Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIP501

Abstract

pIP501 is a 30.2-kilobase (kb) broad-host-range conjugative streptococcal plasmid which encodes chloramphenicol and erythromycin resistance. A smaller conjugation-proficient derivative of pIP501 has been constructed and designated pVA1702. pVA1702 is 25.2 kb, encodes only kanamycin resistance, and confers conjugative ability in an Enterococcus faecalis mating system. The temperature-sensitive transposon delivery vectors pTV1ts and pTV32 carrying Tn917 and Tn917lac, respectively, were used to create insertion mutants of pVA1702 in E. faecalis. These insertions enabled us to localize the determinants conferring conjugation to two separate regions of pVA1702: the 7.5-kb region A and the 8.8-kb region B. A nested set of deletions of the 7.5-kb region was subcloned in Escherichia coli, and the expression of these deletions was examined in an E. coli minicell system. The genes for three conjugal proteins have been located and designated cnjA, cnjB, and cnjC (for conjugation). Two other peptides that are transcribed in a divergent direction from the cnj genes were encoded in the A region, but it is not known whether they are involved in conjugation. These studies indicate that the conjugation genes of pIP501 are arranged in multiple transcriptional units.