Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water

Abstract

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35°C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 108 to 2.0 x 109 CFU/ml) were required. During 1 week of incubation in drinking water, numbers of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media. The possibility of rearrangments complicates risk assessment because antibiotic resistance gene DNA and cloned DNA can become dissociated, making the foreign DNA more difficult to detect.

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

“…These results contrast with in vivo conjugal transfer experiments using mouse, lepidoptera and chicken intestines, which yielded lower frequencies than those obtained in vitro . (Armstrong, Wood & Porteous 1990; Jarret & Stephenson 1990) These authors attributed this to interference by native microbiota, the presence of large amounts of organic matter, pH or relative in situ concentrations of donors and recipients (Sandt & Herson 1991). Similar results on the effects of microbiota on plasmid transfer have been reported by O’Morchoe et al.…”

“…Both public health and ecological reasons emphasize the importance of studying plasmid transfer in aquatic systems. Successful plasmid transfer between aquatic bacteria under laboratory conditions has been demonstrated (Trevors & Oddie 1986; Sandt & Herson 1991), both in vivo and in situ (Gowland & Slater 1984; O’Morchoe, Ogunseitan, Sayler & Miller 1988). An increase in the number of bacteria from aquatic environments carrying R plasmids has also been documented (Bell, Macrae & Elliot 1980; Niemi, Sibakov & Niemela 1983).…”

Gene transfer among bacteria under conditions of nutrient depletion in simulated and natural aquatic environments

“…Matings carried out in rich media resulted in increased mobilisation frequencies. No significant differences to mobilisation frequencies among strains held in drinking water only, were found for drinking water supplemented with 1% trypticase soy broth (TOC 20 mg 1-1) [9]. Although it was claimed that the number of recovered transconjugants increased with increasing temperature, this was marginal, the increase being only about 1.3-times over the range from 15 to 35°C.…”

“…Interestingly, plasmids recovered from transconjugants in these chambers often showed evidence of genetic rearrangements, whereas plasmids recovered from transconjugants produced in the laboratory did not. Using the helper conjugative plasmid R100-1, Sandt and Herson [9] showed mobilisation of a nonselftransmissible genetically engineered plasmid, pHSV106, from E. coli donor strains to an environmental isolate of Enterobacter cloacae in filter sterilised, dechlorinated drinking water (TOC 1.5 to 7 mg 1-1). The transconjugant strains studied showed evidence of genetic rearrangements in pHSV106.…”

“…In the work described above, some of the experiments did not control for the possibility of plasmid transfer occurring between donors and recipients plated on the selection media used for the recovery of transconjugants [6,7,9], and the data presented must be assessed with this in mind. However, in at least two of these cases it is unlikely that transconjugants arose from mating during colony recovery.…”