STREPTOCOCCAL PLASMID PIP501 HAS A FUNCTIONAL ORIT SITE

DNA sequence analysis suggested the presence of a plasmid transfer ori gin-like site (oriT) in the grampositive conjugative plasmid pIP501. T o test the hypothesis that the putative oriT site in pIP501 played a r ole in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompasse d the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobi lized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results ind icated that the oriT-like site was needed for conjugal mobilization. T o demonstrate precise nicking at the oriT site, alkaline gel and DNA-s equencing analyses were performed, Alkaline gel electrophoresis result s indicated a single-stranded DNA break in the predicted oriT site. Th e oriT site was found upstream of six open reading frames (orf1 to orf 6), each of which plays a role in conjugal transfer. Taken together, o ur conjugal mobilization data and the in vivo oriT nicking seen in Esc herichia coli argue compellingly for the role of specific, single-stra nded cleavage in plasmid mobilization. Thus, plasmid mobilization prom oted by pVA1702 (pIP501) works in a fashion similar to that known to o ccur widely in gram-negative bacteria.