IDENTIFICATION AND CHARACTERIZATION OF THE ORIGIN OF CONJUGATIVE TRANSFER (ORIT) AND A GENE (NES) ENCODING A SINGLE-STRANDED ENDONUCLEASE ON THE STAPHYLOCOCCAL PLASMID PGO1

The genes mediating the conjugative transfer of the 52-kb staphylococc al plasmid pGO1 are within a 14.4-kb gene cluster designated trs. Howe ver, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this st udy, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing th e ORF could complement these transposon-inactivated pGO1 mutants for t ransfer. Analysis of the nucleotide sequence of this ORF revealed sign ificant homology between the amino terminus of its predicted protein a nd those of several single-stranded endonucleases. In addition, a 12-b p DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generat e a single-stranded nick at the oriT was demonstrated in Escherichia c oli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by th e presence of oriT and nes could also be mobilized at high frequency i nto staphylococcus aureus recipients with a second plasmid containing only trs, We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, mak e up the minimal staphylococcal conjugative replicon.