GENETIC-ANALYSIS OF A REGION OF THE ENTEROCOCCUS-FAECALIS PLASMID PCF10 INVOLVED IN POSITIVE REGULATION OF CONJUGATIVE TRANSFER-FUNCTIONS

The prgB gene encodes the surface protein Asc10, which mediates cell a ggregation resulting in high-frequency conjugative transfer of the phe romone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis, Previous Tn5 insertional mutagenesis and sequencing analysi s of a 12-kb fragment of pCF10 indicated that a region containing prgX , -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed tha t the positive regulatory region functions in eis in an orientation-de pendent manner (J. W, Chung and G, M. Dunny, Proc. Natl, Acad. Sci, US A 89:9020-9024, 1992), In order to determine the involvement of each g ene in the activation of prgB, Tn5 insertional mutagenesis and exonucl ease III deletion analyses of the regulatory region were carried out, The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immuno blot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Comple mentation analysis demonstrates that a cis-acting regulatory element i s located in the prgQ region and that pCF10 sequences in an untranslat ed region 3' from prgQ are an essential component of the positive-cent ral system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. fa ecalis but that outward-reading transcripts may initiate from within t he ends of Tn5 or from the junction sequences.