Pseudomonas fluorescens survival and plasmid RP4 transfer in agricultural water

Wat. Res. Vol. 24, No. 6, pp. 751-755, 1990 Printed in Great Britain.All rights reserved

0043-1354/90 $3.00+ 0.00 Copyright © 1990PergamonPress plc

P S E U D O M O N A S F L U O R E S C E N S SURVIVAL A N D PLASMID RP4 T R A N S F E R IN A G R I C U L T U R A L WATER J. T. TREVORSl'*, J. D. VAN ELSAS2'*, M. E. STARODUB 1 and L. S. VAN OVERBEEK 2 ~Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2WI and 2Research Institute Ital, P.O. Box 48, 6700 AA, Wageningen, The Netherlands (First received July 1989; accepted in revised form December 1989)

Abstract--Transfer of plasmid RP4 via conjugation between introduced Pseudomonasfluorescens donor and recipient cells was studied in agricultural surface water samples at 22°C over a 10 day period. Transconjugants were recovered when the water was sampled after 3 and 10 days incubation, from non-sterile (unfiltered) surface water that was amended with dilute tryptone-yeast extract (TY) broth and 0.1% (v/v) bentonite clay slurry. RP4 plasmid transfer was not observed in unamended filtered water samples inoculated with donor and recipient cells. However, viable donor and recipient cells were usually present in reduced numbers in most water samples after 10 days. Key words--Pseudomonas, plasmid RP4, conjugation, water, survival

INTRODUCTION An understanding of bacterial movement, survival and genetic interactions between microorganisms in soil and aquatic environments is an essential area of microbiology (Halvorson et al., 1985; Trevors, 1987, 1988; Trevors et al., 1987, 1990; Altherr and Kasweck, 1982; Graham and Istock, 1978; van Elsas et al., 1989, 1990). Gene transfer in aquatic environments has been a scientific concern since resistance(s) to antibiotics were discovered to be mediated via R-plasmids. This area of research has been examined from a public health point of view (Goyal et al., 1979; Grabow et al., 1973) and as a means by which resistance gene pools are maintained by plasmid transfer among microorganisms (Mach and Grimes, 1982; Radford et al., 1981; Trevors, 1987; Trevors and Oddie, 1986; Trevors and Starodub, 1987). Little information is available on bacterial conjugation as well as other gene transfer mechanisms (transformation, transduction, protoplast fusion) in soil and aquatic environments (Trevors et al., 1987). Gene transfer depends on organism survival, plasmid stability, available nutrients and cell numbers; all under the influence of biological, chemical and physical factors that may be changing both in terms of time and spatial distribution (Trevors et al., 1987). Since aquatic environments are natural reservoirs for microorganisms, genetic interactions are possible if appropriate conditions are present. However, at the present time a complete understanding of parameters that influence gene transfer in aquatic environments is not available. This is especially true in environments where essential nutrients may be limiting and/or microbial cell numbers may be relatively low. *Authors to whom all correspondence should be addressed. 751

In addition, mating pairs or aggregates required for bacterial conjugation may not be readily formed under low nutrient and/or specific environmental conditions. Aquatic environments where surfaces (clay, silt, organic matter, rocks) and abundant nutrients (sewage treatment systems) may be present, can promote gene transfer by conjugation (Trevors et al., 1987). Bale et aL (1988) have reported on transfer of the pQM 1 mercury resistance plasmid in undisturbed river epilithon. Transfer frequencies were between 2.2 × 10 -1 and 2.5 x 1 0 - 6 per recipient cell and were dependent on the donor-to-recipient cell ratio. The mating system used filters joined face-to-face; thus bringing donor and recipient cells into contact by manipulation of the system. A limited amount of knowledge has confirmed that gene transfer occurs in marine sediments and water, wastewater treatment plants, rivers, p o n d s and streams (Trevors et al., 1987). Many experiments were conducted in sterilized samples (water, sediment, sewage) in environmental chambers or dialysis sacs in the absence of competition from native organisms. We describe here experiments which studied the effect of nutrients, bentonite clay and indigenous microorganisms on RP4 plasmid transfer between Pseudomonas fiuorescens in sterile (filtered) and nonsterile (unfiltered) agricultural surface water. MATERIALS AND METHODS Organisms and incubation conditions The Pseudomonasfluorescens donor strain was originally isolated from a grassland soil sample in .The Netherlands (van Elsas et aL, 1988). A detailed description Of the organism and the filter mating protocol used to introduce plasmid RP4 from Escherichia coli PC 2366 (RP4) into this strain has been previously reported (van Elsas et al.; 1988). Plasmid RP4 is 36 MDa insize, conjugative and encodes

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resistances to kanamycin (Km), tetracycline (Tc) and carbenicillin (Saunders and Grinsted, 1972; van Elsas et al., 1988). The P. fluorescens donor strain was maintained on King'sB agar slants (peptone, 20g; K2HPO 4, 1.5g; MgSO 4. 7H20, 1.5 g; glycerol, 10 g; agar, 15 g; H20, 1 litre; pH adjusted to 7.2) amended with 80#g/ml Km and 80~tg/ml Tc. The P. fluorescens recipient strain was a plasmidless, rifampicin (Rp) and streptomycin (Sm) resistant mutant of the wild-type strain. It was maintained on King's B agar supplemented with 50 #g/ml Rp and Sm (van Elsas et al., 1990). Long-term maintenance of both strains was in 20% sterile glycerol at -80°C. Donor and recipient cells were grown individually in 50 ml of King's B broth under appropriate antibiotic selection for 18 h at 30°C with shaking at 120 rpm. Cells were centrifuged at 8000g for 10 min at 20°C, washed in sterile distilled water, centrifuged again and resuspended in sterile distilled water. Cell numbers in the inocula were estimated using a Petroff-Hauser counting chamber and a Nikon Labophot microscope at 1000 x magnification. Collection o f surface water Surface water (pH6.0) was collected in a sterile polypropylene container the same day the experiment commenced to avoid any changes due to storage. The surface water was collected from a site approx. 5 cm deep overlying a Guelph loam soil at the Ontario Agricultural College, University of Guelph. The water sample was agitated at room temperature on a magnetic stirrer while 3 ml aliquots were removed and placed in sterile 15 ml Falcon No. 2025 culture tubes (Fisher Scientific, Toronto, Canada). Another set of tubes contained surface water samples that were filtered through a sterile 0.45/~m cellulose acetate membrane. Sterile ultrapure deionized water (Type 1 water, 17.6 Mohm/cm) was used as a control medium that was devoid of all nutrients. One set of filtered and unfiltered surface water samples was amended with 0.1% (v/v) sterile bentonite clay slurry (sterilized by autoclaving) and 100/~1 of TY broth (tryptone, 10 g; yeast extract, 5 g; NaC1, 10 g; H20, 1 litre; pH 7.2) diluted 1/10 strength in sterile distilled water. Bentonite has been previously studied for its influence on plasmid transfer in soil (van Elsas et al., 1988), but not in water samples collected from the environment. Nonamended samples received sterile distilled water. All inoculated tubes received approx. 106-107/ml donor (log 6 to log 7) and 106 to 107 ml recipient cells at the beginning of the experiment. Tubes were incubated statically for 10 days at 22°C under a 12 h dark and 12 h light cycle. Aliquots were aseptically removed after 1, 3 and 10 days incubation and

serially diluted in sterile 0. i % (w/v) sodium pyrophosphate (pH 7.0). Enumeration o f donor, recipient and transconjugant cells Total colony forming units (cfu) were enumerated on TY agar plates as previously described by van Elsas et al. (1988). Donor selective plates were prepared by supplementing King's B agar with 80/zl/ml Km and 80/~g/ml Tc. Recipient selective plates contained 50/~g/ml Rp. Transconjugant selective plates contained Kin, Tc and Rp. Duplicate plates from duplicate experiments were spread with 100/,tl of appropriate dilutions and incubated for 48 h at 30°C in the dark, at which time cfu were enumerated and reported as log values. Plasmid isolation and agarose gel electrophoresis Plasmid DNA was extracted using the alkaline (pH 12.40) sodium dodecyl sulphate method described by Birnboim and Doly (1979). Plasmid preparations were electrophoresed in 0.7% (w/v) agarose gels at 4 V/cm and visualized using u.v. transillumination at 302 nm after staining with an aqueous solution of 0.5/~g/ml ethidium bromide and destaining in deionized water for 1 h. RESULTS AND DISCUSSION R P 4 plasmid t r a n s f e r was examined in surface water samples i n c u b a t e d u n d e r a variety o f conditions (Tables 1-3). T h e R P 4 plasmid a n d its P. fluorescens host have been used in previous studies o n bacterial c o n j u g a t i o n in soil (van Elsas et al., 1988, 1990; Trevors a n d Berg, 1989). Analysis o f five t r a n s c o n j u g a n t isolates for physical presence o f the R P 4 plasmid revealed a single R P 4 plasmid b a n d o n agarose gels, t h a t exhibited electrophoretic mobility identical to the R P 4 d o n o r plasmid (Fig. 1). Previous r e s e a r c h ' u s i n g D N A p r o b i n g o f selected bacterial colonies containing the R P 4 plasmid verified t h a t the plasmid observed in agarose gels, hybridized with p r o b e D N A c o n s t r u c t e d f r o m R P 4 (van Elsas et al., 1989). This confirmed t h a t g r o w t h o n the selective agar m e d i u m can be used as a criterion tO assess physical presence o f the R P 4 plasmid. Sterile Ultrapure w a t e r devoid of n u t r i e n t s a n d trace elements served as a control. A f t e r 1 day

Table 1. Survival of P. fluoreseens donor and recipient cells, and appearance of transconjugant cells in surface water samples after 1 day at 22°C Logcfu/ml* Total Treatments Donors Recipients Transconjugants cfu Sterile ultrapure water (control) 4.17 4.30 [0]'[" ND:~ Surface water Filtered 5.81 7.17 10] ND Filtered, bentonite 5.75 7.13 [0] ND Filtered, nutrients 6.01 7.65 [0] ND Filtered, bentonite, nutrients 5.17 7.40 [0] ND Not filtered 4.52 5.50 [0] 6.66 Not filtered, nutrients 5.17 6.26 [0] 8.50 Not filtered, bentonite 4.80 5.66 [0] 5.95 Not filtered, bentonite, nutrients 5.00 5.90 [0] 8.15 *Values are expressed as means of 4 plate counts from duplicate experiments. t[0]Arithmetic zero: below the limit of detection (about log 1.0 per ml). ~ND (not determined) as donor and recipient cells were added to sterile filtered surface water or ultrapure water. Uninoculated unfiltered surface water samples (controls) at day 1 contained log 6.22 cfu/ml or log 5.82 cfu/ml (bentonite amended), whereas nutrient-amended controls contained log 8.3 cfu/ml. No cfu resistant to Km + Tc or Rp were recovered. Uninoculated filtered surface water samples were sterile.

Bacterial conjugation in water

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Table 2. Survival of P. fluorescens donor and recipient cells, and appearance of transeonjugant cells in surface water samples after 3 days at 22°C

Log cfu/ml* Treatments Sterile ultrapure water (control) Surface water

Donors 3.71

Recipients 3.00

Transconjugants [0]l"

Total cfu ND$

Filtered 5.25 6.83 '[0] ND Filtered, bentonite 5.37 5.99 [0] ND Filtered, nutrients 6.53 4.00 [0] ND Filtered, bentonite, nutrients 4.26 6.04 10] ND Not filtered 3.85 4.24 l0] 6.25 Not filtered, nutrients 6.07 6.28 4.05 8.49 Not filtered, bentonite 4.54 3.06 [0] 6.04 Not filtered, bentonite, nutrients 5.82 5.80 3.30 6.60 *Values are expressed as means of 4 plate counts from duplicate experiments. i'[0]Arithmetic zero: below the limit of detection (about log 1.0 per ml). ~:ND (not determined) as donor and recipient cells were added to sterile filtered surface water or ultrapure water. Uninoculated unfiltered surface water samples (controls) at day 3 contained log 6.66 cfu/ml or log 6.23 cfu/ml (bentonite amended), whereas nutrient-amended controls contained log 8.6 cfu/ml. No cfu resistant to Km + Tc or Rp were recovered.

i n c u b a t i o n in this n u t r i e n t starved e n v i r o n m e n t , d o n o r a n d recipient cell n u m b e r s decreased to log 4.17 a n d 4.30, respectively (Table 1). A f t e r 3 days, respective cell n u m b e r s decreased to log 3.71 a n d 3.00 (Table 2). C o n t i n u e d i n c u b a t i o n u p to 10 days did n o t cause any further decrease in cell n u m b e r s in these samples (Table 3). N o t r a n s c o n j u g a n t s were recovered from these w a t e r samples after 1, 3 or 10 days incubation. Different t r e a t m e n t s were used to assess d o n o r a n d recipient survival a n d a p p e a r a n c e o f t r a n s c o n j u g a n t s over a 10 day period. O f all treatments, transconjugants were detected after 3 days (Table 2) in n o n filtered surface water a m e n d e d with 1/10 s t r e n g t h T Y b r o t h a n d in non-filtered water s u p p l e m e n t e d with 1/10 T Y b r o t h a n d bentonite, a n d after 10 days in non-filtered water samples s u p p l e m e n t e d with bentonite only (Table 3). T h e presence o f 0.1% (v/v) b e n t o n i t e clay caused a slight decrease in the log No. t r a n s e o n j u g a n t s f r o m 4.05 to 3.30 after 3 days (Table 2). However, by day 10, n o t r a n s c o n j u g a n t s

were recovered from this t r e a t m e n t (Table 3). The occurrence o f t r a n s c o n j u g a n t s after 3 days, b u t n o t after 10 days, m a y indicate loss o f the R P 4 plasmid, especially u n d e r s u b - o p t i m a l conditions of temperature a n d nutrients. In a d d i t i o n , absence o f antibiotics which exert a selective pressure o n the organism, m a y lead to plasmid loss. Viable d o n o r cells were recovered, b u t viable recipient cells were n o t recovered. F u r t h e r m o r e , R P 4 plasmid transfer was only detected in unfiltered (non-sterile) surface water (Tables 2 a n d 3). I n addition, the survival o f t r a n s c o n j u g a n t s a p p e a r e d to be less t h a n d o n o r cells. F o r example, in the unfiltered, n u t r i e n t a m e n d e d water samples, the n u m b e r o f d o n o r cells decreased f r o m log 6.07 to 5.22 between days 3 a n d 10 whereas t r a n s c o n j u g a n t n u m b e r s d r o p p e d from log 4.05 to a n undetectable level d u r i n g the same time. A similar t r e n d was also o b s e r v e d for unfiltered samples a m e n d e d with T Y b r o t h a n d bentonite. This suggested t r a n s c o n j u g a n t cells m a y survive for shorter periods o f time unless a d d i t i o n a l nutrients are present

Table 3. Survival of P. fluorescens donor and recipient cells, and appearance of transconjugant cells in surface water samples after 10 days incubation at 22°C Log cfu/ml* Treatments Sterile ultrapure water (eontroO Surface water

Donors 4.38

Recipients 3.34

Transconjugants [0lt

Total cfu ND~

Filtered 4.37 4.67 [0] ND Filtered, bentonite 4.89 4.71 [0] ND Filtered, nutrients 2.40 7.04 [0] ND Filtered, bentonite, nutrients 4.15 5.60 [0] ND Not filtered 2.91 2.15 [0] 5.36 Not filtered, nutrients 5.22 5.09 [0] 5.54 Not filtered, bentonite 5.47 5.21 0.79 7.51 Not filtered, bentonite, nutrients 4.95 [0] [0] 8.06 *Values are expressed as means of 4 plate counts from duplicate experiments. t[0]Arithmetic zero: below the limit of detection (about log 1,0/ml). :~ND (not determined) as donor and recipient cells were added to sterile filtered surface water or ultrapure water. Uninoculated unfiltered surface water samples (controls) at day 10 contained log 6.36 cfu/ml or log 7.07 cfu/ml (bentonite amended), whereas nutrient-amended controls contained log 8.6 cfu/ml. No cfu resistant to Km + Tc or Rp were recovered.

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2

RP4

CHR

Fig. 1. Agarose gel electrophoresis. Lane 1, RP4 plasmid from Pseudomonas sp. donor cell. Lane 2, RP4 plasmid DNA isolated from 1 of 5 different transconjugants isolated after l0 days from an unfiltered water sample amended with bentonite. The 4 other isolates also yielded the same plasmid profile on agarose gels. CHR is chromosomal DNA. in the environment or other factors change to provide optimum conditions for survival and possibly subsequent formation of secondary transconjugants. Filtered surface water lacked indigenous microorganisms, and therefore competition for available nutrients was minimal and cell-to-cell contact required for conjugation should be optimal. However, the lack of competition did not enhance RP4 plasmid transfer, as no transconjugants were recovered from these treatments. Possibly, filtration of water samples removed soil particles and other particulate matter which provided surfaces for cell-to-cell contact to occur on, which is necessary for conjugal RP4 transfer. In the two treatments where transconjugants were detected, good survival of donor and recipient cells was observed during the incubation period. The various treatments were sampled three times over the 10 day period to disturb water samples as little as possible. The 22°C incubation temperature is about 6-8°C below the optimum temperature (28-30°C) for growth and conjugation in the P s e u d o m o n a s strains; however, it is a common environmental temperature in Canada. In a previous study in our laboratories (van Elsas et al., 1988) RP4 plasmid transfer in soil was studied

between the P s e u d o m o n a s donor and recipient strains used in the present study. It was observed that RP4 plasmid transfer was highest in the rhizosphere soil of Triticum aestivum var. Siceo (wheat) plants. The plasmid transfer frequencies and survival of donor and recipient strains decreased significantly at increasing distances (2-10mm) from plant roots. In addition, RP4 plasmid transfer was higher in soil initially inoculated with higher cell numbers. Little information is available on the influence of donor and recipient cell numbers on conjugation in natural environmental samples. In the present study, the inoculum size of log 6-7 represents an intermediate number of cells. The initial introduction of more or less cells and the ratio of donor to recipient cells would have a pronounced effect on the number of transconjugants recovered from water samples (Trevors and Starodub, 1987). No P s e u d o m o n a s transconjugants were detected in similar experiments conducted with log 4 cfu inocula per ml even though donor and recipient cells survived (data not shown). This illustrates the need for relatively high numbers of donor and recipient cells required for cell-to-cell contact and conjugal plasmid transfer. The organism survival trends and numbers of transconjugants observed in water samples provides data only under the conditions used in this study. Different water samples subjected to various conditions of temperature, pH, nutrients, static vs mixed water may provide different results. In addition, different conjugative plasmids and donor/recipient strains would influence the transfer frequencies. The results of this study are interesting in view of the fact that agricultural soils may be fertilized with animal waste and sewage that can contain a high density of bacterial cells that may act as donors of plasmid(s) and recipients in bacterial conjugation (Gealt et al., 1985; Mach and Grimes, 1982). As increasing importance is being placed on the use of microorganisms and/or microbial products in environmental biotechnology, it is necessary to develop procedures, and study natural environments where gene transfer may occur (Stotzky and Krasovsky, 1981). Only limited research has been completed on microbial survival (Trevors et al., 1989, 1990) (especially with genetically engineered organisms) and the influence of the environment on survival of introduced organisms and acquisition of genes by microorganisms (Stotzky and Krasovsky, 1981; Trevors et al., 1987). The present investigation also demonstrated the ability of bentonite clay and nutrients to enhance RP4 plasmid transfer in unfiltered (non-sterile) surface water samples. The initial nutrient status of the surface water samples was not assessed in this study. However, it is likely that little available carbon was present because the small amount of TY broth added to water samples stimulated the number of transconjugants detected in unfiltered samples. At the present time, a paucity of knowledge exists on aspects

Bacterial conjugation in water o f ecology, physiology and genetics o f microbial species that inhabit soils and waters. The use of non-sterile environmental water samples offers a degree of realism compared to nutritionally rich culture media or chemostat experiments which are often used to study organism survival, plasmid transfer and stability. Furthermore, selective plating which is not useful in detecting viable but non-culturable cells was adequate in this study for enumeration o f donor, recipient and transconjugant cells. Viable cfu counts is only one approach used in the study o f microbial ecology. Methods such as total D N A extraction, D N A : D N A hybridization probing, most probable number (MPN) analysis and immunofluorescence are valuable in microbial ecology research (Trevors and van Elsas, 1989). By using a variety o f detection/enumeration techniques more complete data sets will be forthcoming on genetic interactions and organism survival in the environment. Acknowledgements--This research was supported by a NATO Collaborative Research Grant awarded to J.T.T. and J.D.v.E. Sincere appreciation is expressed to S. Sprowl for typing this manuscript. REFERENCES

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