APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1991, p. 3020-3027

0099-2240/91/103020-08$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 57, No. 10

Conjugational Transfer of Recombinant DNA in Cultures and in Soils: Host Range of Pseudomonas putida TOL Plasmids MARIA-ISABEL RAMOS-GONZALEZ, ESTRELLA DUQUE, AND JUAN L. RAMOS* Consejo Superior de Investigaciones Cientificas, Estacion Experimental del Zaidin, Unidad de Bioquimica Vegetal, E-18080 Granada, Spain Received 1 April 1991/Accepted 23 July 1991

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10-2 transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10-3 to 1o-7 transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.

Genetically engineered microorganisms have been widely used in industry for many years. These organisms have been produced by mutagenesis and selection of improved strains. The current state of recombinant gene technology allows a high degree of specificity in the construction of microorganisms of potential industrial interest. The assessment of the possible risks involved in the deliberate or accidental release of in vitro recombinant microorganisms in open environments requires an understanding of both the potential of introduced organisms to transfer genetic material to resident microorganisms and the capability of resident microorganisms to gain information from the recombinant organisms. Conjugation is probably the most efficient way to transfer genetic information (31), although other mechanisms, such as transformation (4, 8, 9) and transduction (18), also play important roles. Conjugational intra- and intergeneric gene transfers have been demonstrated under a variety of laboratory culture conditions. However, the number of studies of gene transfer in natural habitats or microcosms is rather limited. Intrageneric plasmid transfer in soils and aquatic microcosms has been demonstrated for bacteria such as Escherichia coli, Pseudomonas spp., Bacillus spp., and Streptomyces spp. (5, 11, 17, 18, 31, 35). Intergeneric plasmid transfer has also been observed in aquatic and soil microcosms (19, 30). Although the interpretation of the results of such studies is sometimes complex, the data support gene exchange in plausible ecological situations and suggest that in some cases the frequency of gene exchange can be explained by selection. Self-transmissible Pseudomonas putida TOL plasmid pWWO encodes the enzymes that are required for mineralization of toluene and related aromatic hydrocarbons. TOL catabolic pathways have been *

used as a model system for the expansion of the range of aromatic hydrocarbons that a bacterium can degrade. Recombinant TOL plasmid pWWO-EB62 allows a host bacterium to grow on p-ethyltoluene and was generated by introducing three successive mutations in TOL plasmid pWWO (1, 24). Interest in the environmental safety of genetically engineered microorganisms has highlighted the importance of the gene transfer profiles of the recipients. The studies described in this paper were undertaken to determine whether recombinant TOL plasmids were transferred to a number of gram-negative bacteria and whether they expressed catabolic functions under laboratory and soil conditions. Our results support the hypothesis that TOL plasmids can be transferred to a limited number of gram-negative bacteria, including pseudomonads belonging to rRNA group I, Escherichia coli, and Erwinia chrysanthemi. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains used in this study are shown in Table 1. The bacteria were grown at 30°C on LB or M9 minimal medium (15) supplemented with one of the following carbon sources: glucose (0.5%, wt/vol), acetate (10 mM), succinate (10 mM), or benzoate (5 mM). The antibiotic-resistant strains constructed in this study were spontaneous mutants, which were selected on LB plates supplemented with the appropriate antibiotics. When double mutants were obtained, the mutations were introduced successively. Antibiotics were used at the following concentrations: ampicillin, 100 ,ug/ml; kanamycin, 50 ,ug/ml; nalidixic acid, 10 to 20 ,ug/ml; piperacillin, 30 jig/ml; rifampin, 20 to 50 pug/ml; streptomycin, 25 to 100 pug/ml; and tetracycline, 10 ,g/ml. Plasmids pWWO-EB62 (IncP9 p-ethyltoluene+ m-xylene+

Corresponding author. 3020

TOL PLASMID TRANSFER

VOL. 57, 1991

TABLE 1. Strains used in this study Relevant

characteristicsa

Source or reference

Prototroph, RestPrototroph, Rest' Rifr Smr derivative of 2440 Rif' derivative of 2440 PTr derivative of 2440 Nalr Smr TrpPrototroph, Rif' Nalr Prototroph, Kmr Prototroph, Rif' Smr Prototroph, Rif' Smr Kmr Prototroph, Kmr Rif' Smr Prototroph, Rif' Smr

6 D. Springael Our laboratory Our laboratory Our laboratory S. Harayama Our laboratory 11 This study This study This study This study

Prototroph, Nalr Smr

This study

Rif' Met- Trp-

Our laboratory

Rif Thi- Thr- Leu-

This study

RecA- Smr

15

RecA-

16

Prototroph, Rif' Nalr

This study

Prototroph, Smr Nalr

This study

Prototroph, Rif' Smr

This study

Strain Pseudomonads P. putida 2440 P. putida 29 P. putida EEZ3 P. putida EEZ19 P. putida EEZ15 P. putida PaW340 P. fluorescens EEZ20 P. aeruginosa 7NSK2 P. stutzeri EEZ22 P. acidovorans EEZ23 P. solanacearum EEZ24 P. diminuta EEZ25 Other bacteria Escherichia coli K-12 strain EEZ8000 Escherichia coli K-12 strain LE392R Escherichia coli K-12 strain C600R Escherichia coli K-12 strain HB101 Escherichia coli K-12 strain CC118 Acinetobacter calcoaceticus EEZ26 Alcaligenes sp. strain EEZ27 Erwinia chrysanthemi EEZ28

a Abbreviations: Rest-, restriction negative; Rest', restriction positive; Nalr, nalidixic acid resistance; Rif, rifampin resistance; Smr, streptomycin resistance; Kmr, kanamycin resistance; PTr, phosphinothricin resistance.

p-methylbenzoate+ m-methylbenzoate+ Mob' Tra+) (1), pRK2013 (Kmr helper plasmid) (29), pUT-Km (R6K replication origin, Apr Kmr Mob' Tra-) (10), and pUT-TC (R6K replication origin, Apr Tcr Mob' Tra-) (10) have been described previously. Plasmids pWWO-EB62K (Kmr m-xylene+) and pWWO-EB62T (Tcr m-xylene-) were constructed in this study and are described below. Filter matings. Cultures (0.5 ml) of donor and recipient bacteria that were grown overnight were mixed, centrifuged in a bench centrifuge (12,000 rpm, 2 min), washed with 1 ml of LB medium, and resuspended in 25 ,ul of LB medium. The cells were dropped onto a nitrocellulose filter (pore size, 0.2 ,um) that was placed on the surface of an LB agar plate. Controls consisting of donor and recipient bacteria that were not mixed were always included and incubated under similar conditions. After 12 to 16 h at 30°C, the cells were suspended in 1 ml of 50 mM phosphate buffer (pH 7.0) and serially diluted. The selective plates used for donor bacterium P. putida EEZ15 bearing pWWO-EB62 or pWWO-EB62K contained M9 minimal medium supplemented with 5 mM alkylbenzoate and 400 ,ug of phosphinothricin per ml, as well as kanamycin in the case of pWWO-EB62K. The selective plates used for P. putida EEZ15(pWWO-EB62T) were prepared as described above except that they contained 10 mM acetate as a C source and tetracycline. The recipient bacteria were selected on M9 minimal medium containing an appropriate C source and the antibiotics to which they were resistant. The transconjugants were selected on the same plates as the recipients, except that p-ethylbenzoate was

3021

used to select transconjugants bearing pWWO-EB62 and 5 mM m-methylbenzoate and kanamycin were used to select

transconjugants bearing pWWO-EB62K. Tetracycline was used to select transconjugants bearing pWWO-EB62T. Preparation of plasmids from transconjugants. A 0.5-ml portion of an overnight culture was harvested, and the resulting pellet was resuspended in 0.2 ml of 25 mM TrisHCl-25 mM EDTA (pH 8.0) containing 20% (wt/vol) sucrose and 1 mg of lysozyme per ml. The cells were lysed by adding 0.1 ml of a freshly prepared solution containing 0.3 N NaOH and 2% (wt/vol) sodium dodecyl sulfate. After gentle mixing, the resulting viscous suspension was incubated at 55°C for 5 min to minimize the chromosomal DNA (14). After phenol extraction, 50 ,u1 of the aqueous phase was mixed with 8 ,ul of loading buffer (15), and the preparation was run on 0.7% (wt/vol) agarose gels at 10 V/cm for about 4 h. DNA hybridization. Total DNA was prepared basically as described by Robson et al. (27). The methods used for DNA restriction, separation of DNA fragments by electrophoresis on agarose gels, transfer to nitrocellulose membranes, and DNA fixation have been described previously (15, 28). The membranes were soaked in the hybridization buffer recommended by Boehringer, Mannheim, Germany (catalog no. 1093657, except that N-lauroyl sarcosine was not added and the blocking reagent was used at a concentration of 5% [wt/vol]) containing 50% (vol/vol) formamide and 0.5 mg of herring sperm denatured DNA in 10 ml of solution for 6 to 8 h at 42°C. Then 200 ng of denatured digoxigenin-dUTPlabeled probe (see below) was added, and incubation was continued for 14 to 16 h at 42°C. The digoxigenin-labeled hybrid DNAs were detected by using an enzyme immunoassay according to the manufacturer's instructions (Boehringer). Labeling DNA probes. The approximately 1.8-kb BamHI restriction fragment from plasmid pJLR156 containing the entire xylS gene (23) was randomly labeled with digoxigenindUTP according to the manufacturer's instructions (Boehringer). Soils used. The soils used in this work were sandy loam soils (pH 7.5 to 8.0). One of the soils, a fluvisol soil, was relatively rich in organic matter (2.25%, wt/wt) and had a low CaCO3 content (6.3%, wt/wt); the other soil, a cambisol soil, was relatively poor in organic matter (0.63%, wt/wt) and had a high CaCO3 content (23.4%, wt/wt). Other characteristics of these soils have been described previously (22). Before use the soils were sifted through a 4-mm-mesh metal sieve and sterilized under a vapor stream (120°C, 1 h) three times; the mass was allowed to cool completely between sterilization treatments. Cells were introduced into the soils at a concentration of about 108 CFU/g of soil unless otherwise indicated. Matings in soil. For mating experiments, donor and recipient bacteria were introduced successively into soils to avoid formation of conjugational bridges. The donor bacterium was always P. putida EEZ15 bearing pWWO-EB62 or pWWO-EB62K, while recipient bacteria were prototrophs that were resistant to two antibiotics. Controls consisting of unmixed donor and recipient bacteria were always kept under similar incubation conditions. The temperatures in these experiments were 15 to 17°C. To estimate the numbers

of culturable cells (colony-forming units) of donors, recipients, and transconjugants, 10 g of soil was added to 90 ml of 50 mM phosphate buffer, and the preparation was shaken for 60 min at 30°C. This was the first dilution, and while still being shaken, this dilution was used to prepare a range of dilutions so that we could detect between 10 and 108 CFU/g

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RAMOS-GONZALEZ ET AL.

of soil. For each determination at least three different dilutions were spread in duplicate onto selective plates. The selective plates used for the donor, recipient, and transconjugant bacteria were the same as the plates used for filter matings (see above). RESULTS Construction of model recombinant TOL plasmids bearing antibiotic markers. The construction of recombinant TOL plasmid pWWO-EB62 has been described previously (1, 24). Derivatives of this plasmid bearing antibiotic resistance markers were generated by inserting within the TOL plasmid a transposaseless mini-TnS that encoded resistance to kanamycin and tetracycline (10). A triparental mating involving P. putida EEZ19(pWWO), Escherichia coli CC118 lambdaPIR bearing Mob' Tra- plasmid pUT-Km (10), and Escherichia coli HB101 bearing helper plasmid pRK2013 was performed. Since pUT-Km does not replicate in P. putida (a lambda-PIR- bacterium), Kmr P. putida EEZ19 transconjugants were selected on minimal medium containing rifampin, kanamycin, and m-xylene as the sole C source. More than 95% of the clones were piperacillin sensitive, which suggested that they had resulted from true transposition rather than cointegration of plasmid pUT-Km with host replicons. To find clones bearing mini-Tn5 in the TOL plasmid, all Kmr p. putida EEZ19 transconjugants were mixed and mated with phosphinothricin-resistant (PT r) P. putida EEZ15. Cotransfer of the kanamycin marker and the m-xylene degradation capability were selected in phosphinothricin-resistant transconjugants; m-xylene+ Kmr PT-r clones were sensitive to rifampin, a finding that confirmed the presence of the kanamycin cassette in the TOL plasmid. One of the transconjugants, which was designated P. putida EEZ15(pWWO-EB62K), was selected for further experiments.

Another TOL derivative was generated in a similar way by inserting a mini-TnS marker that encoded resistance to tetracycline. The source of the mini-TnS::Tc marker was pUT-Tc (10). The mutant TOL plasmid was designated pWWO-EB62T. The mini-TnS marker was inserted on the xylE gene, which made the bacterium unable to grow on m-xylene and m-toluate, although it transformed these aromatic compounds into m-methylcatechol (21). Transfer of TOL plasmids to other bacteria on plates. The potential recipients (Table 1) for TOL plasmid pWWO-EB62 were prototrophic bacteria that were resistant to one or two antibiotics and were not able to grow on alkylbenzoates as sole sources of carbon and energy. Matings between donor bacterium P. putida EEZ15(pWWO-EB62) and the recipients were performed for about 16 h at 30°C on LB plates with donor-to-recipient cell ratios of 1:1, 10:1, and 1:10, where 1 represented about 108 cells. With restriction-positive and restriction-negative P. putida strains as recipients, the frequency of transconjugants that were able to grow on alkylbenzoates was on the order of 1 to 10-1 transconjugant per recipient. Intrageneric transfer was assayed with pseudomonads belonging to different RNA groups (20). High frequencies of transfer and expression of TOL were found with Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri (group I); in these cases the gene transfer frequency was on the order of 1 to 10-2 transconjugant per recipient (Table 2). With Pseudomonas solanacearum (group II), Pseudomonas acidovorans (group III), and Pseudomonas diminuta (group IV), no transfer of the TOL plasmid was observed (Table 2).

TABLE 2. Transfer of recombinant TOL plasmids from P. putida EEZ15 to different gram-negative bacteria Recipient

Intrageneric transfer P. putida EEZ3 (Rest-) P. putida 29 (Rest') P. fluorescens EEZ20 P. aeruginosa 7NSK2 P. stutzeri EEZ22 P. acidovorans EEZ23 P. solanacearum EEZ24 P. diminuta EEZ25 Intergeneric transfer Alcaligenes sp. strain EEZ27 Escherichia coli EEZ8000 Erwinia chrysanthemi EEZ28

Frequencies of transconjugants with the following donors': pWWOEB62

pWWOEB62K

pWWOEB62T

1-1o-'

l-lo-l

1-io-1 1-10-1 10-2

ND ND ND

i-io-I

1-io-1