Transconjugants of plasmid pJP4, originating from an agar plate mating of a Pseudomonas putida donor with an activated sludge-derived microbial community, were isolated and identified by partial 16S rDNA sequencing. The transconjugant strains belonged to a variety of genera of the α-, β-, γβ- and γ-classes of the Proteobacteria, mostly to the families Rhizobiaceae and Comamonadaceae and the genus Stenotrophomonas. Only P. putida and Delftia spp. strains were able to grow on 2,4-D as the sole carbon source.
Even for well-characterized plasmids, knowledge about their possible distribution in a microbial community is limited. Since the possible success of a mobile genetic element within a certain environment is not only determined by its own genetic traits, but also by the physiological properties of its momentary host, more information about the possible natural distribution of these elements is desirable. For example, many pWW0 and pJP4 plasmid hosts have been determined by successful transfer from a donor to a pure recipient culture [1,2]. But the actual in situ host range may differ considerably from laboratory results [3,4]. In the case of catabolic plasmids carrying genes for xenobiotic degradation, expression of these genes and subsequent development of the degradation capabilities will in most cases be restricted to a subgroup of host species. This may be due to lack of promotor recognition or translation of the produced mRNAs, failure to import the substrate, substrate toxicity and concentration, or incomplete assembly of the pathway if the host needs to provide additional catabolic genes . Nevertheless, non-degrading transconjugants may serve as a plasmid-reservoir, which might facilitate the adaptation of the community upon encountering the corresponding xenobiotic compound. Additionally, they may permit transfer of the plasmid to recipients which were not accessible by the initial donor strain. We tried to gain some information about the host range of the IncP-1 plasmid pJP4 by performing a conjugation experiment under conditions leading most likely to high transfer rates, i.e., on a nutrient-rich agar surface, but with a complex, activated sludge-derived recipient community analogous to an approach used by Dong et al.  for the transfer of heavy metal resistance genes.
Materials and methods
Donor strain construction
A plasmid donor strain carrying a dsRed-tagged derivative of the conjugative broad-host range, IncP-1 plasmid pJP4  was constructed. This strain was Pseudomonas putida SM1443 carrying a chromosomally encoded lacIq gene , which was chromosomally labelled with two copies of a gfp gene under control of a psbA promotor by transposon mutagenesis with plasmid pUTgfp2x  according to Christensen et al. . To label plasmid pJP4, transposon mutagenesis of Ralstonia eutropha JMP134  with a pUTgm plasmid carrying a Plac-dsRed expression cassette  was carried out, and dsRed-tagged plasmids were selected by transfer to gfp-labelled SM1443 cells . The resulting strain showed a constitutive green and an IPTG-inducible red fluorescence, carried chromosomal resistances against rifampicin and kanamycin, and plasmid-encoded resistances against gentamycin and mercury chloride. Under non-induced growth conditions without IPTG, expression of the lac promotor-controlled dsRed gene was repressed by LacI. In this way, green fluorescent donor cells could be easily distinguished from red fluorescent transconjugant cells. In contrast to the plasmid-free strain SM1443, the constructed donor strain was able to grow on MMN medium  containing 1 mM NH4Cl and 5 mM 2,4-D as the sole carbon source.
For the conjugation experiment, mixed liquor from the aeration basin of a wastewater treatment plant (Grüneck near Munich, Germany) was collected, washed with PBS, adjusted to 30% glycerol and stored at −70 °C. An aliquot of this glycerol-stock corresponding to 1 ml of the original sample was cultivated overnight at 30 °C in 50 ml synthetic wastewater  without benzyl alcohol. Four milliliters of this culture was mixed with 1 ml overnight culture of the donor strain, grown in LB medium , washed and resuspended in 100 μl PBS, and spotted on the surface of an R2A  agar plate. A control experiment where the donor was omitted from the sludge culture was also performed. The plates were incubated for 16 h at 30 °C and then stored at 4 °C for 14 days to account for the slow maturation of DsRed  and to ensure the development of red fluorescence in transconjugants. Microscopic investigation of cell samples from both plates showed red fluorescent cells in the sample with donor added, but not in the control sample without donor addition.
Isolation of transconjugants
The entire mating patch was resuspended in 1 ml of PBS and subjected to preparative flow cytometry targeting red fluorescent cells. Since no red fluorescent cells were observed in the control sample, this sample was not further investigated. For transconjugant isolation, samples of the resulting fraction were plated onto R2A agar containing HgCl2 (10 μg/ml). The developing colonies were streaked on R2A containing gentamycin (25 μg/ml) and microscopically checked for red fluorescence. Finally, 56 mercury- and gentamycin-resistant, red fluorescent isolates were obtained, which were considered to be transconjugants.
Phylogenetic analysis of transconjugants
For genetic screening of transconjugants, extracts of heat-denatured cells were used as template DNA in a PCR with primer (GTG)5 according to Couto et al. . Twenty-four isolates showing different profiles were selected. Their 16S rRNA genes were partially amplified by PCR using primers 341F and 907R according to Muyzer et al. , and custom-sequenced by MWG-Biotech AG (Ebersberg, Germany). Sequences were aligned with the most closely matching DNA sequences found in the EMBL database by FASTA search, and, together with a selection of sequences of known pJP4 transconjugant species, phylogenetic trees were constructed using Clustree at the GENIUSnet HUSAR computer (http://genome.dkfz-heidelberg.de/biounit/) and Treeview . The partial 16S rRNA gene sequences obtained in this study have been deposited in the EMBL nucleotide sequence database (Accession Nos. AJ550273–AJ550296). To exclude the possibility that the same strain had been isolated more than once, isolates having identical or nearly identical 16S sequences were shown to be genetically different by screening with a primer BoxA1R . Additionally, growth of the 24 sequenced isolates on MMN medium  with 1 mM NH4Cl and 5 mM 2,4-D and a 2,4-D indicator broth  was tested.
To ensure that the ability to degrade 2,4-D had not been lost due to rearrangements of the plasmids in the transconjugants, the presence of a part of the tfdB gene was shown by a tfdB-targeted PCR using conditions and primers described in  for all sequenced isolates. Additionally, plasmids from all sequenced transconjugants were transferred back to the plasmid-free donor strain SM1443::gfp by conjugation on R2A and selection of plasmid-carrying SM1443::gfp cells by plating on MMN medium containing 0.2% sodium citrate, 1 mM NH4Cl, 25 μg/ml of kanamycin, 25 μg/ml of gentamycin, and 50 μg/ml of rifampicin. Cells grown on these plates were then checked for growth on 2,4-D by inoculation into 2,4-D indicator broth.
Results and discussion
Fig. 1 shows a phylogenetic tree constructed from partial 16S rDNA sequences of transconjugants isolated in this study, closely related sequences and sequences of previously described pJP4 transconjugant species [1,5,19–25]. The pJP4 transconjugants isolated and sequenced in this experiment showed a broad taxonomic distribution within the α-, β-, γβ-, and γ-Proteobacteria, and belonged mainly to Rhizobiaceae, Comamonadaceae families, and genus Stenotrophomonas.
Most transconjugants showed a sequence similarity of >99% to the most closely related GenBank sequence. Exceptions were TK41 (98.5% to sequence AF078773 of Comamonas sp. 12022) and TK44 (98.7% to sequence AB004754 of Klebsiella oxytoca JCM1665). The sequence of TK14 was 100% identical with that of isolate HAMBI2402 (AF501340), assigned as ‘Ochrobactrum anthropi’, but the two sequences formed a cluster separated from the clade of O. anthropi and Ochrobactrum tritici. In addition, the sequences from TK57 and ‘Ultramicrobacterium’ (AB008507) were nearly identical, as well as the sequences from TK59 and the ‘Xanthomonas group bacterium’ (AF513452), but a more detailed taxonomic characterization of these branches is lacking.
The ability to utilize 2,4-D as the sole source of carbon and energy was restricted to just a few of the isolated transconjugants. Only strains TK20 and TK38, affiliating with P. putida, and TK31 and TK36, affiliating with Delftia acidovorans, were able to grow in the applied test media. Integrity of the plasmids with respect to 2,4-D degradation, especially in the non-degrading transconjugants, was indicated by two observations: tfdB-PCR showed the presence of a part of the tfdB gene in all transconjugants and plasmids from all non-degrading transconjugants could be transferred back to strain SM1443::gfp (the initial plasmid donor), which then was able to grow in 2,4-D indicator broth, in contrast to the plasmid-free strain.
No transconjugants belonging to Burkholderia or Ralstonia were isolated in this study. Strains of these genera and of Pseudomonas have been most frequently found as 2,4-D degrading pJP4 hosts in soil studies [5,20,21,24]. In a recent study by Goris et al. , strains of Stenotrophomonas maltophilia were found to be 2,4-D degrading pJP4-transconjugants. We also obtained several isolates showing sequence identity with S. maltophilia, but they were not able to grow on 2,4-D as sole carbon source. In general, our results support earlier findings , namely that Inc-P1 plasmids have a highly diverse natural host range.
The differences in species distribution, as compared to the results obtained in the studies mentioned above, likely reflect the differences in species diversity of the two environments (soil and activated sludge) but may also have to do with the fact that the conjugation experiment in our study was conducted in an environment without 2,4-D as selective compound. The type of media (R2A) and the choice of plate mating over in situ mating in liquid activated sludge may have had an impact on the transconjugant diversity as well, as has recently been shown in another study by Goris et al. . Since our methodology relies on growth of the transconjugants on solid media and on expression of DsRed within a transconjugant cell, non-culturable cells and strains which do not express DsRed have been missed by this approach. Vice versa, species expressing DsRed at a high level might have been preferentially targeted by fluorescence-based detection and sorting techniques and therefore have been favoured over other transconjugant species. This could have been the case with α-proteobacteria, since our α-proteobacterial isolates displayed intense fluorescence under microscopic observation and formed brightly pink colonies on R2A agar, in contrast to, e.g., the P. putida strains we obtained. Additionally, 15 other of the initially isolated strains gave (GTG)5 profiles which had a high similarity to those of strains TK4, TK7, and TK29, indicating that 23 out of 56 isolates possibly belonged to the α-Proteobacteria. Also, expression of the mercury and gentamycin resistance genes residing on our pJP4 derivative which have been used for isolation of transconjugants might be host-dependent, again giving a possibility for selection of certain transconjugant groups.
In conclusion, our study shows the highest transconjugant diversity of a self-transmissible broad-host range plasmid characterized in a single experiment to date. The results also indicate that Inc-P1 plasmids may be promising candidates for genetic engineering of mobile genetic elements for bioremediation purposes, as proposed by Top et al. .
We thank S. Molin for the donation of P. putida SM1443, S. Molin and L. Eberl for the pUTgm plasmid carrying the Plac-dsRed expression cassette, and J.K. Jansson for pUTgfp2x. This work was supported by Grant Ha3164/2-1 from the German Research Foundation (DFG) to M.H. and S.W.