APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1992, p. 3380-3386 Vol. 58, No. 100099-2240/92/103380-07$02.00/0Copyright © 1992, American Society for Microbiology

Expression and Transfer of Engineered Catabolic PathwaysHarbored by Pseudomonas spp. Introduced into

Activated Sludge MicrocosmsKLAUS NUBLEIN,t DORIS MARIS, KEN TIMMIS, AND DARYL F. DWYER*

National Research Center for Biotechnology, Molecular Microbial Ecology Group,Department ofMicrobiology, 3300 Braunschweig, Germany

Received 12 March 1992/Accepted 30 June 1992

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120)and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosmsthat had the level of aeration, nutrient makeup, and microbial community structure of activated sludgereactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encodeddegradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate(4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateraltransfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 106 to 107 bacteria per mlof activated sludge declined to stable population densities of 104 to 10' bacteria per ml. FR120 degradedcombinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenousmicroorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB wasdegraded only after the concentration of 4MB was much reduced. The indigenous microbial community waskilled when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosmscontaining FR120, the microbial community maintained a normal density of viable cells. Indigenous microbesreadily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. Infilter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P.putida UWC1 was not detectable (<10-7 transconjugants per donor cell). In contrast, pWWO-EB62, a TOLplasmid derivative which contains all functions necessary for conjugation, transferred to P. putida UWC1 at afrequency of 10-1 transconjugant per donor cell. Within the microcosms, pWWO-EB62 transferred readily toP. putida UWC1; transconjugants reached a density of approximately 103 bacteria per ml, appearing twice asfast with 4EB present as in its absence. Transconjugants arising from transfer of pFRC20P to P. puia UWC1were rarely observed.

Large quantities of industrial products and by-productsare present in the environment. Various regulatory agencies,such as the U.S. Environmental Protection Agency, havedesignated many of these compounds as toxic substanceswhich need to be removed from contaminated sites (14).Methods commonly used to accomplish this, including phys-ical containment of affected material, chemical conversions,and burning, are costly, not always effective, and sometimescreate additional pollutants. An alternative is to use thenatural biodegradative capabilities of microorganisms to de-grade pollutant compounds (16). Unfortunately, pollutantswith novel chemical structures or with substituents which arerarely or never found in nature (xenobiotics) are often resis-tant to microbial degradation. It is possible to geneticallymodify certain microorganisms to enable them to degradesome problem chemicals. This potentially important applica-tion for genetically engineered microorganisms (GEMs) haslong been appreciated (24), and several GEMs with modifiedcatabolic pathways have been constructed (27, 28).

Successful application of this technology may require thatpollutant-degrading GEMs be used in situ. Although prog-ress has been made in designing GEMs which function safely

* Corresponding author.t Present address: Department of Microbiology, Michigan State

University, East Lansing, MI 48824.

in the environment (9, 12), both the efficiency of using GEMsto degrade pollutants and their effects on the environmentneed better evaluation. Issues relevant to this evaluationinclude the ability of GEMs (i) to survive, (ii) to stablymaintain new genetic information and accomplish the task ofdegrading pollutants, (iii) to transfer novel genetic materialto other organisms, (iv) to spread beyond the target environ-ment, and (v) to negatively affect the ecosystem (21).Two bacteria, Pseudomonas sp. strain B13 FR1

(pFRC20P) (FR120) (28) and Pseudomonas putida KT2440(pWWO-EB62) (EB62) (27), were designed to degrade sub-stituted aromatic compounds. Substituted aromatics areoften present in industrial sewage, in which they can disruptthe normal biodegradative functions of indigenous microor-ganisms, thereby lowering the efficiency of waste disposalprocesses (15). For this reason, activated sludge was chosenas a target environment in which degradation of aromaticcompounds by the two modified pseudomonads was evalu-ated. We report here on their introduction into activatedsludge microcosms and on the analysis of their survival,expression of the engineered catabolic pathways, and trans-fer of plasmids containing genes of the engineered pathwaysto recipient microorganisms. Microcosms were chosen asinvestigative tools since they allow experiments with GEMsto be performed under conditions in which environmentalcomponents necessary for expression of ecosystem pro-

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FATE AND ACTIVITY OF GEMs IN MICROCOSMS 3381

TABLE 1. Bacterial strains with aromatic compounds used as sole carbon and energy sources and antibiotic resistance markersa

Pseudomonas strain Carbon source(s) Resistance Source ormarker reference

FR120 3CB, 4CB, 4MB, 4CP KM (c)b 28IE (p)c

EB62 3MB, 4MB, 3EB, 4EB, 3,4DMB 27UWC1 Benzoate RE (c) 19UWC1(pFRC20P) Benzoate BE (c) This study

TC (p)UWC1(pWWO-EB62) Benzoate, 4.I RE (c) This study

a Underlined aromatic compounds (5 mM) and antibiotics (final concentrations as follows: tetracycline, 15 jg/ml; rifampin, 100 jig/ml) were used for selectivegrowth of the respective bacteria. 3,4DMB (3,4-dimethyl benzoate) was used to enumerate EB62, since activated sludge contained fewer bacteria (10 bacteria perml) which used 3,4DMB as a carbon and energy source than bacteria which used 4EB (103 bacteria per ml). 4CB, 4-chlorobenzoate; 4CP, 4-chlorophenol; 3MB,3-methyl benzoate; 3EB, 3 ethyl benzoate; KM, kanamycin; TC, tetracycline; RF, rifampin.

b c, chromosomally encoded.c p, plasmid encoded.

cesses are present (35), while the GEMs are still maintainedin contained situations.

MATERIALS AND METHODS

Bacterial strains and plasmids. FR120 metabolizes 3-chlo-robenzoate (3CB) and 4-methyl benzoate (4MB) via a hybridortho degradation pathway (28). Although most of the intro-duced genes specifying enzymes of the hybrid pathwayhave been integrated into the bacterial chromosome, a 26-kbDNA fragment encoding the gene for the 4-methyl-2-enelac-tone isomerase ofAlcaligenes eutrophus JMP134 was clonedinto a pLAFR3 vector. This hybrid plasmid was designat-ed pFRC20P. EB62 metabolizes 4-ethyl benzoate (4EB)through a modified toluate degradation pathway (27).pWWO-EB62 is a conjugation-proficient TOL plasmid deriv-ative containing mutated genes for catechol 2,3-dioxygenaseand the XylS transcriptional regulator which allows thecatabolic pathway to transform 4EB. The parental strains ofthe two GEMs are, respectively, Pseudomonas sp. strainB13 (8) and P. putida KT2440(pWWO) (2). P. putida UWC1(UWC1) is a plasmid-free, restriction-negative, spontaneousrifampin-resistant derivative of P. putida KT2440. Bacteriaand plasmids are listed in Table 1.Media and culture conditions. For routine growth of bac-

terial strains and their enumeration via selective plating, M9minimal medium (18) was employed with the appropriatearomatic compounds (5 mM) as the sole sources of carbonand energy plus antibiotics (Table 1). Purified agar (OxoidL28) was used in selective media to minimize backgroundgrowth by indigenous microorganisms. Synthetic sewage(22) used in microcosms consisted of urea (30 mg/liter),peptone (160 mg/liter), and meat extract (110 mg/liter) sup-plemented with H3BO3 (0.075 mg/liter), ZnCl2 (0.012 mg/liter), MnCl2 4H20 (0.008 mg/liter), CoCl2- 6H20 (0.05 mg/liter), CuCl2 2H20 (0.002 mg/liter), NiOCl2. 6H20 (0.005mg/liter), Na2MoO4. 2H20 (0.008 mg/liter), MgSO4. 7H20(0.024 mg/liter), and NH4Fe-citrate (0.6 mg/liter) and wasbuffered (pH 7.2) with M9 buffer (18). For every experiment,bacteria were obtained from cultures maintained in glycerolat -70°C.Microcosm experiments. Microcosms consisted of 2.5-liter

glass bottles containing sterile synthetic sewage (900 ml) plusactivated sludge (100 ml) freshly obtained from a municipalsewage treatment plant and hence had a nutrient composi-tion similar to that of sewage and a microbial communityderived from an activated sludge digester. The sludge mix-ture was vigorously aerated with air passed through a sterile

filter (0.2-,um pore size; Sartorius, Gottingen, Germany) at aflow rate of 120 ml/min to accomplish activated digestion andefficient mixing. Sterile synthetic sewage was added at adilution rate of 0.05/h with a peristaltic pump. The micro-cosms were maintained at room temperature. Each type ofmicrocosm was done in duplicate; experiments were alsorepeated with activated sludge obtained on two differentdates. The data in the figures are from one representativemicrocosm.FR120 and EB62 were grown for 18 h (log phase) in

selective media (Table 1), pelleted by centrifugation (5,000rpm, 1 min), and resuspended in Luria broth (LB) (18) beforebeing added to individual microcosms to a density of 106 to107 bacteria per ml. The microcosms were then allowed toequilibrate for 7 h, after which inflow of sterile syntheticsewage began. When appropriate, the microcosms and feedwere amended with substituted benzoates, either 1 mM or4.0 mM (each) 3CB and 4MB (for experiments involvingFR120) or 2 mM 4EB (for experiments involving EB62).Microcosms without GEMs were set up in parallel as con-trols. Transfer of modified plasmids was tested by combiningGEMs with UWC1 (inoculum densities of 106 bacteria perml).GEMs, UWC1, and transconjugant bacteria were enumer-

ated as total CFU per milliliter in microcosm samples (3 ml)following dispersion of flocs by vigorous vortexing andplating out appropriate dilutions onto selective media (Table1). Gene probes were used in colony hybridization proce-dures to identify the presence of pFRC20P and pWWO-EB62 in putative transconjugant bacteria. The diversity ofprotozoans in the microcosms was monitored by phase-contrast microscopy. Heterotrophic bacteria were enumer-ated with antibiotic medium 3. Oxygen levels were deter-mined with a glass oxygen electrode (Uniprobe). Theconcentrations of aromatic compounds were determined byhigh-pressure liquid chromatography (HPLC).Colony hybridization procedures. The nucleic acid probe

for pFRC20P was a 1.4-kb fragment from the gene encoding4-methyl-2-enelactone isomerase of A. eutrophus JMP134cloned into the HindIII site of pUC18. The probe forpWWO-EB62 was a 0.5-kb fragment from the xylE geneencoding catechol 2,3-dioxygenase from EB62 cloned intothe SalI site of pUC19. DNA fragments were obtained bycutting plasmid DNA with HindlIl or Sall followed byseparation by agarose gel electrophoresis. DNA fragmentswere excised, electroeluted (18), and end labelled with[a-32P]dCTP (110 TBq/mmol; Amersham Corp.) (30 min,

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Time (days)FIG. 1. Experiments with FR120. Survival of FR120 (-) and heterotrophic bacteria (A) in activated sludge microcosms. (A) FR120 alone

was added to microcosm; (B) FR120, 3CB (E), and 4MB (-) were added to microcosm; (C) 3CB and 4MB alone were added to microcosm.

25°C) with the Klenow fragment of Escherichia coli DNApolymerase I (Bethesda Research Laboratories).

Bacterial colonies putatively containing pFRC20P andpWWO-EB62 were transferred to selective agar. Isolatedcolonies were transferred to biodyne nylon membranes (Pall,New York, N.Y.) and lysed, and the released DNA wasdenatured according to the manufacturer's instructions. Themembranes were dried and treated with prehybridizationsolution. Labelled probe was added and allowed to hybridizeto the membranes, which were then washed, dried, andexposed to X-ray film. Procedures were according to thosedescribed by Maniatis et al., with slight modifications (18).Plasmid stability and transfer of pFRC20P and pWWO-

EB62. FR120 and EB62 were grown in LB (18) and dilutedevery 24 h for 16 days into fresh medium; after approxi-mately 150 generations, dilutions of the bacterial suspensionwere spread on LB agar plates. Single colonies (200) weretested on media selective for the modified catabolic path-ways (Table 1) to determine the number of GEMs retainingthe modified plasmids.

Transfer of pFRC20P and pWWO-EB62 to UWC1 wasdetermined by filter matings. FR120(pFRC20P), EB62(pWWO-EB62), and UWC1 were grown to late log phase onselective medium, collected by centrifugation (5 x 103 rpm,1 min), and washed three times in LB. GEMs were individ-ually mixed with UWC1 (500 pl each), and the mixtures werethen centrifuged. The resulting pellets were suspended in LB(50 ,ul) and transferred to sterile membrane filters on LBagar. Following incubation (12 h at 30°C), the filters werewashed with LB (5 ml); the resulting bacterial suspensionswere serially diluted and plated on selective media (Table 1)to enumerate donor, recipient, and transconjugant bacteria.

Transfer of plasmids was also monitored in microcosmsystems that contained only sterile synthetic sewage (1 liter)and that had been inoculated with overnight cultures ofeither FR120 plus UWC1 or EB62 plus UWC1 (50 ,ul ofeach). Bacteria were enumerated by growth on selectiveagar media (Table 1).HPLC analyses. Microcosm samples were filtered

(0.22-,um pore size; Millipore), frozen (-20°C), and storeduntil analysis. The concentrations of 3CB, 4MB, and 4EB inthe samples were determined by comparison with standardsolutions by using a Beckman system gold HPLC with a C18column (Ultrasphec ODS) and UV detector (235 nm). Thesolvent was water-methanol (60:40) at a flow rate of 1

ml/min. The minimum concentrations of 3CB, 4MB, and4EB that were detectable were 1 ,uM.

RESULTS

Microcosm characteristics. In control microcosms withoutGEMs, the number of heterotrophic bacteria and the mor-phological diversity of microorganisms remained constantthroughout each experiment (data not shown). The indige-nous microbial community included a variety of protozoansdominated by Vorticella spp. and free-swimming ciliates.The pH and oxygen concentration remained at 7.5 and 3.4mg/liter (+0.6 mg/liter), respectively. Addition of GEMs,UWC1, and substituted benzoates did not affect the numberof heterotrophs (Fig. 1 and 2) or the diversity of protozoans.

Survival and growth of added bacteria. Microcosms wereinoculated with FR120 and EB62 to densities of 106 to 107bacteria per ml; densities thereafter declined to relativelystable levels of 104 to 105 bacteria per ml in microcosms towhich selective substrates were added (Fig. 1B and 2B) andin microcosms which received only synthetic sewage (Fig.1A and 2A). For the following two reasons, we concludedthat selective enumeration gave relatively accurate densitiesfor introduced microorganisms and transconjugants: (i) ap-proximately 80% of GEMs and transconjugants could bereisolated immediately following introduction into micro-cosms and (ii) the densities of indigenous bacteria in controlmicrocosms which grew on selective agar were at least 3 logunits lower than the densities of GEMs and of the transcon-jugant UWC1(pWWO-EB62) in test microcosms. Therefore,it was unlikely that indigenous microorganisms contributedto the counts of GEMs and transconjugants.

Degradation of substituted benzoates. In microcosms with-out FR120, an 8-day adaptation period was required beforeindigenous microorganisms began to degrade 3CB and 4MB(1.0 mM each) (Fig. 1C). Initially, only 4MB was degradedand by day 10 the compound was not detected (<1 ,uM)although it was continuously added via the feed. At thispoint, degradation of 3CB became noticeable, but at acomparatively lower rate, so that by day 16 the concentra-tion was reduced to only 0.2 mM. Thus, although microor-ganisms with the ability to degrade 4MB and 3CB developedfrom the indigenous microbial community, their degradativeabilities were limited in that 3CB was not degraded when4MB was present in detectable concentrations.

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FIG. 2. Experiments with EB62. Survival of EB62 (Cl), UWC1 (M), and heterotrophic bacteria (A) in activated sludge microcosms. (A)EB62 and UWC1 were added to microcosm; (B) EB62, UWC1, and 4EB (X) were added to microcosm; (C) 4EB alone was added tomicrocosm. Transconjugant bacteria (0) were formed by transfer of pWWO-EB62 from EB62 to UWC1 in the microcosms represented bypanels A and B.

FR120 had a profound effect on the degradation rates of3CB and 4MB in the microcosms. Both compounds weresimultaneously degraded, and the concentrations of bothwere less than 0.2 mM by day 5 (Fig. 1B). Thereafter, 4MBwas not detected and the concentration of 3CB remained lessthan 0.1 mM. Thus, FR120 functioned in the task for whichit was designed. Furthermore, the GEM also degraded shockloads of both chemicals. When 3CB and 4MB (4 mM each)were added to a GEM-free microcosm after 2 days of growthon synthetic sewage, black, polymeric metabolites accumu-lated, followed by an overnight decrease in the density ofviable microorganisms to <105 CFU/ml (compared withnormal densities of 107 to 108 CFU/ml; see reference 15 foran explanation of the toxic effects of combinations of chloro-and methyl-substituted aromatic compounds). When FR120was present in a parallel experiment, polymers were notevident and the microbial community remained viable atnormal densities (Fig. 3).

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FIG. 3. Survival of FR120 (-) and heterotrophic bacteria (A) inan activated sludge microcosm which was given a shock load of 3CBand 4MB (4 mM each) on day 2 (.4).

In contrast to studies with 3CB and 4MB, indigenousmicroorganisms degraded 4EB (2 mM) rapidly; degradationstarted on day 3+ and by day 9 4EB was no longer detected(Fig. 2C), although it was present in the incoming feed.When EB62 was present (Fig. 2B), 4EB was degraded onlyslightly faster than in the control microcosms not containingthe GEM.

Stability and transfer of pFRC20P and pWWO-EB62. Plas-mid stability in FR120(pFRC20P) and EB62(pWWO-EB62)was assessed in vitro by using nonselective conditions toobtain an indication of whether plasmid loss from bacteria inthe microcosms could be expected. Following approxi-mately 150 generations of growth in LB, 100 and 96% ofbacterial colonies from broths inoculated with FR120 andEB62, respectively, retained the ability to metabolize 4MBand 4EB. Thus, potential loss of plasmids from host cells inthe microcosms was considered insignificant, especially incases in which microcosms contained substrates selectivefor plasmid-encoded degradative pathways.

Frequencies of plasmid transfer (transconjugants per do-nor cell) were obtained by filter mating between plasmid-containing GEMs and the recipient microorganism, UWC1.The frequency of transfer of pFRC20P was undetectable(< 10-7), whereas that for pWWO-EB62 was 10-1. This wasexpected, since pWWO-EB62 is a broad-host-range plasmidwhich transfers to a variety of gram-negative bacteria (4),whereas pFRC20P, a pLAFR3 derivative, does not containthe functions necessary for independent conjugation (32).Within microcosms, transconjugants formed by transfer of

pWWO-EB62 from EB62 to UWC1 were readily detected(Fig. 2A and B). Randomly chosen colonies of putativetransconjugants all tested positive for the gene for catechol2,3-dioxygenase following colony hybridizations with thegene probe; colonies of UWC1 and 4EB-degrading hetero-trophic bacteria obtained from GEM-free control micro-cosms tested negative. When 4EB was resent, transconju-gants occurred at a density of 103 to 104bacteria per ml byday 2 (Fig. 2B), but without 4EB, similar densities were notreached until day 4 (Fig. 2A). This could be due to fastergrowth of transconjugants with 4EB as the substrate. Inter-estingly, in each case the density of transconjugants (103 to

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FIG. 4. Formation of transconjugant bacteria (0) due to conju-gative transfer of pWWO-EB62 from EB62 (a) to UWC1 (M) inmicrocosm systems to which activated sludge was not added.

104 bacteria per ml) remained below that (104 to 105 bacteriaper ml) of the parent strains, EB62 and UWC1.

Conjugative transfer of pWWO-EB62 in microcosm sys-

tems to which activated sludge was not added was alsomeasured. EB62 and UWC1 were inoculated to densities of104 bacteria per ml; by 10 h, they had increased to 107bacteria per ml. Transconjugants began to appear at 30 h andreached a final density similar to that of the parent bacteria(Fig. 4). This result is in contrast to that observed inmicrocosms containing activated sludge, in which the den-sity of transconjugants was always well below that of theparent strains, and suggests that activated sludge microor-ganisms detrimentally affected either transfer of pWWO-EB62 to UWC1 or growth of the transconjugants.

Putative transconjugants formed by transfer of pFRC20Pfrom FR120 to UWC1 were detected in microcosms atdensities approximating 102 bacteria per ml. Colonies were

randomly chosen throughout the experiment and probed todetermine whether they contained pFRC20P. Of 153 colo-nies that were probed for the gene for 4-methyl-2-enelactoneisomerase, 35% were positive. Colonies ofPseudomonas sp.

strain B13 and heterotrophic bacteria obtained from controlmicrocosms did not hybridize to the probe. The remaining65% of the colonies were of bacteria which used benzoate as

a carbon and energy source and which were resistant torifampin and tetracycline; these microorganisms were

present within control microcosms at the same frequency as

that in test microcosms (data not shown).

DISCUSSIONBefore GEMs can be used to degrade environmental

pollutants in situ, it is necessary to obtain informationconcerning their fate, their efficiency in degrading pollutants,and the possible effects that they may have on the environ-ment. In many cases, these investigations can be made byusing appropriate microcosms (20, 35). Microcosms for soil(5-7, 23, 36), aquatic (1), sediment (38), aquifer (13), andsewage (17) ecosystems have been used to predict the fate ofGEMs introduced into these environments. Analysis ofthese studies, as well as the results obtained in our work,suggests that microcosms may retain enough of the complex-

ity of the target ecosystem to allow for appropriate investi-gations of specific factors related to environmental introduc-tions of GEMs.The natural environment contains abiotic (e.g., tempera-

ture and nutrient availability) and biotic (e.g., predation andcompetition) stresses which limit the survival and growth ofintroduced bacteria (29, 33). The activated sludge micro-cosms used in the present study maintained important bioticstresses which could limit colonization and survival of theintroduced GEMs (protozoan predators as well as possiblecompetitors, including bacteria able to degrade 3CB, 4MB,and 4EB). In spite of these limitations, FR120 and EB62established growing populations. The densities of FR120 andEB62 in microcosms containing selective substrates weresimilar to those occurring in microcosms to which thesesubstrates were not added. Thus, the GEMs survived inconditions in which they had no obvious competitive advan-tage over indigenous microorganisms.The experiments also verified that the GEMs degraded

aromatic compounds via expression of engineered catabolicfunctions under in situ conditions. FR120 performed well bysimultaneously degrading 3CB and 4MB, thereby protectingthe microbial community of the activated sludge from shockloading of the compounds. In a recent report (24a), FR120degraded 3CB and 4MB in whole-core sediment micro-cosms, demonstrating that the degradative activity can beexpressed in other environments as well. In our experi-ments, EB62 did not greatly increase the rate of degradationof 4EB because of effective degradation by microorganismswhich were indigenous to activated sludge. On the otherhand, if we look at soil microcosms to which EB62 has beenadded, the GEM degrades 4EB while indigenous microbeslack the ability to do so (26). When taken together, theseresults suggest that well-designed GEMs can be potentiallyuseful agents for degrading pollutants in ecosystems such aswaste streams and polluted soils. In a recent publication(19), it was reported that the GEM P. putida UWC1(pD10)failed to degrade 3CB in a model activated sludge unit,whereas it degraded 3CB in vitro. In this case, the degrada-tive activity observed in vitro is presumably not expressed innature. These experiments emphasize that microcosms havea use in screening GEMs and other bacteria for use inbioremediative applications.The present study also gives insight into the type of

conditions necessary for active in situ degradation of 3CBand 4MB by FR120. In the microcosms, degradation oc-curred following a lag (Fig. 1), which contrasts with rapiddegradation in vitro (28) and suggests that the GEM prefer-entially used nutrients initially available in the syntheticsewage and that this suppressed utilization of substitutedbenzoates until after the microbial community became sta-bilized and the nutrient influx became growth limiting. Asimilar phenomenon is observed when microbial inocula areused to enhance the biodegradation of chemicals in theenvironment (10). Other factors which can inhibit biodegra-dation of chemicals in the environment include chemicalconcentrations which are too low to induce enzymes ofcatabolic pathways and insufficient densities of microbialdegraders (10, 25, 39). These factors probably did not play arole in the present work since (i) the degradation pathwayswere initially induced by growth of bacterial inocula onminimal medium containing substituted benzoates and (ii)the inoculum density (106 bacteria per ml) was more thansufficient for degradation (compare with Fig. 1B [degrada-tion of 3CB and 4MB at densities of 104 to 105 bacteria perml]).

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Transfer of pWWO-EB62 from EB62 to UWC1 occurredduring filter matings (frequency of 10-1) and in activatedsludge microcosms. Selective pressure for pWWO-EB62, inthe form of adding 4EB to the microcosms, caused transcon-jugants to appear earlier (Fig. 2B) than in microcosmswithout addition of 4EB (Fig. 2A). This was possibly due toan increase in the rate of either plasmid transfer or thegrowth of transconjugants. In either case, the final density ofthe transconjugants was always one log unit less than that ofthe parent bacteria. In contrast, the final density of thetransconjugants in microcosm systems containing only cul-tures of EB62 and UWC1 was the same as that of the parentstrains (Fig. 4). This suggests that the microbial communityof the microcosms stabilized the densities of EB62, UWC1,and transconjugants in a ratio different from that found insystems containing only the three bacteria. Thus, environ-mental conditions can affect bacterial growth dynamics andinteractions in ways which may not be evident from in vitrostudies.pFRC20P is not self-transmissible, and hence its transfer

in a filter mating to UWC1 in the absence of other organismswas not detectable. The infrequent transfer of pFRC20P toUWC1 which was observed in the microcosms presumablyoccurred after acquisition by FR120 of mobilizing plasmidsfrom indigenous microorganisms. Mobilizing plasmids arepresent in activated sludge microorganisms, and similarresults have been reported elsewhere (19).The results from this study relate to three of the five issues

requiring evaluation when considering the use of GEMs inthe environment. (i) The GEMs were able to survive andgrow under environmental conditions, and (ii) most impor-tantly, they degraded the target chemicals via modifiedcatabolic pathways. (iii) In addition, transfer of recombinantgenes in the form of the modified plasmid pWWO-EB62 wasreadily observed. In situ horizontal transfer of plasmid DNAhas been demonstrated via mobilization (11, 17), conjugation(3, 34, 37), and transduction (30). Such observations have ledto the conclusion that as long as horizontal transfer isperceived to constitute a risk, recombinant DNA needs to beincorporated into GEMs in ways which reduce the potentialfor its transfer (9). The results reported here reinforce thisconcept and emphasize the necessity of developing newmethods for this purpose (e.g., see reference 12).The present study was not specifically designed to discern

potential impacts by GEMs on the ecosystem, although onecan argue that transfer of genetic material from the GEMs toindigenous organisms constitutes an impact (20). Ecologicalimpacts may also arise as alterations in the structure ofindigenous microbial communities or of ecological pro-cesses. In recent reports (31, 38), GEMs introduced intomicrocosms did not discernibly affect the various functionalgroups of indigenous microbes. Microcosms can be espe-cially useful in analyzing the potential actions of GEMs sincesuch factors are best studied with contained and easilymanipulated systems.

ACKNOWLEDGMENTS

This work was supported by a Biotechnology Action Programgrant from the European Community (BAP-0417-D), the GermanMinistry of Research and Technology (BMFT Vorhaben 0319-433A), and the Fonds der Chemischen Industrie.We thank J. Egestorff for help with the HPLC analyses, R. Pipke

and K. Davies for the gene probes, and U. Karlson for help with thefigures.

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