil~to~2"g~(Zeph~et~al7Tl988). When lysogenic strains of £. coli are used as
the source of phage PI, the same numbers of lysogenic and recipient J?, coli cells are
-------
added..' Control studies with soil inoculated with' the same numbers of only phage PI or
only E. coli donors or recipients must be conducted, to determine both survival and
mutation rates.
The addition of nutrients can be achieved most easily by diluting the host and
transducing phage in fresh LCB before their addition to soil. Amendment of the. soil with
LB or other nutrient solutions can be made periodically, to measure the effects of
nutrients on transduction and on the multiplication and survival of the phages and host
cells. The potential for transduction in the absence of exogenous nutrients can be
determined most easily by diluting the host and phage inocula in saline before their
addition to soil. For example, inocula of coli and phage PI (10® to 10® CFU or
PFU/hil) raised in LCB are diluted 1: 1000 in saline, and a final inoculum of 105 to 10®
CFU or PFU/g soil is added. Concentration and washing in saline of the host cells can be
accomplished simultaneously by centrifugation at 5000 £ for 10 min. However, as the
viability of the inoculum could be affected by centrifugation, viable counts of the washed
suspensions should be determined.
Incubation and sampling. Soil samples containing phage and bacteria should be
incubated at approximately 2S C (the average temperature of soils in temperate zones)
and maintained at their -33 kPa water tension in a high-humidity incubator and on .the
basis of periodic weight measurements and the addition of sterile distilled water (sdH20)
when necessary. Three to four samplings of the soil for the enumeration of recipient and
donor bacteria, phage,' and jtransductants should be made during the first week (e.g., on
days 0,1, 3, and 7), and then sampling should be at least weekly until there art no
significant changes in these populations. If sufficient personnel and funds are available,
the numbers of total bacteria, fungi, protozoa, and other indicators of species diversity
should be evaluated concurrently.
In studies on the transduction of E. coli by phage PI in soil, experiments were
conducted for 28 days (Zeph et ah, 1988). Individual soil tubes were sacrificed at each
vt-i
-------
I
sampling by the addition of 18 ml of sd^O to duplicate or triplicate soil tubes that
contained 2 g soil (i.e., a 1: 10 dilution), and subsequent serial decade dilutions were
spread-plated on selective media. Plates for the enumeration of £. coll donors,
recipients, and transduotants were incubated at 30 C; for phage PI at 42 C; and for total
and gram-negative indigenous soil bacteria at 25 C.
Enumeration. The efficacy of the selective media used for the enumeration of the
various groups of bacteria (i.e., transductants, donors, recipients, and indigenous) in
nonsterile soil is an important parameter to establish before the start of ?n experiment,
especially as the numbers of bacteria transduced in soil can be relatively low. In studies
on the transduction by phage PI Cm cts::Tn501 of Cm- and Hg-resistance to E. coli, the
resistance markers on the phage genome were utilized to monitor the number of
transduced E. coli in soil, and the antibiotic-resistances coded on nonphage plasm ids'
enabled following the fate of the recipient or donor (lysogenic) £. coli populations. E.
coli transductants were enumerated on MAC containing Cm (90^ig/ml; MAC-Cm) or Hg
(20 or 30^iM; MAC-Hg), as the lactose-utilizing strains of E. coli form distinctive dark
red colonies on MAC. Recipient £. coli J53(RP4) arid W3110(R702) were enumerated on
MAC containing Tc (25>ig/ml; MAC-Tc), resistance to which is encoded on the plasmids.
In studies in nonsterile soil with lysogenic _E. coli donors and nonlysogenic
recipents, the donor E. coli J53(P1 Cm £ts) was enumerated on MAC-Cm, the recipient
E. coli W3110(R702) on MAC-Tc,' and the transduced E. coli W3110(R702)(P1 Cm cts) on
MAC containing both Cm and Tc at the concentrations shown above. The lowest limit of
2 1
detection of E. coli donors, recipients, and transductants in nonsterile soil was .10 to 10
CFU/g soil, as growth of indigenous soil bacteria resistant to the concentrations of Cm,
Tc, and Hg used interfered with accurate detection of lower numbers of £. coli (Zeph e£
al., 1988). The problem of interference by indigenous soil bacteria resistant to
antimicrobial agents is discussed below.
When lysogenic E. coli J53(RP4)(P1 Cm cts;:Tn501) donors and nonlysogenic E. coli
-------
W3110 were added, to sterile soil, Hg-resistant transductants of E. coli W3110 (i.e.,
W3110(P1)) were enumerated on M9 minimal agar containing 30^iJW Hg (M9M). The
auxotrophic E. coli J53(RP4)(P1) donor does not grow on minimal agar, and it was
enumerated o,n MAC-Tc. MAC was used to enumerate total E. coli |e. coli J53(RP4MP1).
W3110, and W3110(Pljj, which enabled the numbers of E. coli W3110 to be calculated
from the difference between the total E. coli and, the E. coli J53(RP4MP1) plus W3110(P1)
counts.
Auxotrophic recipients can also be utilized to study transduction in soil. For
example, a Sm-resistant coli K-12 recipient that was auxotrophic for threonine and
leucine was used to study transduction by phage PI in nopsterile soil (Germida and
Khachatourians, 1988); Lysates of phage PI were raised on prototrophic E. coli. The
' i 1 h |
minimal salts agar medium (MSA), which contains Sm, Cy to suppress fungi, and eosin and ,
i x • '
methylene blue to suppress the growth of bacteria other than enterobacteria, was
satisfactory for the isolation of E. coli transductants prototrophic for threonine and
leucine and resistant to Sm.
Minima] media can also be used to counterselect an auxotrophic E. coli lysogen, as
discussed above (Zeph et al., 1988). Nevertheless, the use of markers for resistance to
antimicrobial agents is more efficient for monitoring transduction in soil,,as many soil
bacteria will often grow more readily on minimal media than on media containing
antimicrobial agents. Moreover, when using a minimal agar medium to select for
transductants, incubation of the plates for more than 3 or 4 days can result in the
syntrophic growth of the auxotroph and the formation of background lawns on the
plates. This makes it difficult to differentiate between cells that have been transduced
to prototrophy and cells of the auxotrophic recipient.
Detection of tranaductants in nonstertle soil. The detection of transductants in
nonsterile soil is difficult, as there are no efficient methods for the enumeration of low
levels of transduced bacteria. Chromosomally-borne genes for resistance to Nx and
-------
other antibiotics or to heavy metals can be exploited in the isolation of a host bacterium
containing either or both of these groups of genes. However,, isolation of specific hosts
based on resistance, either chromosomal or vector-borne, to many commonly used,
antibiotics (e.g., Sm, Cm, Tc) or some heavy metals (e.g., Hg) has limitations, as many
bacteria in many soils now exhibit resistance to these antimicrobial agents. The limit of
detection for most methods is, at best, between 10 to 10° transductants/g soil, and it is
often several orders of magnitude higher. In some soils, the "background" resistance to
many common antimicrobial agents can be as high as 50% of the isolatable bacteria.
i
Increasing the level of resistance in the host bacterium through mutation and selection or
concurrently using a second antibicftic- or heavy metal-resistance marker often results.in<
reduced recovery, as some bacteria are not able to express readily'the antimicrobial-
resistance phenotype after exposure to the stressful conditions in soil (Devanas €it al.,
1986; Pettibone et al., 1987). Furthermore, resistance to antimicrobial agents may be
, ' i
lost if the resistance genes are located on a plasmid that.may disappear through
segregation during the period of the soil study. A "viable but nonculturable"
physiological state, in which the cells remain viable but are so debilitated that they will
not form colonies on selective media (e.g., Devanas et al., 1986; Roszak and Colwell,
1987), has been suggested as one cause of the poor recovery of some introduced bacteria
from soil. However, by initially plating the soil dilutions on nonselective media, to allow
"resuscitation" of the stressed bacteria, and then replica-plating to selective media,
specific donors, recipients, and transductants can be enumerated (see Section VII).
Recovery of phages from aoLL Difficulties can be encountered in the recovery of
phages from soil, as adsorption of phages on soil particulates, especially on clay minerals,
can result in apparent decreases in phage titers in soil. For example, phage PI adsorbs
strongly on soil components, probably on the clay fraction, as over 95% of the added
phage Pi pelleted with the soil particulates after centrifugation of soil amended with
this phage (Zieph and Stotzky, unpublished). The usual technique for the recovery of
VI -7
-------
phage particles from soil, which is filtration of appropriate soil dilutions through a 0.22
or 0.45^m membrane filter, removes phage PI from the dilutions, as the phage binds on
soil constituents present in the dilutions and impacted on the membrane.
An alternative technique involves the use of nonfiltered soil dilutions and plaque-
assay lawns of antibiotic-resistant _E. coli grown on media containing the appropriate
antibiotic at concentrations inhibitory to soil bacteria (Zeph et al., 1988). For example,
100^ig/ml Tc is added to LCA bottom and top agar, which allows the growth of confluent
lawns of E. coli Jl>3(RP4) and the formation of plaques by phage PI. To enumerate free
phage PI in soils inoculated with _E. coli lysogenic for the phage, chloroform should be
added to a concentration of 2% (vol/vol) to the initial soil dilution for S min, with
periodic vortexing, to kill the lysogens and to prevent their prophages from forming
plaques on. the assay lawn. Once the chloroform has settled to the bottom of the initial
dilution tube (usually after approximately 1 min), dilutions are prepared for plaque assay
of the phage, as described above. The use of chloroform should be restricted only to
chloroform-resistant phages, such as phage PI.
As the recovery of some phages from soil can be low, specific eluents have been
used to desorb various phages from soil particulates. For example, the substitution of
i
skim milk for dl^O as the initial diluent increases the recovery of phage PI from less
than 10% (dHjO) to 60% {skim milk) of the original number of phage particles added to
soil (Zeph and Stotzky, unpublished). Other common eluents used to desorb phages from
soil are casein, nutrient broth, beef extract, glycine, EDTA, and egg albumin (Bitton,
1980). However, recovery of phage PI with the eluents so far tested has been less than
100%.
Suppression of fungal growth on selective media. Fungal growth on selective
media containing dilutions of nonsterile soil can be suppressed by the addition of Cy to
-the-media;- Theconcentration~aTCy'tou$e depends on thepopulation level of fungi in
the specific soil and the soil dilutions to be plated. A concentration of 50 to 200^tg/ml
-------
Cy will usually inhibit essentially all fungal growth.in tl.e 10"* dilution of nonsterile soil
(Tremaine.and Mills, 1988; Wollum, 1982).
. Transduction frequency. The calculation of transduction frequencies in situ is
complicated by the fact that studies in soil are usually conducted for periods of days or
weeks. Inasmuch as transduced bacteria may multiply in soil and produce cells with the
same phenotype, it is not possible to distinguish between multiplication and
transduction., Consequently, estimates of transduction frequency are best obtained early
in a soil study before significant multiplication of cells occurs^ The transduction
frequency is expressed as the number of transductants divided by the number of recipient
bacteria originally added.
t A
-------
Stepwise Summary of Procedures for Studying Transduction in Soil
-¦ Prepare soil (e.g., sieve; amend with clays, CaCO^, etc.; condition; add to appropriate
microcosm).
- Prepare suspensions of cells of recipient and lysogenic bacteria or phage lysate.
- Wash cell suspensions by dilution or centrifugation.
- Adjust suspensions for appropriate multiplicity of infection (MOD for transduction
(MOI = 1 to 5).
- Add cells of recipient and lysogen or phage lysate sequentially to soil with sufficient
water (or a nutrient solution) to bring the soil to its -33 kPa water tension.
- Incubate soil, preferably in a high-humidity chamber.
- Sample soil periodically (either by sacrificing multiple microcosms or from a batch
system).
- Prepare appropriate serial decade dilutions of the soil.
- Plate dilutions on selective media suitable for growth of recipient, lysogen, and
transductant, as well as of indigenous soil bacteria. Plaque assay the dilutions for free
phage.
- Incubate plates, and record numbers of colony-forming and plaque-forming units that
develop on each medium.
- Verify presumptive recipient, lysogen, and transductant cells for phenotype and/or .
genotype (see Section VII).
VI-10
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VII. Identification, Characterization, and Confirmation of Recombinants
Introduction
The isolation of genotypically-identical bacteria, whether donors, recipients, or
recombinants, for identification is most, commonly and easily done by separating
individual cells on a solid nutrient medium. Streaking or spreading serial dilutions of soil
directly on the surface of the medium or suspending the dilutions in molten agar before
making pour plates are means of separating cells so that they produce separate colonies
(i.e., aggregates of cells) that presumably arise from each individual cell. However, even
the formation of a single colony does not guarantee that the colony is a pure culture, and
it is advisable tp restreak the colony of interest until colonies are produced that are
identical to one another, both macroscopically and microscopically. Even pure cultures
can exhibit some variation, e.g.Vsmooth-rough colony morphology, presence and absence
of spores, and variability in the gram stain. Microbiology laboratory manuals describe in
detail'the methods for obtaining and maintaining pure cultures from isolated colonies
(Atlas et al., 1988; Ausubel et al., 1987; Benson, 1986).
Selective media
Conventional methods of enumeration and identification depend on the growth and
"phenotypic expression" of measurable traits of an organism under conditions that
restrict or inhibit organisms without the traits, e.g., selective media. Many methods can
be used to select or enrich for specialised, individual phenotypes, and most microbiology
laboratory manuals provide descriptions of various selective and differential media and
their applications. When sampling soil environments, specialized media will be necessary
for the enumeration of typical groups of soil microbes (e.g., nitrifying, nitrosofying,
denitrifying, sulfate reducing, and cellulolytic bacteria; fungi; protozoa), if knowledge of
the spectrum of soil microbes present is desired. A soil extract-mediunv-will be required
to recover oligotrophic microbes that are not recovered on more copiotrophic standard
VI1-1
-------
laboratory media (Wollum, 1982) (see Appendix).
Media for the detection and differentiation of GEMs on the bases of their
metabolic and cultural requirements are useful for the detection, identification, and
enumeration of riovel genes in soil. The use of strains that are labeled or "tagged" with'
genes that enable a particular metabolic activity (e.g.t toluene degradation, use of X-gal)
or confer resistancie to an antimicrobial agent (e.g., Tc or Hg) facilitates the detection of
the GEM containing the novel genes. If the strains are to be released to the
environment, it is essential to choose antibiotics that are hot used for treatment of
humans or other animals, as microbial resistance to clinically-useful antibiotics is a
major health problem.
Genes that are carried extrachromosomally may be unstable unless there is
i i
selection pressure, as the plasmids may be lost through segregation (see Section II).
Therefore, to monitor the persistence of introduced novel genes, it is preferable to use
' ' ' ' ,' .
genes that are located on the chromosome rather than on a transposable element that
readily recombines with other sites or elements, such as transmissible plasmids. It may
be beneficial to use a strain with two genetic markers, so that a spontaneous mutation
will not eliminate the detectability of the phenotype when it is plated on media
containing ebeh selective agent individually, as well as on media containing both agents.
The additive effect of a second antibiotic or heavy metal in the selective medium may
also reduce the background level of contaminating indigenous microbes, as autochthonous
microbes are not usually multiply-antibiotic resistant (see Section V).
The use of microbes tagged with genes that code for metabolic activities that can
be linked to a chromogenic substrate in a selective medium is a distinct advantage in
their detection and enumeration. For example, the insertion of genes for lactose
fermentation into a nonenteric bacterium will facilitate its enumeration on media that
are selective for coliforms, e.g., Eosln methylene blue agar, MAC agar, Endo-agar, X-gal
(5-bromo-4-chloro-3-indolyl-^-D-galactoside) agar, IPTG (isopropylthio^-D-galactoside,
VI1-2
-------
an analogue of lactose) agar, as the chromogenic substrates aid in the visualization of
colpnies of tagged organisms (Jain et al., 1988). Various cloning vectors have been
developed that use the lacZ ^-galactosidase) gene as a marker, e.g., phage M13
(Messing, 1979) and plasmid pUC(Vieiraand Messing, 1982, 1987). These ONA sequences
that code for lactose metabolism have been integrated into the chromosome of the host
to pirovide a marker for monitoring the GEM in the environment (Drahos et al., 1986).
The xyIE gene from the TOL plasmid of P. aeruginosa has been cloned into other genera 1
of bacteria, which then produce a yellow color on catechol-containing media, as the
result of the activity of catechol oxygenase (Walter et al., 1988b).
Some strains of Pseudomonas (e.g., P. aeruginosa, P. fluorescens) release
fluorescent water-soluble pigments, that are easily visualized under ultraviolet light.
Various types of Pseudomonas isolation media are commercially available.
"Breakthrough" of indigenous microbes
Many indigenous soil microbes are inherently resistant to the antibiotics and/or
heavy metals routinely used in selective media for the isolation of cells containing novel
genes. The use of additional antibiotics in the medium to reduce the growth of
indigenous bacteria.is sometimes effective. The upper limits of tolerance of the novel
genes and the hosts in which they may reside to the selective antibiotics and/or heavy
metals must be defined, so that the maximum levels of the antimicrobial agents can be
used in the recovery medium to reduce the growth of the indigenous microbiota.
Prolonged incubation of plated soil dilutions, even on media containing multiple
antibiotics, may also permit the "breakthrough" growth of indigenous microbes,
particularity of fungi (Krasovsky and Stotzky, 1987). Hence, shorter incubation periods
and the enumeration of small colonies may be necessary to estimate the populations of
recombinants before they are overgrown by indigenous microbes. Morphological
differences, e.g., in colony size, shape, texture, and pigmentation, between the colonies
of the GEM and those of indigenous microbes sometimes enable the distinction between
VI1-3
-------
introduced experimental and autochthonous strains.
The high numbers of indigenous soil bacteria (10® to 109 CFU/g soil) will
sometimes have a "sparing" effect on the antimicrobial action of the selective agents.
Under these conditions, the next'higher dilution of the soil sample may reduce the
numbers of indigenous microbes sufficiently to reduce or eliminate the sparing effect
andi, thereby, breakthrough.
Indigenous soil fungi may frequently break through the selective recovery medium
and obscure or inhibit the growth of the bacterial strains of interest. The antifungal
agent, Cy, should be incorporated into the recovery medium at concentrations from 50 to
200 >»g/ml, depending on the soil, to curtail fungal growth with no effect on the growth of
bacteria.
ViaMe but nonculturable bacteria
, ' > >
A phenomenon referred to as "viable but rionculturable" was first described for
aquatic bacteria that could not be recovered or detected on complex synthetic laboratory
media but which retained the ability to produce a virulent infection in laboratory animals
(Colwell etal., 1985; Roszak and Colwell, 1987). The inability to detect some introduced
GEMs directly from soil on initial isolation media has also been reported (Devanas et al.,
1986). If the host-novel gene systems to be studied show a tendency towards "viable but
nonculturable", it may be advisable to use initially a less selective recovery medium
(e.g., MAC) rather than one that may be inhibitory (e.g., MAC + Tc) to the microbes
stressed in the soil environment, even though the £>henotype of the host-gene system is
for resistance (e.g., Tcr). Colonies recovered on the less selective medium are then
tested on a more stringent selective medium for expression of the resistance phenotype.
In other words, a "resuscitation" step is introduced for the "stressed" experimental GEMs,
after which the complete resistance profile is expressed (see Section V).
Oligotrophic media may be necessary for the recovery of indigenous soil organisms
that are not accustomed to the relatively high levels of nutrients contained in standard
VI1-4
-------
laboratory media. Various oligotrophic media have been suggested for the recovery of
microorganisms from soil (Wollum, 1982).
Gene transfer on recovery media vs. in situ
One of the purposes of developing methods for the monitoring, isolation, and
identification of GEMs is to detect the transfer of their novel genes in situ. If the
concentration of parentals in the soil dilutions, is high, there is a possibility of cell
contact between the parentals on the recovery medium. Consequently, there is the
possibility that any gene transfer observed, particularly by conjugation, did not occur in
soil but only on the recovery medium. Several, methods have been suggested to
determine whether the presence of recombinants on the recovery medium is a result of
gene transfer in situ or in vitro (Curtiss, 1981). In one method, the recipient strain is.
selected to be resistant to Nx, which is incorporated into the medium along with other
selective agents, for the recovery of the recombinant.' Don^r cells will be incapable of
participating in gene transfer on this medium, as Nx inhibits the DMA gyrase necessary
for the transfer of DN A (Curtiss, 1981; Walter et al., 1987). Cells that grow on the Nx-
containing medium and have the selectable phenotype for tHe novel genes are assumed to
be recombinants that resulted from gene transfer in soil.
DNA "fingerprinting'' and plaatnid profiles
Restriction endonuclease analysis or DNA "fingerprinting" can be used to detect
'1 »
and monitor the fate of specific genes. Restriction endonucleases recognize specific
nucleotide sequences in double-stranded1 DNA and cleave the DNA at a specific sequence
that is usually four to eight base pairs long. Different restriction endonucleases
recognize different sequences, and depending on the frequency of the recognition
sequence, a specific endonuclease will cleave the DNA numerous times, which will result
in a number of different-sized DNA fragments that can then be separated according to
their molecular mass (Mr) by agarose~gel electrophoresis. The Mr of each fragment is
estimated by comparing the migration of the DNA fragments to DNA standards of known
-------
Mr. barkers of Mr that are commonly used to produce a standard curve are restriction
endonuclease digests of DNA from coliphage lambda or plasmid pBR322, and many DNA
fragments and plasmids of known Mf are commercially available. The distribution of
different-sized fragments resulting from the cleavage by different endonucleases is
unique for each chromosome or plasmid analyzed; hence, a "fingei^rint" of the specific
DNA is obtained (Ausubel et al., 1987).
The bacterial strains that express the specific phenotype determined by the genes
of interest are isolated in pure culture, as described above. The total DNA is'extracted,
from these cultures by any of several procedures (Ausubel et al., 1987; Crosa and Falkow,
1981; Maniatis e£ al., 1982). Chromosomal DNA is separated from plasmid DNA by
linearization of chromosomal DNA by mechanical shearing followed by differential
centrifugation techniques, e.g., cesium chloride-ethidium bromide density gradient'
centrifugation, or by agarose gel electrophoresis. The choice of procedure will depend on
the information desired from the isolated DNA. The appropriate DNA containing the
gene of interest (i.e., either on the chromosome or on a plasmid) is treated with a series
of endonucleases, which are commercially available (e.g., International Biotechnologies
Inc., New Haven, CN; New England Biolabs, Beverly, MA; Bethesda Research
Laboratories, Gaithersburg, MD) (Ausubel e^aL, 1987).
Crude but rapid screening of intact plasmid preparations can be used to evaluate
i i
hundreds of colonies a day for the presence of plasmid DNA by gel electrophoresis (Kado
and Liu, 1981). This is a well-established method for the characterization of plasmids.
Plasmid profiles (number and size per strain) have been used to identify bacterial
populations and to study plasmid epidemiology in natural environments, including soil
(Jain £t al., 1988).
DMA probes
Introductibo.~DN A-DNA ftybridization caiTbe used'to detect specific gene
sequences in GEMs that have been added to soil or other natural environments and in
-------
indigenous bacteria to which the genes may have been transferred. A highly specific and
unique DN^l sequence in the chromosome of the host or in the vector (e.g., a plasmid or a
bacteriophage), preferably in the novel DNA, is isolated and purified, as described above,
and labeled with either a radioactive nuclide (e.g., 32P) or a chromogenic agent (e.g.,
biotin-streptavidin-alkaline-phosphatase-dyes) (Ausubel et al., 1987). DNA probes are
often prepared from genes cloned in plasmid vectors so that the probe DNA can be
conveniently produced in sufficient quantities. This DNA probe is hybridized with DNA
from bacteria (usually those that express the specific phenotype on selective media) that
have been isolated from soil and immobilized and lysed on a nitrocellulose or nylon filter
membrane. Bacteria whose DNA hybridizes with the probe are then located on the filter.
One advantage of hybridization with DNA probes is the relatively high degree of'
specificity when compared with classical microbiological plating methods on selective
mediA. Another advantage is that microorganisms can be detected directly in soil
' without prior growth on selective media. Techniques for DNA hyoridization in studies of
microbial ecology include Southern blot, dot or slot blot, and colony hybridization (see
below).
Methods that isolate total bacterial DNA frofn soil and then hybridize this DNA
with specific DNA probes may be valuable in: 1) monitoring the survival, establishment,
and growth of, and gene transfer by, GEMs in soil (see Holben and f iedje, 1988; Steffan et
al., 1988); 2) the detection of groups of microorganisms capable of performing the same
function (e.g., containing njf genes); 3) monitoring gene flow among indigenous bacteria;
and 4) the detection of nonculturable microorganisms (Jain et al., 1988). These direct
DNA methods, however, have a detection sensitivity of only about 4 x 10* cells/g soil
(Holben et al., 1988), whereas some selective isolation methods have a sensitivity
approaching 2 x 10* cells/g soil (Devanas e£ah, 1986). Moreover, direct methods for the
detection of novel genes in soil, including DNA hybridization, do not distinguish between
viable and nonviable microorganisms. The choice between using DNA hybridization or
VI1-7
-------
classical microbiological methods will depend on the information and the sensitivity
required. In all probability, both methods, in various combinations, will be'used.
Furthermore, DNA hybridization methods that are more sensitive, specific, and rapid, as
well as less expensive, will undoubtedly be developed.
Types of DNA probes. The probe DNA must be labeled with a tag that will enable
its detection when hybridized to homologous (i.e., target) DNA sequences in the cells
being probed. The first labeling technique for DNA probes was the use of radioactive
nuclides, e.g., P, which, were incorporated into the nucleotides of the probe DNA
(Ausubel et al., 1987; Maniatis et al., 1982). 32P is preferred to 35S or 3H, as it has a
higher specific activity and, therefore, results in the greatest sensitivity. Moreover, the
activity of enzymes that act on DNA labeled with 32P is not affected, as 32P causes only
small changes in the structure of DNA. After hybridization to its target bNA, the probe
DNA is visualized by exposure of the hybridization membrane to X-ray film, which is
1 32
sensitive to the gamma rays emitted by P.
An alternative to autoradiography are chromogenic techniques that use DNA
labeled with nonradioactive molecules, such as biotin. The biotin-labeled DNA is
visualized by the application of a streptavidin-alkaline phosphatase (Leary et al., 1983) or
streptavidin-peroxidase (Syvanen et al., 1986) conjugate, followed by enzymatic
conversion of a chromogenic substrate to produce a color, which identifies the target
DNA with which the probe has hybridized on the filter.
Preparation of DNA probes. Labeling of tne probe DNA with radioactive
nucleotide triphosphates can be accomplished by nick translation. DNA polymerase I
adds labeled nucleotides to the 3'-OH terminus at nicked sites along one strand of double-
stranded DNA. This enzyme also has exonuclease activity and removes unlabeled
nucleotides that are then replaced with radiolabeled nucleotides, which results in DNA of
high specific activity, e.g., greater than 108 cpm/>jg of DNA when labeled with 32P
(Maniatis etal., 1982). Nick translation is not the only method for radiolabeling DNA for
VI1-8
-------
use as probes.1 End-labeling of small DNA fragments with { / -^P)nucleotides, using
polynucleotide kinase from coliphage T4, or labeling with (0f-^P)oIigonucleotides, using
random primers and the Klenow fragment of E. coli polymerase 1 or phage T4
polymerase, can be used to obtain radiolabeled DNA with specific activities that are
often higher than those obtained by nick translation (Bentjen et al., 1989). Procedures for
the isolation of DNA, probe preparation, hybridization with target DNA, and detection of
the hybridization signal are detailed in Ausubel al. (1987) and Maniatis etal. (1982).
Three different techniques are available for the preparation of nonradioactive
DNA probes, i.e., nick translation, chemical labeling, and photolabeling. Commercially-
prepared Wits are available for all three methods of DNA labeling, which result in
nucleotides labeled with biotin.
Nick translation is the most frequently used method of labeling DNA with biotin
(Brigati et al., 1983). Kits, in which biotin-labeled dATP or dUTP and enzymes'are
provided for the nick translation reaction, are available from Bethesda Research
Laboratories and Enzo Biochem, Inc. (New York, NY). Procedures outlined by the
manufacturer or in Ausubel et al. (1987) and Maniatis et al. (1982) can be used to label
DNA with biotinylated nucleotides; as the nick translation reaction is the same as used
with radionucleotides.
Chemical labeling of the DNA probe involves the insertion of an antigenic sulfone
group into the cytosine moieties of the denatured DNA (Renz.and.Kurz, 1984). A
specific monoclonal antibody is then bound to the sulfone group, followed by the
application of an enzyme-anti-immunoglobulin'conjugate that catalyzes a chromogenic
I
substrate system and enables visualization of the labeled probe DNA. Kits are available
from FMC Byproducts (Rockland, ME)-
Procedures for the direct photolabeling of DNA with biotin have been described by
Forster et al. (1985). This Is a nonenzymatic method in which the probe DNA is labeled
by irradiating photoactivated biotin with a high intensity lamp in a mixture containing
-------
the DNA.' Photolysis of an azide group on photobiotin results in covalent bonding to the
base group on the nucleic acids. Visualization is achieved by the application of a
I
streptavidin-enzyme conjugate and a chromogenic substrate, as described above. Highly
purified DNA must be used, as the highly reactive photoactivated biotin will bind to
proteins, tris buffer salts, and polyethylene glycol and cause nonspecific signal
development. A commercial kit that supplies the photobiotin and procedures for labeling;
is available from Sigma Chemical Co. (St. Louis, MO) or Bethesda Research Laboratories.
Hybridization techniques.
. 1 ¦ 1
Southern hybridization. Hybridization of DNA probes to target DNA that has been
separated on agarose gels by electrophoretic techniques and transferred to a
nitrocellulose or nylon filter membrane is termed Southern hybridization (Southern,
197S). Details of the procedures used for Southern hybridization are presented in
Ausubel et al. (1987) and Maniatis et al. (1982).
Two distinct protocols for isolating the target DNA from soil samples for Southern
hybridization have been developed: 1) direct extraction of DNA from soil samples
(Ograin et al., 1987); and 2) extraction of the bacterial population from soil, followed by
lysis of the bacterial cells (Holben et al., 1988). Direct extraction of nucleic acids from
soil is more efficient than the recovery of intact bacterial cells. However, direct ,
extraction suffers from the limitations that a heterogeneous DNA sample is obtained, as
eucaryotic and cell-free DNA are also extracted, and that humic acids, among other soil
constituents, contaminate the DNA sample, which could result in decreased hybridization
of the DNA probe to target DNA. Although the recovery of intact bacterial cells by
I
differential centifugation is a more lengthy process and results in the recovery of only
approximately 80% of the total bacteria presumably present in soil (Holben et al., 1988),
the DNA sample i$ not contaminated with humic acids and nonbacterial DNA, and it
undergoes less shearing, so that the sample contains DNA of more uniform size than that
obtained by direct extraction. Treatment of the purified DNA extract with restriction
i _irt
-------
enzymes before hybridization enables the comparison of the size of the labeled fragment
with a known restriction map of the DNA from the GEM of interest and, thereby, easier
identification of restriction fragments that contain the novel gene (Jain et ah, 1988). .
Moreover, restriction analysis of DNA isolated from, soil, either directly or indirectly,
» , ,
can detect gene rearrangements that may have occurred in soil (Holben et al.t 1988).
Genie arrangements and deletions have also been detected in freshwater, suggesting that'
genetic instability may be enhanced in natural habitats (O'Morchoe et ah, 1988).
Slot/dot blot hybridization. Slot or dot blotting techniques of DNA hybridization
involve the placement of bacterial cells, viruses, or extracted DNA at specific locations
on nitrocellulose or nylon filters. The cells or viruses are then lysed, and the DNA is
»
immobilized on the filter and hybridized with the DNA probe. For example, dot blots are
prepared by placing aliquots (5 to 10 >il) of target DNA directly on'the filter with a
micropipettor, forming a circular dot of bacteria, phage, or DNA for hybridization.
Alternatively, slot blot apparatuses are available in which'samples are placed in slots (30
to 40 samples per filter are possible) and immobilized on the hybridization filter by the
application of a vacuum (Steffan and Atlas, 1988). Slot blot hybridization with
radiolabeled DNA probes has been used to detect novel genes in GEMs added to a
nonsterite sediment microcosm (Sayler et al., 1986; Steffan and Atlas, 1988), and dot blot
hybridization with a biotinylated DNA probe has-been-used to evaluate presumed£. coli
transductants isolated from nonsterile soil that wqs inoculated with E. coli and phage PI
(Zeph and Stotzky, 1989).
Colony hybridization. The hybridization of a DNA probe to target DNA released
from bacterial colonies impregnated into nitrocellulose or nylon filters is termed colony
hybridization (Grunstein and Hogness, 1975). Descriptions of the methods employed in
colony hybridization with DNA probes can be found in Ausubel et ah (1987) and Maniatis
et al. (1982). Colony-hybridization-techniques have been used to detect specific gene
sequences naturally present in soil bacteria (Barkay et al., 1985) and novel genes
v 1 I . 1 1
-------
introduced into bacteria that had been added to soil (Rafii and Crawford, 1938).
( i
Normally, dilutions of soil are plated .on selective media, and plates with well separated
bacterial colonies are used as (he source of colony DNA. The colonies are transferred to
a filter membrane for lysis arid hybridization. Alternatively, the screening of large
numbers of bacteria for the presence of novel DNA can be accomplished it lawns of
bacteria (approximately 105 to 10® cells/plate) are used'in colony hybridizations with
^2P-labeled probes (Sayler et ah, 1985), particularly II the isolation of the organism of
interest from discrete colonies is not desired. However, when this procedure is used with
biotin-labeled DNA probes, problems can arise with nonspecific signal development
during the probe visualization step (see below).
The advantage of the colony hybridization technique is that it of ten enables a
more rapid screening of environmental samples for the presence of microorganisms
containing a novel genets) than Southern hybridization or slot/dot blot techniques.
However, the microbes must grow oh the isolation media, to produce sufficient target.
DNA for hybridization, in contrast to the Southern hybridization method. For some
purposes, e.g., monitoring gene transfer from an introduced GEM to the indigenous
bacterial population, colony hybridization is the preferred method, as growth of the
introduced GE.M can be inhibited on selective media, thereby allowing hybridization of
the probe with colony DNA from only the indigenous population. Jain et al. (19S8)
emphasized that preliminary testing of the DNA hybridization protocol is important to
avoid false positives when attempting to detect GHMs in environmental samples: a good
selective medium must be used to prevent growth of indigenous bacteria that can give
false positive hybridization signals; and the specificity of the DNA probe for the novel
gene must be ascertained before its use in the monitoring of GEMs in the environment.
Sensitivity and specificity of PNA probes. The sensitivity of radiolabeled DNA
probes is generally greater than that of nonradioactive probes, Zwadyk et al. (1986)
reported that a ^P-labeled probe was 100 times more sensitive than biotinylated probes
VII-12
-------
in detecting plasm id pBR322 DNA with Southern hybridization, and a minimum of 22 ng
I
of target DNA was necessary for detection with biotinylated plasmid pBfc.322 DNA
probes. However, when purified target DNA was spotted on nitrocellulose filters,
Zwadyk et al. (1986) detected as little as 9.7 pg of target DNA with a 32P-lrbeled probe
and 39 pg with a biotjnylated probe prepared with a kit (Bethesda Research
Laboratories). Zeph and Stotzky (1989) detected 50 pg of target DNA from phage PI
using the same biotinylated probe kit. Leary et al. (1983) reported greater sensitivity
with a biotinylated DNA probe made from linearized rather than from intact plasmid
DNA, and they detected 3.1 pg of target DNA by Southern hybridization.
Another expression of sensitivity is the minimum number of cells containing
target DNA that can be detected in abackground of indigenous bacteria on enumeration
plates by colony hybridization. This is useful in comparing DNA probes on the basis of
their ability to detect GEMs added, to natural environments that may contain high
numbers of indigenous bacteria. Sayler et ah (1985) utilized ^P-labeled DNA probes to
detect P. putida added, to sediment microcosms at a sensitivity of 1 colony of P. putida in
a background of 10® indigenous bacterial colonies that contained nonhomologous DNA.
The minimum number of cells containing homologous DNA that can be detected is
also used as a measure of the sensitivity of DNA probes. For example, Holben et^al.
(1988) were able to quantitate Bradyrhizobium japonicum, added to nonsterile soil, at
levels of 4.3 x 10* cell&'g soil, which was equivalent to 0:2 pg of target DNA per 1 yg of
total purified DNA isolated from soil bacteria. Frederickson et al. (1988) used the most
probable number (MPN) method, combined, with DNA hybridization of DNA isolated from
individual MPN tubes, to detect P. putida. added to nonsterile soil, at concentrations as
low as 10 cells/g soil and Rhizobium leguminosarum at 100 cells/g soil. The application
of DNA probe techniques to studies of microbial ecology has been reviewed by Holben
and Tiedje (1988).
VII-13
-------
The relatively low sensitivity of DNA probes in detecting and enumerating
bacteria containing homologous DNA in soil can b$ overcome, in part, through the
amplification of the target DNA sequences. The polymerase chain reaction (PCR)
procedure has been used to increase probe sensitivity by using the hybridized DNA probe
as a primer for'chain elongation beyond the target DNA sequence (Somerville et al.,
1988; Saiki et al., 1988). After hybridization of the probe to target DNA. DNA synthesis
is initiated from the 3'-OH terminus of the probe DNA strand with radiolabeled
i 1 •
nucleotides and DNA polymerase. The PCR is repeated 20 to 30 times by remelting the
DNA strands and hybridizing with fresh probe DNA, followed by another chain elongation
reaction, which results in an enhancement of the sensitivity of the DNA probe by at least
,3 orders of magnitude. The specificity of the probe depends on the initial hybridization
to target DNA, and the sensitivity is enhanced by DNA synthesis from the free 3'-OH end
into adjacent genes (Colwell et al., 1988). Steffan and Atlas (1988) employed the PCR
method to increase the sensitivity of a probe for the detection of catabolic genes in
Pseudomonas cepacia AC1100 that was added to sediment microcosms.
The use of biotinylated DNA probes has an inherent problem, as contamination of
the sample with protein will result in nonspecific color development caused by the
binding of the avidin-enzyme conjugate to protein components in the cell lysate, in
addition to binding to the biotin moiety on the labeled probe. Consequently, all protein
i
must be removed from the hybridization filters when using biotinylated DNA probes,
particularly when whole bacterial cells are lysed on the filter, as in slot/dot blots or
colony hybridizations. Treatment of the filter with'chloroform, phenol, and proteolytic
enzymes will remove significant amounts of cell debris and protein (Haas and Fleming,
1986; Rafii and Crawford, 1988; Zeph and Stotzky, 1989).
In summary, radiolabeled DNA probes have the advantage of being more sensitive
in many applications than biotinylated probes, although sensitivity is being improved.in^=
the latter. However, biotinylated DNA probes are less hazardous than radiolabeled DNA,
VI1-14
-------
have a much longer storage life (one year or more compared to two weeks for ^P-
labeled DNA), and have a lqwer cost.
Serological techniques
Serological methods, based usually on the expression of specific surface antigens,
require the isolation of the bacteria of interest on media that are relatively selective
(i.e., that eliminate or reduce the growth of unrelated soil bacteria). The bacteria are
purified and then challenged with a spectrum of polyclonal or monoclonal antibodies
raised against the bacteria of interest. The reactions between the bacteria and the
antisera (usually polyclonal) are then scored as being either: 1) nonreactive; 2) cross-
reactive with many sera without distinctive features and common to all isolates; 3)
cross-reactive with distinctive features between isolates and a few antisera; or 4) a
i
specific reaction with a single antiserum. Although this method can be highly specific in
some instances,-the surface antigens of some bacteria change during growth, and they
may be different in soil than in the pure cultures that were used to raise the antigens.
Moreover, serological data do not always agree with data obtained with DI4A probes and
with other characteristics of the bacteria (e.g., Schofield et al., 1987).
Although polyclonal antisera are easier to prepare, they are less specific that
monoclonal antibodies. The use of labeled monoclonal antibodies provides a highly
specific technique for the direct detection of GEMs in the environment. However, there
are significant limitations in this methodology: e.g., the presence of the novel DMA in
the microbe is only presumptive and must be verified by other methods; the sensitivity is
low when employed in soil studies; it is not possible to differentiate between viable and
nonviable cells (Colwell et al., 1988). The detection of GEMs in the environment with
monoclonal antibody techniques has been reviewed by Omenn (1986).
Heat induction of prophages
Verification of the transfer of novel genes by transduction is possible with some
phages by induction of the lytic cycle of replication of the prophage. Zeph et al. (1988)
VI1-15
-------
confirmed by heat induction of lysis that presumptive E. coli transductants. isolated from
soil inoculated with E. coli and phage PI on the basis of the antimicrobial-resistance
phenotype coded for on the phage PI genome, contained the phage (Table 2). The phage'
PI strain used had a temperature-sensitive mutation in the cl gene, which codes for a
repressor protein that maintains lysogeny. The presumed E. coli transductants were
grown in LCB at 30 C, with shaking, until log phase (optical density of 0.2 at 600 nm),
followed by induction of lysis by incubation at 42 C. The presence of phage PI virions in
the culture was demonstrated by spotting aliquots of the heat-induced lysate on lawns of
£. coli. It might be possible to use this technique to confirm the presence of any
prophage DNA in presumed transductants, as incubation of lysogenic bacteria at
temperatures above their optimum for growth should inactivate or impair the activity of
the repressor protein that maintains lysogeny. Moreover., other agents that can induce
the lytic cycle (e.g., UV radiation, mitomycin C) should be evaluated'with lysogens that
do not respond to elevated temperatures.
When attempting to demonstrate gene transfer to indigenous soil bacteria by
transduction by phage PI, isolates of soil bacteria were grown at 25 C until log phase,
exposed to 42 C for 30 min, and then reihcubated at 25 C to allow lysis of the'bacteria by
,the phage. Continued incubation was not at 42 C, as most soil bacteria have temperature
optima for growth between 20 and 30 C and may not grow at the 42 C used to induce
prophage PI.
VII-16
-------
VIIL Quality Assurance/ Quality Control
I
Sample representativeness and custody
All soil samples should be contained in separate and well-identified (e.g;, labeled
with waterproof ink) bags or plastic-lined garbage pails. All bacterial cultures should be
maintained at 4 C in pure culture on slants in wellridentified test tubes and transferred
regularly. Purity and phenotype should be determined after each transfer! Soil stocks
should be located in a separate locked room, and stocks of bacteria, phages, media, clays,
etc. should be in locked laboratories to which only personnel of the laboratory and
appropriate security personnel have access. These, stocks should be under the direct
supervision of the Principal Investigator.
Sampling procedures
Stocks of soils, clays, bacteria, phages, media, etc. should be thoroughly mjxed
1 . » 1 i
before each experiment and before subsampling' to ensure maximum homogeneity. The
number of subsamples will vary from experiment to experiment, depending on results of
previous experiments, and should be based on significance between treatments a,t a
probability level (P) of less than 0.05.
Comparability
Microbiological data should be reported as number of CFU or PFU/g soil, oven-dry
equivalent. Frequency of recombination is expressed as a per cent of donor, recipient, or
donor plus recipient, depending on the study. The denominator used to obtain the
percentage must be clearly indicated. All other data (e.g., physicochemical soil
characteristics; nomenclature and phenotype of bacteria, plasmids, an'* bacteriophages;
Mp.of DNA fragments) should be reported in standardized units.
Calibration procedures and frequency
All calibration activities should be thoroughly documented. Calibration records
should be on permanent file in bound notebooks.
V1IM
-------
Instrumental. Thermometers for monitoring temperatures of refrigerators,
I ,
freezers, incubators, laboratories, etc. should be verified biannually against a
thermometer traceable to the National Institute of Standards and Technology. Buffer
solution for the.calibration of pH meters'can be obtained from commercial sources (2 to '
3 pH values that bracket the working range of pH should be evaluated by this method, in
addition to electronic resistance methods). Pipettors and glass pipette? should be
calibrated every six months by gravimetry; random samples of disposable pipettes should
i ' •
also be checked periodically for accuracy. Spectrophotometers should be calibrated
biannually with decade dilutions of several sources or types of DNA, protein, and
bacteria. Calibration of agarose gel electrophoresis is conducted with commercial DNA
Mr standards (these standards should be run with all unknown preparations). Microscopes
should be calibrated annually with decade dilutions of standardized polystyrene latex
particles. For instruments used routinely, a check list should be fastened on or near each
instrument, and each user should initial and date the list after routine calibrations have
been made before and after a^lysis of samples.
Analytical. 32p_ and Ov.jr DNA probes should be verified for specificity with the
cDNA against whifch they were prepared. Similar verification can be made for probes for
gene products. These verification procedures can also serve to test the reactivity of the
X-ray films and the efficacy of the film developer solutions, the enzyme color_
development reaction for nonradioactive DNA probes, and the sensitivity of monoclonal
antibodies in serological techniques.
Microbiological. The pKenotype of all novel bacteria and the infectivity of the
phages should be verified in pure culture before experiments are conducted in soil,
furthermore, studies in sterile soil can provide another level of verification for studies in
nonsterile soil. Spectrophotometry calibration of bacterial numbers should be correlated
periodically with microscopic and plate counts. The efficacy o_f the sterilization of soils must be
determined by plating on several media, ranging from nutrient-rich to nutrient-poor.
VII1-2
-------
, Analytical procedures
Quality assurance must be established by numerous replicates per experiment and repeat
experiments. When more than one genetic marker is contained in the novel DNA, direct or
replica plating should be to selective media specific for the detection of each marker, either
individually or in combinati^. Furthermore, DNA probes should b$ used when necessary (e.g.,
when there appears to be a loss or segregation of the novel DNA). When analytical procedures
developed by others are used, the verification procedures should be those of the developers. It
must be emphasized that the study of gene transfer in soil is a relatively new area of research,
and sufficient published data are not available for routine verification and quality assurance.
Therefore, verification of gene transfer by novel bacteria by several techniques is required.
Experimental design and statistical analyses
The experimental design and the statistical analysis to be applied to the data must be
determined before the start of each study. The.assistance of a statistician is helpful at this
stage. The number of replicates to be used should be sufficient to detect a P of less than 0.05.
This number of replicates is determined in preliminary studies, with the application of
appropriate statistical theory and formulas. At the end of each experiment, the data must be
statistically analyzed'(e.g., means t standard errors of the means, Student's t-test, ANOVA,
regression analyses) and plotted or tabulated (Atlas and Bartha, 1981). As mentioned above,
data shoud be expressed in.standard terms, or if not, the dimensional units should be clearly
defined.
Experimental errors are detected by abnormally high coefficients of variation (greater
than 50%) between replicates and by unanticipated and unexplainable deviations in time-
response curves. Inasmuch as experiments must not only be sufficiently replicated and
statistically analyzed, but also repeated, errors will be readily detected. In the event of such
errors, replicated experiments should be repeated. Although statistically significant differences
-may-be-observed in some-experiments, the potential ecological significance of these differences-
must be further evaluated (e.gM in repeat experiments or in more complex microcosms),
VIII-3
-------
especially if the differences arej transitory.
Data analysis and reporting
Data should be recorded in ink on appropriate data sheets, in duplicate, in permanent,
bound laboratory notebooks. All entries should be initialed. One copy should.reside with the
research technician collecting the day-to-day data, and the other should reside with the Project
Manager. A sample data sheet is included (see Appendix). '
Internal quality control checks
The routine quality controls of a microbiology laboratory should be followed. For
example, periodic checks (at least monthly) must be conducted on laboratory water for
microbiological quality (less than 1Q4 CFU/ml) and conductance (less than 2^S/cm); the
temperature of refrigerators (^2 C), freezers M C), incubators (^2 C), and autoclaves (121 C)
should be continually monitored (usually twice op a measurement day); pH and quality checks
should be made on media lots; sterility checks on -nedia lots, water, soils, and other autoclaved
or radiation-sterilized materials stould be made by plating. A log book of internal quality
controls should be maintained. These internal quality control checks are routine in basic
research laboratories.
Preventative maintenance
All major pieces of laboratory equipment should be monitored regularly and serviced
routinely, as individually required, through service contracts with outside contractors, a£ well as
internally.
Corrective action
If any experiments or parts of experiments have been inappropriately designed,
conducted, or analyzed, the entire experiment must be repeated. It must be emphasized,
however, that gene transfer among bacteria in soil is essentially an unexplored area and that the
development of appropriate experimental design, conduct, and analyses is an intrinsic part of
the research.
Vltl.1
-------
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26
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APPENDIX
Media Composition (all media prepared with distilled water)
1., Luria Broth (LB) (Maniatis et al., 1982)
tryptone 10 g
yeast extract 5 g
glucose 1 g
sodium chloride S g
dH20 1 L
2. Luria Broth with calcium and.magnesium (LCB) (Zeph etal., 1988)
. LB containing:
10 rnM MgS04-7H20
2 mJW anhydrous CaCl-2
3. Luria Agar (LA) (.Maniatis ££aL, 1982)
LB containing:
Bacto-agar 15 g/L
4. Luria Top Agar (Maniatis etal., 1982)
LB containing:
Bacto-agar 7.5 g/L
1
-------
5. Minimal Salt Agar (MSA) (Germida and Khachatourians, 1988).
glucose (or lactose)
10 g
vita.nin B12
0.1 g
tetracycline
0.1 g
streptomycin
0.2 g
eosin
0.4 g
methylene blue
0.07 g
leucine
0.02 g
threonine
0.08 g
Bacto-agar
15g
dH20
1 L
6. VO Minimal Agar containing. HgC^ (M9M) (Zeph et ah, 1988)
Na^'.POj 6g
KH2P04 3 g
NaCl 0.5 g
NH4C1 1 g
Bacto Agar 15 g
dH20 1 L
after autoclaving, filter-sterilized solutions of the following are added to
yield the indicated final concentrations: 0.2% glucose, 2 m>f MgS04"7H20,
0.1 mVt CaCl2, and 30^i\f HgCl2.
2
-------
7., Minimal Liquid (ML) iCurtiss, 196S)
NH4C1
5g
nh4no3
U
Na2S04
2g
k2hpo4
9g
kh2po4
5g
MgS04.7H20
0.1 g
Glucose
-5 g,-
dH20
1 L
Autoclave separately at 121 C at IS pa; pH = 6.8.
8. Minimal Agar (MAM Curt iss, 1965).
salts in ML
Glucose (autoclave separately) 3 g
Bacto-agar 15 g
dH20 1 L
pH = 6.8
3
-------
9. Minimal Mating Medium (3M) (Curtiss et al., 1968)
NH4a 5 g
nh4no3 1g
Na2S04 2 g
K2HP04 4.9 g
KH2P04 6.3 g
Glucose (autoclave separately) 5g
dH20 1 L
pH = 6.3
10. Soil Extract .Agar (SEA; (Allen, 1957)
KH2P04 0.5 g
Glucose lg
Soil extract 100 ml
Tap water 90.0 ml
adjust to pH 7.0
Preparation of soil extract:
1.0 kg of garden soil is heated with 1000 ml tap water in the autoclave
(121 C, 15 psi) for 30 min. A small amount of CaCO^ is added, and the soil
suspension is filtered several times through a double paper filter (Whatman No. 1)
until no turbidity is evident. This solution is bottled in 100 ml quantities
and autoclaved before storage.
4
-------
Antimicrobial Agents Commonly Used in Selective Media *
Approx
Special
Initial
Cone
- stability at
Cone
ml to t
Agent
Abbrev
instructioas
diluent
(mg/ml)
4 C (days)
Oig/inl)
to 1 I.
impicillin
Ap
Filter®
dll2Oc
25
?
200
8
Carbenicillin
Cb
Filter
dlloO
50 -
14
100
2
tephalothin j
Ceph
Filter
0.1 M PH
12.5
5
25
2
Chloramphenicol
Cin
Filter
25
30
25
1
*hlortetracycjine
CTc
Use foil
50% KtOll"
10
5
15
1.5
'yclohexiinide
cy
Use foil
d»2o
12.5
5
25
2
ientamicin
Gn
Filter
dll20
12.5
30
25
2
lanainycin j
Kn
Filter
dli20
25
30
50
2
lercuric chloride
Hg
Filter
dlloO
40
30
40
1
lafcillin n
Nf
Filter
dlloO
1
5
2
2
lalidixic acidf
Nx
Filter
0.1 M NaOII
16
30
32
2
tifampicin j:
Itf
Use foil
MeOII
25
5
100
4
treptomycin
Sm
Filter
dH20
12.5
30
?5
2
etracychne
Tc
Use foil
50% EtOII
10
5
15
1.5
obramycin
Tb
Filter
dll20
5
30
10
2
trimethoprim
Tp
Filter
15% 0.06 M
2
30
50
25
HCL
All concentrated stock solutions are to be labeled with name of agent, concentration, date, and analyst's initials.
Filter sterilized
Store in dark |covered with foil
Distilled water
Phosphate buffer, pll 6
Ethanol 1
Methanol
ourtesy of A. Porteous
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Experiment No.:
ncubation time:
unendment: >i
)ilution
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