Vol. 56, No. 8APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1990, p. 2471-24790099-2240/90/082471-09$02.00/0Copyright ©) 1990, American Society for Microbiology

Gene Escape Model: Transfer of Heavy Metal Resistance Genesfrom Escherichia coli to Alcaligenes eutrophus on

Agar Plates and in Soil SamplesEVA TOP,1'2 MAX MERGEAY,2* VERSTRAETE'

Laboratory of Microbial Ecology, Faculty ofAgricultural Sciences, State University of Ghent, Coupure Links 653,B-9000 Ghent,' and Laboratory of Genetics and Biotechnology, Studiecentrum voor

Kernenergie (S.C.K.), Boeretang 200, B-2400 Mol,2 Belgium

Received 22 February 1990/Accepted 29 May 1990

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding forplasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soilsamples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipientstrain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavymetals represents escape of the czc genes. The two plasmids into which this DNA fragment has been clonedpreviously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 andthe nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30°C, the direct mobilizationof pDN705 occurred at a frequency of 2.4 x 10-2 per recipient, and the mobilization of the same plasmid bymeans of the IncPl conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or

in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x i0- and 2 x 10-5 per

recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a

frequency of 10-7 to 10-8 and only by means of plasmid pULB113. The direct mobilization of pDN705 was

further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20°C were

highest in the sandy-loam soil (10-5 per recipient) and were enhanced in all soils by the addition of easilymetabolizable nutrients. For nonsterile soils, transfer of pDN705 at 28 and 20°C was observed in thesandy-loam soil, only when the soil was amended with nutrients. Frequencies varied between 1.5 x 10-8 and1.5 x 10-6 per recipient. The results demonstrate that even genes incorporated into nonmobilizable plasmidscan be exchanged between two different genera and that the presence of broad-host-range plasmids in putativerecipients among soil bacteria could increase the risk of gene dissemination in case of release of geneticallyengineered microorganisms. The results also reveal that in certain soils, environmental conditions andparticularly nutrient levels are conducive to gene transfer.

Recently, considerable attention has been given to theintroduction of genetically engineered microorganisms(GEMs) into soil ecosystems. Possible applications are thecontrol of plant diseases and insect pests and the removal ofxenobiotic compounds (53). The deliberate release of theseorganisms may have unpredictable consequences for theenvironment, and extensive scientific research studying mi-crobial interaction and especially the fate of introducedstrains and their recombinant DNA is necessary. On theother hand, accidental release of GEMs from laboratories(mainly Escherichia coli strains) and industrial installationsmust be taken into account as well. Concern has beenexpressed not only about the survival of the GEMs in theenvironment but also about the dissemination of their genet-ically engineered DNA sequences. Indigenous microorgan-isms acting as recipient strains for the genetically engineeredDNA sequences acquire new information, and the acquisi-tion of plasmids sometimes seems to cause physiologicalalterations (19, 47). Both intra- and interspecific transfer ofgenetic information have been extensively demonstrated invitro, and plasmids or other translocatable elements havebeen found in many genera of soil bacteria (12, 17, 28, 30,35). However, the link between these two observations-invitro gene transfer and the presence of transferable extra-chromosomal genetic information in different genera-has

* Corresponding author.

been pointed out only a few years ago. An early study (61)observed plasmid transfer between E. coli strains in sterilesoil. Schilf and Klingmuller (41) noted a transfer frequencyof 10' in matings between E. coli and the indigenousbacterial populations of an agricultural soil. During the lastthree years, more studies demonstrating gene transfer in soilhave been reported. Conjugation is the most profoundlystudied mechanism of gene transfer in soil and will also beexamined in this study. Most model systems assayed in-traspecific or intrageneric gene transfer. Conjugation be-tween E. coli strains in sterile and natural soils has beendemonstrated several times (20, 51, 55, 61). Intragenericplasmid transfer has also been observed between bacilli insterile and nonsterile soil (56, 58), between pseudomonads insoil (54, 57, 58, 59) and the rhizosphere (57, 58), and betweenStreptomyces strains in sterile soil (2, 36, 37). Less informa-tion is available on conjugal gene transfer between bacteriaof different genera in soil: intergeneric plasmid transfer hasbeen observed in sterile soil (13, 38), and plasmid mobiliza-tion from a genetically engineered E. coli strain to theindigenous soil microorganisms has been detected in naturalsoil (14).The aim of this study was to establish a model system for

assaying the intergeneric transmission and expression ofcloned genes in both sterile and nonsterile soil samples. Thiswas carried out by conjugation with broad host range plas-mids as cloning vectors and as mobilizing plasmids. E. coli

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TABLE 1. Bacterial strains

Strain Relevant markers Plasmid Reference(s)(parental strain) or source

A. eutrophusAE 104 Plasmid-free derivative of A. eutrophus CH 34, 30

CdS Zns CoBS CoAs Nis Aut+, prototrophicAE 703 (AE 104) RP4 This workAE 704 (AE 104) pULB113 This work

E. coli K-12CM 214 (MXR) F- lac pro thiA recA galE pULB113 60CM 140 (J53) pro met (X) RP4 C. I. KadoCM 469 (S17/1) pro thi hsdS rpsL::RP4 (tra+) pDN705 44, 33CM 485 (HB101) pro leu rpsL lacY recA hsdR hsdM pMOL149 3, 33CM 746 (BHB2600) met hsdR hsdM pDN705 This work

K-12 was used as the donor strain in this simulation of theaccidental release of laboratory strains. Alcaligenes eutro-phus was chosen as the recipient strain, because it is a goodcolonizer of polluted soils and thus perfectly apt to survive ina variety of harsh conditions (6, 7). For the cloned genes, czcwas used, a 9.1-kilobase (kb) fragment conferring resistanceagainst cobalt, zinc, and cadmium and originating frompMOL30 (240 kb), one of the two megaplasmids of A.eutrophus var. metallotolerans CH 34 (6, 30, 33, 34). Asthese czc genes are not expressed in the donor strain of thismodel, E. coli K-12, but are expressed as soon as they haveentered the recipient, A. eutrophus AE 104, the assaysimulates the escape of originally unexpressed genes into anappropriate recipient strain. By means of this model, theinfluence of parameters like storage of the cloned genes (inmobilizable and nonmobilizable vectors), type of soil, andaddition of nutrients on gene transmission was investigated.

MATERIALS AND METHODS

Bacterial strains and plasmids. The strains used in thisstudy, with their plasmids and main characteristics, arelisted in Table 1; the plasmids themselves are listed in Table2. Bacterial strains were maintained in liquid nitrogen. Theczc fragment is a 9.1-kb EcoRI fragment of pMOL30 confer-ring resistance to cadmium, cobalt, and zinc (30, 33, 34) andhas been cloned into the EcoRI site of pRK290 and pBR325(Table 2). Strains AE 703 [AE104(RP4)] and AE 704[AE104(pULB1113)] were obtained by matings with E. coliCM 140 and CM 214, respectively.Media. Liquid cultures of all strains were grown in 869

broth (27) supplemented with tetracycline (20 ,ug/ml) when

TABLE 2. Plasmids

Plasmid Characteristics Refer-ence

RP4 60 kb, broad host range, Tra+, IncPl, TcR 5KmR ApR

pULB113 68 kb, broad host range, Tra+ IncPl, TcR 60KmR ApR, Mu3A

pDN705 29 kb, broad host range, Tra- Mob', 33IncPl, TcR; CdR ZnR CoR (not expressedin E. coli); equal to pRK290::czc

pMOL149 21.2 kb, Tra- Mob- TcR ApR, CdR ZnR 7CoR (not expressed in E. coli); equal topBR325 with a 16-kb fragment ofpMOL30 (four EcoRI fragments includingthe 9.1-kb czc sequence)

appropriate. Donor E. coli cells were enumerated on 869agar plates (27) with tetracycline (20 ,ug/ml). The plates wereincubated at 42°C to counterselect A. eutrophus. The me-dium of Schatz and Bovell (40) supplemented with 0.2%azelate was used to enumerate A. eutrophus AE 104 at 28°C.AE 703 and AE 704 were grown on the same mediumcontaining tetracycline (20 Fxg/ml). Transconjugants in platematings and in sterile soil were enumerated on Tris medium(30) supplemented with 0.2% azelate and 1 mM Zn2+. Aselective medium that completely prevented growth of theindigenous soil bacteria and still revealed transconjugantswas not found. The most suitable medium appeared to beTris azelate with 2 mM Zn2+ and 0.8 mM Cd2 . Cyclohex-imide (200 ,ug/ml) was added to suppress fungal growth.Nevertheless, replica plating on Schatz and Bovell azelatewith tetracycline and on Tris azelate with Co2+ 0.6 mM wasneeded to distinguish between indigenous resistant organ-isms and real A. eutrophus transconjugants. All stock solu-tions (tetracycline, kanamycin sulfate, cylcoheximide, ampi-cillin [10 mg/ml], ZnCl2 or ZnSO4 7H20 [1 M], CdCl2 H20or CdSO4- H20 [1 M], and CoCl2 6H20 [1 M] orCoSO4- 7H20[0.1 M]) were filter sterilized into sterile testtubes.

Soils. Three types of soil were used in this study. Tillegemsoil is a sandy soil (particle size, >50 ,um, 92.7%; 2 to 50 pLm,2.7%; 0 to 2 ,Im, 3.6%; organic matter, 3.11%; CaCO3, 0%[pH = 5.2]) with a moisture content at field capacity of16.5% for dry soil. Tiegem sandy-loam soil (organic matter,3.4% [pH = 7]) has a moisture content at field capacity of24% on dry soil. Merapi is a clay soil with a clay content of±30% and pH - 6.3 (organic matter, ±3%; moisture contentat field capacity, 50% for dry soil).

Freshly collected soil was stored in closed plastic bagsduring the experiments. Before use, soils were sievedthrough a 2-mm-pore-size sieve. To sterilize them, soils wereautoclaved twice, with a 1-day interval. All experimentswere performed at a moisture content equal to 75% of therespective field capacities. The nonsterilized soil wasbrought to this moisture level 2 weeks before the start of theexperiment to avoid a sudden extensive growth of theindigenous organisms at the beginning of the test.

Plate matings. Parental strains that harbored a plasmid (allplasmids code for Tc resistance) were grown overnight in 5ml of 869 broth supplemented with 20 ,ug tetracycline per ml.Plate matings were performed as previously described (22).

Verification of transconjugants. Plasmid transfer to therecipient strains was first verified by testing all plasmid-bound resistances with appropriate selective media. After

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TRANSFER OF PLASMID-BORNE HEAVY METAL RESISTANCE GENES

TABLE 3. Transfer of czc genes in plate matings

Expt no. Vector and donor Helper E. coli Recipient TransferE. coli strain strain (plasmid) A. eutrophus strain frequency'

pDN705 (Tra- Mob')la CM 469 AE 104 2.4 x 10-2lb CM 469 AE 104 4.1 x 10-42a CM 746 CM 140(RP4) AE 104 6.0 x 10-42b CM 746 CM 214(pULB113) AE 104 1.0 X 10-33a CM 746 AE 104(RP4) 1.9 x 10-53b CM 746 AE 104(pULB113) 1.8 x 10-54 CM 746 AE 104 <3.3 x 10-8

pMOL149 (Tra- Mob-)5a CM 485 CM 140(RP4) AE 104 <5.6 x 10-95b CM 485 CM 214(pULB113) AE 104 3.2 x 10-76a CM 485 AE 104(RP4) <5.2 x 10-96b CM 485 AE 104(pULB113) 2.0 x 10-87 CM 485 AE 104 <5.1 x 10-9

a Number of transconjugants (Zn+) per number of recipients (average of two to three experiments).b Performed at 20°C.

this purification, transconjugants were screened for thepresence of plasmids by plasmid isolation. Plasmids wereisolated by a modified method of Kado and Liu (16). Theovernight-grown culture was centrifuged in an Eppendorfcentrifuge (12,000 x g, 3 min) and the pellet was suspendedin 0.1 ml of E buffer. Cells were lysed by adding 0.2 ml oflysing solution and gently mixing till the suspension becamereally viscous. The solution was heated at 65°C for 60 to 90min in a water bath. A 0.6-ml quantity of phenol-chloroformsolution (1:1, vol/vol) was added. The solution was brieflyshaken and centrifuged at 4°C in an Eppendorf centrifuge(12,000 x g, 15 min).Agarose electrophoresis was performed in a horizontal

system for submerged gel electrophoresis (gel contained 8%agarose) at 5 V/cm for about 4 h. Photographs were takenwith a Polaroid camera, type 55 film, and a shortwave UVlight source.

Soil experiments. Liquid cultures of both donor and recip-ient, grown overnight in 869 broth, were centrifuged (9,000x g, 10 min), the supernatant was decanted, and the cellswere suspended in 0.85% saline or in 869 broth if enrichmentof the soil was intended. If necessary, soil moisture contentwas adjusted with sterile deionized water. Transfer andsurvival studies were performed in sterile 200-ml glass potswith 100 g of soil, and the pots were covered with aluminiumand polyethylene foil and stored in a larger container inwhich a free water surface caused a relative humidity of100%. The whole was incubated at constant temperature. Atsubsequent times, soil samples (1 g) were taken and dilutedin saline. The first dilutions (1/10 or 1/5) were vigorouslyagitated for at least 30 s on a Vortex mixer; all subsequentdilutions were agitated for 5 s. The appropriate dilutionswere plated out in duplicate on the media described above.Duplicate soil samples were used for each treatment.COD measurement. The soil suspension (100 g in 150 ml of

deionized water) was shaken for 1 h and centrifuged. Thesupernatant was filtered through a folded filter (Schleicher &Schuell, Inc., 595 1/2) and the soluble chemical oxygendemand (COD) of the filtrate was measured by the standarddichromate method previously described (49). The Student ttest was used to calculate significant differences.

RESULTSTransfer of the heavy metal resistance genes in plate mat-

ings. All matings listed in Table 3 were performed in order to

calculate the frequencies of transfer of the czc genes. Thedirect mobilization of the Tra- Mob' plasmid pDN705 wascarried out by using the donor strain S17/1 (Table 1) harbor-ing the tra functions of plasmid RP4 in its chromosome. Thisstrain can mobilize pDN705 at a high frequency (Table 3).Mobilization of the plasmid was also performed by usinganother E. coli donor strain lacking functional tra genes. RP4and RP4::Mu3A (pULB113) were used as mobilizing plas-mids. Mobilization of pDN705 in a triparental cross wasinvestigated by using E. coli CM 140 or CM 214, containingthe plasmids RP4 and pULB113, respectively.pDN705 could also be transferred from donor to recipient

in a biparental cross if the recipient harbored the mobilizingplasmid RP4 or pULB113. Those recipient strains [AE104(RP4) and AE 104(pULB113) (Table 1)] were able toacquire the heavy metal resistance coded by pDN705 bycapturing the whole plasmid. This phenomenon has beencalled retromobilization, retrotransfer, or shuttle transfer(29, 42, 50). The low transfer frequency in comparison withthe direct mobilization frequency (Table 3) seems to berelated to incompatibility between the IncPl-derivedpDN705 and the RP4 (or RP4::Mu3A) plasmids.

If the czc genes were inserted in the Tra- Mob- plasmidpBR325 (the resulting plasmid was called pMOL149), theycould be transferred from donor E. coli to recipient A.eutrophus by means of the mobilizing plasmid pULB113(Table 3). Because pMOL149 lacks a mob site, transmissionof the czc sequence could only be reached by integration ofthe DNA fragment in pULB113. Since the donor strain usedin this experiment, E. coli HB101, is a recA host, homolo-gous recombination is excluded. Agarose electrophoresis ofplasmid DNA in the transconjugants (Fig. 1) demonstratedthat the resulting plasmids have become larger thanpULB113, and the differences in size increment suggest atransposition of the czc genes from the recombinant vectorinto pULB113 rather than a cointegration between the twoplasmids. This could explain the rather low transfer fre-quency (Table 3), formation of cointegrates being morefrequent than transpositions requiring specific deletions ofthe vector (31). The transposition element Mu3A-derivedfrom phage Mu (9)-seems to be responsible for the trans-position of the czc genes, as mobilization could not bedemonstrated with RP4 as the conjugative plasmid (Table 3).Again, mobilization was investigated in a triparental matingand in a retromobilization configuration (Table 3).

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FIG. 1. Agarose gel electrophoresis after extraction of plasmidDNA by the method of Kado and Liu (17). Lanes: 1 and 7, CM214(pULB113); 2 and 3, AE 104 transconjugants from the tri-parental mating [AE 104 x CM 485(pMOL149) x CM 214(pULB113)] with enlarged pULB113; 4 and 5, CM 485(pMOL149);6 and 8, AE 704 retrotransconjugants from the mating [AE 704(pULB113) x CM 485(pMOL149)] with enlarged pULB113; 9,AE 704(pULB113).

As shown in Table 3, transfer frequency of the resistancegenes was much lower if they were situated on a Mob-plasmid. Nevertheless, these results demonstrate that evengenes from a Mob- plasmid in a recA host can escapetowards and become expressed in other genera.

Transfer of pDN705 from E. coli CM 469 to A. eutrophusAE 104 was further investigated in sandy, sandy-loam, andclay soils.

Transfer of the heavy metal resistance genes in sterile soil.All samples were incubated at 20°C after inoculation of theparental strains.

(i) Sandy soil (Tillegem). Figure 2 shows that the recipientpopulation survived very well and that additional nutrientswere needed to prevent E. coli from dying off. Plasmidtransfer occurred only in the presence of added metaboliz-able nutrients. Transconjugants were not detected within 1day after inoculation of both parental strains, but they wereobserved after 4 days. Transfer frequency was very low(Table 4) in comparison with that for the plate mating at thesame temperature (20°C) (Table 3). Addition of broth notonly enhanced the nutrient level of the soil but also raisedthe pH from 5.2 to 5.7, which is more favorable for conju-gation.

(ii) Sandy-loam soil (Tiegem). In Tiegem soil (Fig. 3), inboth the absence and the presence of added nutrients, theintroduced donor and recipient populations proliferated dur-ing the first 2 weeks (Fig. 2). After 5 h of incubation, notransconjugants were detected in any of the samples; how-ever, after 24 h, transfer of plasmid pDN705 was detected ata frequency of 10-5 (Table 4), which was not much lowerthan in plate matings at the same temperature (20°C) (Table3). The number of transconjugants increased slightly untilday 7 and remained roughly stable during further incubation(37 days). Addition of nutrients had no significant effect onthe transfer frequency. The growth of both strains afterinoculation in the unamended soil can be explained by theavailability of nutrients which had been set free after steril-ization of the soil sample. The COD after autoclaving washigher than before autoclaving by a factor of 10 (Table 5).

(iii) Clay soil (Merapi). As in the sandy soil, the recipientstrain survived very well in the sterile unamended soil, butthe donor strain declined rapidly during 2 weeks. (Fig. 4). No

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FIG. 2. Transfer of plasmid pDN705 and survival of donor,recipient, and transconjugant cells in sterile unamended (A) andnutrient-amended (B) sandy soil (Tillegem) at 20°C. Symbols: *, AE104; 0, CM 469; O, transconjugants.

transconjugants could be detected during this period. Theaddition of nutrients, which did not alter the pH of the soil,had a significant effect on the survival of the donor strain E.coli, and as a result, transconjugants could be isolated 1 dayafter inoculation. The COD of the unamended soil wassignificantly lower (P < 0.005) than the COD of the sandyand sandy-loam soils (Table 5).

Transfer of the heavy metal resistance genes in nonsterilesoil. (i) Sandy soil (Tillegem). Since 28 to 30°C appeared to bethe optimum temperature range for plate matings between E.coli and A. eutrophus, this temperature range was alsoconsidered most favorable for gene transfer in soil. Unlike inthe sterile soil, no plasmid transfer was detected when theautochtonous microbiota were present (Table 6). Figure 5demonstrates the poor survival of the donor strain E. coli.Even in the nutrient-amended soil, donor cells showed arapid decline in viable numbers and could not be isolatedafter 2 weeks. This poor survival may be the cause of thevery low probability of transfer of pDN705 in this soil (Table6).

(ii) Sandy-loam soil (Tiegem). The first experiment wascarried out at 28°C. The recipient-donor ratio in the inocu-lum was 20. In the unamended soil, no transfer was observedduring 2 weeks and the donor population also declinedrapidly from the first day. The addition of nutrients (1.24 mgof COD per gram of dry soil) caused growth of bothintroduced parental strains and gave rise to detection of asmall number of transconjugants after 1 and 2 days of

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TRANSFER OF PLASMID-BORNE HEAVY METAL RESISTANCE GENES

TABLE 4. Transfer of plasmid pDN705 after 1 day in different types of sterile soil at 20°C

Soil typeo

b Transfer frequency by:and nutrient statusa Donor Recpent Transconjugantsonor Recipient

Sandy (Tillegem)4.6 x 105 7.1 x 106 <2.5 x 101 <5.4 x 10-5 <3.5 x 10-6

+ 1.3 x 106 6.4 x 106 <1.2 x 101 <9.2 x 10-6 <1.9 x 10-6+ 1.9 x 106c 1.0 x 108c 5.0 x 101c 2.6 x 10-5c 5.0 x 10-7c

Sandy-loam (Tiegem)5.0 x 106 5.9 x 107 5.5 x 102 1.1 x 10-4 0.9 X 10-5

+ 1.0 X 107 1.0 X 108 1.0 X 103 1.0 X 10-4 1.0 X 10-5

Clay (Merapi)1.8 x 105 1.6 x 106 <2.5 x 101 <1.4 x 10-4 <1.6 x 10-5

+ 1.4 x 106 6.9 x 107 2.3 x 102 1.6 x 10-4 3.3 x 10-6a The amounts of nutrients added to the three soils (sandy, sandy loam, and clay) were 0.86, 1.45, and 1.45 mg of COD per g of dry soil, respectively (1 ml

of 869 broth equals 17.2 mg of COD). +, Nutrients added; -, nutrients not added.bCFU per gram of soil.c Observed after 4 days incubation of the soil.

incubation (Fig. 6; Table 6). Transfer frequency at 28°C wasvery low (1.5 x 10-8) in comparison with the frequencies inthe sterile soil at 20°C (1.0 x 10-5) and in plate mating at280C (2.4 x 10-2). To simulate better the environmentalconditions, the same mating experiment was carried out at20°C. Soil was amended with nutrients (2 mg of COD per g ofdry soil), and the recipient-donor ratio was 3.0. As was

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FIG. 3. Transfer of plasmid pDN705 and survival of donor,recipient, and transconjugant cells in sterile unamended (A) andnutrient-amended (B) sandy loam soil (Tiegem) at 20°C. Symbols: *,AE 104; 0, CM 469; 0, transconjugants.

found in the experiment at 28°C, the number of both parentalstrains increased after inoculation (Fig. 7); transfer fre-quency was 1.5 x 10-6 (Table 6). The experiment withnutrient-amended soil was repeated simultanuously at 28°Cand at 20°C to verify the effect of temperature on the transferfrequency. Soil was amended with 2.64 mg of COD per gramof dry soil, and the recipient-donor ratio in the inoculum was2.3. At 28°C, a rapid increase in number of parental strainsoccurred along with a rapid increase in transconjugants,while at 20°C, at which donor and recipient strains grewmore slowly, transconjugants could be isolated only on day5 and still at a very low level (Fig. 8; Table 6).

DISCUSSION

The plate matings have demonstrated that even genesincorporated into a Tra- Mob- plasmid like pBR325 canescape from E. coli K-12 to A. eutrophus, although at lowfrequencies (10-7 to 10-8). For a long time, such nonmobi-lizable plasmids have been considered safe in connectionwith the release of recombinant DNA (23, 32). Mobilizationof Mob- vectors like pBR322, pBR325, and pHSV101 hasalready been demonstrated in triparental matings (11, 24, 26)and in biparental matings with the conjugative plasmid in thedonor strain (23). All these matings were, however, per-formed with members of the same family (Enterobac-teriaceae). Our model uses strains belonging to differentfamilies, even to different sections (21). In addition, thephenomenon of retromobilization creates a new possibilityfor gene dissemination. Donor bacteria, harboring IncPlplasmids and more precisely, RP4::Mu3A, can acquire chro-mosomal markers (29, 42) or markers of Mob- plasmids (thisstudy) from the recipients at frequencies almost similar tothose observed for the mobilization from donor to recipient.This hermaphroditic property-for the host can act both as afemale recipient and as a male donor-shared by IncPl

TABLE 5. COD and water pH for three types of soil

COD (mg/g of dry soil) in:Soil type Water pH

Sterile soil Nonsterile soil

Sandy (Tillegem) 5.2 0.815 0.100Sandy loam (Tiegem) 7.0 0.707 0.075Clay (Merapi) 6.3 0.274 0.081

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FIG. 4. Transfer of plasmid pDN705 and survival of donor,recipient, and transconjugant cells in sterile unamended (A) andnutrient-amended (B) clay soil (Merapi) at 20°C. Symbols: *, AE104; 0, CM 469; O, transconjugants.

plasmids must be considered in the problematics of generelease, because it offers the bacteria a kind of gene-

capturing device. Moreover, these IncPl plasmids may playan important role in polluted environments (31); at least twocatabolic plasmids were classified as broad-host-range IncPlplasmids: pJP4 (8) (catabolism of the herbicide 2,4-dichlo-rophenoxyacetic acid) and pSS50 (43) (involved in the deg-radation of some chlorinated biphenyls).

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recipient, and transconjugant cells in nonsterile unamended (A) andnutrient-amended (B) sandy soil (Tillegem) at 28°C. Symbols: *, AE104; 0, CM 469; CL, transconjugants.

The direct mobilization of plasmid pDN705 in both sterileand nonsterile soil has been shown to be greatly dependenton the type of soil. As stated by Trevors et al. (53), sterilesoil does not simulate the normal soil environment butrepresents, however, a compromise between strict labora-tory conditions and in situ experiments.The results obtained with sterile soils in this study provide

some interesting information. In sterile conditions, the re-

TABLE 6. Transfer of plasmid pDN705 after 1 day in nonsterile sandy soil (Tillegem) and sandy-loam soil (Tiegem)

Soil type Temp Donor" Recipientb Transconjugants Transfer frequency by:and nutrient status" (OC) DonoreciiRecipientnjgan

Sandy (Tillegem)28 6.0 x 104 1.2 x 107 <2.5 x 101 <4.2 x 10-4 <2.1 x 10-6

+C 28 1.1 X 106 1.3 x 108 <2.5 x 101 <2.3 x 1o-5 <1.9 X 10-7Sandy loam (Tiegem)

28 2.5 x 107 1.0 x 108 <1.2 x 101 <4.8 x 10-7 <1.2 x 10-7+C 28 6.5 x 107 7.8 x 10" 1.3 x 101 1.8 x 10-7 1.5 Xl-8+d 28 6.2 x 107 1.4 x 108 1.4 x 102 2.3 x 106 1.0 x 10-6+ 20 1.1 x 107 5.1 x 107 <8 <7.3 x 10-7 <1.6 x 10-7+ 20e 1.2 x 107 1.2 x 108 8 6.9 x 10-7 6.9 x 10-8+f 20 3.0 x 107 1.5 x 108 2.3 x 102 7.7 x 10-6 1.5 x 10-6

c+, nutrients added; -, nutrients not added.bCFU per gram of soil.c 1.24 mg of COD per g of dry soil.d 2.65 mg of COD per g of dry soil.eAfter 5 days of soil incubation.f 2.00 mg of COD per g of dry soil.

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Time (days)FIG. 6. Transfer of plasmid pDN705 and survival of donor,

recipient, and transconjugant cells in nonsterile unamended (A) andnutrient-amended (B) sandy-loam soil (Tiegem) at 28°C. Symbols: *,AE 104; 0, CM 469; 0, transconjugants.

cipient strain A. eutrophus, which is a normal soil inhabitant,seemed to survive well in the three types of soil. E. coliK-12, on the contrary, demonstrated very good survival onlyin the sandy-loam soil Tiegem (up to 40 days), not in the twoother soils. The poor survival of E. coli in the unamendedTillegem sandy soil is in correspondence with the findings of

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Time (days)FIG. 8. Transfer of plasmid pDN705 and survival of donor,

recipient, and transconjugant cells in nonsterile nutrient-amendedsandy-loam soil (Tiegem) at 20°C (A) and 28°C (B). Symbols: *, AE104; 0, CM 469; O, transconjugants.

Klein and Casida (18), who assumed lack of nutrients to bethe main cause of the quick die-off of E. coli. Although a lotof studies have demonstrated the protective effect of clayminerals (20, 46, 48), the Merapi clay soil did not turn out tobe an environment suitable for E. coli. The lack of nutrientsin this clay soil-the COD of the clay soil was significantlylower (P < 0.005) than the COD of the sandy-loam soil-appeared to counteract the otherwise favorable effect of ahigh clay content. A correlation between donor survival andthe number of transconjugants formed, was obviously dem-onstrated. Transfer frequency was highest (10-5) in thesterile Tiegem sandy-loam soil. It was also the only soil inwhich transfer was detected in the absence of added nutri-ents. Richaume et al. (38) also detected intergeneric plasmidtransfer in a sterile sandy-loam soil at 20°C. On the otherhand, Trevors (51) could observe plasmid transfer betweenE. coli strains in an autoclaved sandy-loam soil only afteraddition of nutrients. Transfer of conjugative plasmids andmobilization of nonconjugative plasmids in both triparentaland biparental matings between Streptomyces strains havebeen observed in nutrient-amended as well as unamendedsterile silt loam (2, 36, 37).

In nonsterile soil, the E. coli population declined in boththe sandy soil and the sandy-loam soil, but the rate of declinewas much higher in the sandy soil. The poor survival incomparison with survival in the sterile soils is most probablycaused by antagonism and competition with the indigenousmicroorganisms, by parasitism, and by predation by nema-

2 4 6 8 10 12 14

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todes and soil protozoa (1, 18). The poor survival of thedonor strain resulted in lack of detection of plasmid transferin both the amended and unamended sandy soils and in theunamended sandy-loam soil. The observed gene transfer inthe nutrient-amended Tiegem sandy-loam soil at both 20 and28°C indicates that under certain conditions, plasmid trans-fer can occur in natural soil. In spite of the fact that E. coliis not a normal soil inhabitant, it appears to multiply andtransfer its plasmid in soil, thereby using the availabledegradable nutrients. However, our model system has beendesigned to define an upper limit of gene release in soil (aworst-case scenario, as stated before [14]); with realisticconditions, e.g., involving eucaryotic genes, a better con-tainment of cloning systems, no addition of an appropriaterecipient strain, and a lower amount of GEMs released, adecrease in probability of transfer of several orders ofmagnitude could be reasonably foreseen.

In this gene escape assay, only gene transmission towardsA. eutrophus was investigated. Probably, plasmid pDN705was also transferred to indigenous soil bacteria, but thisevent can hardly be observed by means of the plate-countingtechnique. Firstly, the czc genes have to be expressed in thenew host; secondly, the minimal medium used in this studysuppresses growth of all auxotrophic transconjugants; andfinally, not all soil microorganisms are culturable on micro-biological media (39). In situ plasmid transfer from anintroduced GEM to indigenous soil microorganisms, by theplate-counting method, has been observed (14), but alltransconjugants appeared to be Pseudomonas fluorescens,indicating that this technique probably excludes potentialtransconjugants that are not culturable on the selectivemedium. The monitoring of gene dissemination towards theindigenous microbial population requires other detectionmethods. DNA probe methods have been developed andallow the detection of specific DNA sequences in noncultur-able organisms also, but they are not sensitive enough todetect a few transconjugants per gram of soil (10, 15, 25, 45,52). The polymerase chain reaction, followed by hybridiza-tion with a specific probe, allows detection of picogramamounts of DNA (4, 45).The czc fragment is an interesting tool in the study of

transmission of recombinant DNA. This gene escape assayis sensitive enough to detect transfer of genetic informationstored in Tra- Mob- plasmids (lo8 per recipient in platematings), but the study of transfer of chromosomal czc genesremains to be completed. The plate-counting technique, witha minimal medium supplemented with heavy metals, isselective enough to detect transfer of genes cloned in Tra-Mob' plasmids in nonsterile soil (10-6 to 10-8 per recipient).In addition, the experiments reported here focus attention onthe transfer properties (in particular the ability of retrotrans-fer) of IncPl plasmids, and thus on the emergence ofadditional possibilities of gene dissemination if such plas-mids are present in a biotope where release is likely to occur.In order to assay retrotransfer in soil, the czc fragment willbe cloned into a Tra- Mob' IncQ vector, which is compat-ible with the IncPl mobilizing plasmids and is thus expectedto be transferred in soil at detectable frequencies. This geneescape assay, simulating the accidental release of laboratorystrain E. coli K-12, could be extended to simulate deliberaterelease of soil bacteria by using, for example, Pseudomonasputida as the donor of the czc genes. Fluorescentpseudomonads do not express the czc fragment either.Transfer of the czc genes will also be investigated in soilpolluted by heavy metals to assess the influence of pollutant

stress on the spread of genes conferring resistance to thisstress factor.

ACKNOWLEDGMENTS

This work was partially supported by the EEC BAP programsBAP 0366-B and 0367-B. E. Top is Research Assistant of the BelgianNational Fund for Scientific Research.We thank M. Hofte for useful discussion and L. Diels for

comments on the manuscript.

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