Conjugal and genetic analysis of the IncHI1 plasmid, R27

by Sherburne, Craig K.

The complete DNA sequence of R27 has been compiled and analyzed. The l8O,361 bp plasrnid contains 210 open reading Crames, 70 of which have been previously identified or contain significant homology to other plasmid or prokaryotic open reading kames. 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. typhimtirium LPS mutant recipients and donors, were assessed for the IncHIl plasmids R27 and pDT2454, and the incHI2 plasmid R478. It was observed that only a specific tmcation of the outer core, by mutation of the rfF LPS biosynthesis gene, increased conjugal transfer. Stabilization and initiation of conjugal transfer is, in most cases, negatively af'fected by LPS truncation. The major component of the R27 conjugal pilus was identified as a 7.6kDa peptide, likely generated by the trM 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 obrained. It is possible that insertion events in nhA 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 containhg ORFOI 8, 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 theoretidy 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 similarîty to the IncP plasmids, a separation of the transfer regions similar to the hcP 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 anceston of many different plasmid systems forming the unique IncHIl system observed today.