Conjugal and genetic analysis of the IncHI1 plasmid, R27

The complete DNA sequence of R27 has been compiled and analyzed. The 180,361bp plasmid contains 210 open reading frames, 70 of which have been previously identified or contain significant homology to other plasmid or prokaryotic open reading frames. Two transfer regions separated by over 64kb are observed, with the gene order and individual genes in transfer region 2 (Tra2) displaying significant homology to the F plasmid transfer region;The frequency of transfer and ability to form mating aggregates, using isogenic S. typhimurium LPS mutant recipients and donors, were assessed for the IncHI1 plasmids R27 and pDT2454, and the IncHI2 plasmid 8478. It was observed that only a specific truncation of the outer core, by mutation of the rfaF LPS biosynthesis gene, increased conjugal transfer. Stabilization and initiation of conjugal transfer is, in most cases, negatively affected by LPS truncation.;The major component of the R27 conjugal pilus was identified as a 7.6kDa peptide, likely generated by the trhA gene. Although visualized in a cell-free radiolabeled expression system, the trhA gene product is resistant to denaturation and protein staining in SDS-PAGE analysis. No mutations of the trhA gene in R27 through antibiotic cassette insertion were obtained. It is possible that insertion events in trhA destabilize R27 by an unknown mechanism.;The gene responsible for entry exclusion in R27 has been identified. R27 transfer has been shown to significantly decrease into recipient cells containing ORF018, encoding a 14.1 kDa protein. The protein contains no significant homology to entry exclusion proteins of other systems, although it does contain a strong N-terminal transmembrane sequence theoretically sequestering it to the bacterial membrane.;R27 contains a large number of elements in common with the F plasmid family, although it encodes a pilin with strong similarity to the IncP plasmids, a separation of the transfer regions similar to the IncCP plasmids, and an entry exclusion gene which shows no homology to either system. These results demonstrate the apparent hybrid nature of R27, with its current state appearing to be the result of genetic exchanges between ancestors of many different plasmid systems forming the unique IncHI1 system observed today.