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 organism (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 rearrangements complicates risk assessment because antibiotic resistance gene DNA and cloned DNA can become dissociated, making the foreign DNA more difficult to detect.