Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonucl ease cleavage patterns within their tra regions. Complementation analysis a nd sequence data presented here indicate that these two plasmids encode ess entially identical conjugal DNA-processing proteins. This region contains t hree genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin ( oriT). Three corresponding proteins were visualized by sodium dodecyl sulfa te-polyacrylamide gel electrophoresis, and complementation analysis confirm ed that this region contains three tm complementation groups. All three pro teins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropa thy profile of TraJ suggests a transmembrane topology similar to that of se veral homologous proteins. Both traK and traI were required for efficient i nterplasmid site-specific recombination at oriT, while traJ was not require d. The leading region of pKM101 contains three genes (stbA, stbB, and stbC) , null mutations in which cause elevated levels of plasmid instability. Pla smid instability was observed only in hosts that are proficient in interpla smid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.