CHARACTERIZATION OF 6 LINKED OPEN READING FRAMES NECESSARY FOR PIP501-MEDIATED CONJUGATION

pIP5O1 is a broad-host-range gram-positive conjugative plasmid. Previo us transposon mutagenesis of a pIP5O1 derivative, pVA1702, identified two regions, A and B, that were involved in conjugation proficiency. R ecently, we reported that the A region contained a functional oriT sit e similar to those found on plasmids of gram-negative origin (Wang and Macrina, 1995). We report here the nucleotide sequence of the complet e A region. Analysis of this DNA sequence (8121 bp) revealed six conti guous open reading frames (orf1-6) immediately downstream from the ori T site. ORF1 was similar to,sram-negative plasmid nickase proteins kno wn to play a role in the initiation of conjugal transfer. The remainin g ORF proteins (2-6) were unique. Complementation studies suggested th at the gene products of ORFs 3-6 were able to function in trans. A sin gle available insertion mutant in orf2 could not be complemented in tr ans. Thus. although the conjugative apparatus that initiates transfer (oriT and nickase) appears to be conserved between this class of sam-p ositive plasmid and gram-negative plasmids, most of the A region genes of pIP5O1 involved in transfer appear unique and their cellular and m olecular roles are undefined. (C) 1995 Academic Press, Inc.