IDENTIFICATION AND CHARACTERIZATION OF THE GENES OF ENTEROCOCCUS-FAECALIS PLASMID PCF10 INVOLVED IN REPLICATION AND IN NEGATIVE CONTROL OF PHEROMONE-INDUCIBLE CONJUGATION

The prgB gene of the Enterococcus faecalis pheromone-inducible conjuga tive plasmid pCF10 encodes the surface protein Asc10. This protein med iates cell aggregation and its expression results in high-frequency tr ansfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation of prgB expression, target p lasmids were constructed containing a recently identified positive con trol region and a prgB ::lacZ transcriptional fusion; expression of pr gB in cells carrying these plasmids was thus verified by beta-galactos idase assay. The target plasmids were used in genetic studies with com patible plasmids containing cloned pCF10 genes supplying putative nega tive control functions to define the minimum region of pCF10 required for shutdown of prgB expression in the absence of exogenous pheromone. The minimum segment required for negative control, as identified by d eletion analysis, was a 6.9-kb region extending from the 5' end of a g ene called prgN, through a previously identified gene, prgX. The DNA i n this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid repl ication genes were identified. Genetic analysis indicated that the prg N, -Y, and -X genes are involved in negative control; prgW may also pl ay a role in negative control, since it appeared to be required for ex pression of prgY. prgX, or a closely adjacent DNA sequence, acted in c is. The region of pCF10 containing negative control genes was also sho wn to function as an autonomous replicon in E. faecalis. (C) 1996 Acad emic Press, Inc.