Characterization of six linked open reading frames necessary for pIP501-mediated conjugation.

pIP501 is a broad-host-range gram-positive conjugative plasmid. Previous transposon mutagenesis of a pIP501 derivative, pVA1702, identified two regions, A and B, that were involved in conjugation proficiency. Recently, we reported that the A region contained a functional oriT site similar to those found on plasmids of gram-negative origin (Wang and Macrina, 1995). We report here the nucleotide sequence of the complete A region. Analysis of this DNA sequence (8121 bp) revealed six contiguous open reading frames (orf1-6) immediately downstream from the oriT site. ORF1 was similar to gram-negative plasmid nickase proteins known to play a role in the initiation of conjugal transfer. The remaining ORF proteins (2-6) were unique. Complementation studies suggested that the gene products of ORFs 3-6 were able to function in trans. A single available insertion mutant in orf2 could not be complemented in trans. Thus, although the conjugative apparatus that initiates transfer (oriT and nickase) appears to be conserved between this class of gram-positive plasmid and gram-negative plasmids, most of the A region genes of pIP501 involved in transfer appear unique and their cellular and molecular roles are undefined.