Streptococcal plasmid pIP501 has a functional oriT site.
DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized 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 indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.
Wang
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Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298-0678, USA.
Macrina
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FL
D001483:Base Sequence; D002874:Chromosome Mapping; D003227:Conjugation, Genetic; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D013291:Streptococcus / Q000235:genetics*