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 aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis 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 that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease 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 (immunoblot) 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. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.
Chung
J W
JW
Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108, USA.
Bensing
B A
BA
Dunny
G M
GM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D013293:Enterococcus faecalis / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007763:Lac Operon; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008565:Membrane Proteins; C071729:Asc10 protein, Enterococcus faecalis