Identification and Characterization of the Genes ofEnterococcus faecalisPlasmid pCF10 Involved in Replication and in Negative Control of Pheromone-Inducible Conjugation

Abstract

TheprgBgene of theEnterococcus faecalispheromone-inducible conjugative plasmid pCF10 encodes the surface protein Asc10. This protein mediates cell aggregation and its expression results in high-frequency transfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation ofprgBexpression, target plasmids were constructed containing a recently identified positive control region and aprgB::lacZtranscriptional fusion; expression ofprgBin cells carrying these plasmids was thus verified by β-galactosidase assay. The target plasmids were used in genetic studies with compatible plasmids containing cloned pCF10 genes supplying putative negative control functions to define the minimum region of pCF10 required for shutdown ofprgBexpression in the absence of exogenous pheromone. The minimum segment required for negative control, as identified by deletion analysis, was a 6.9-kb region extending from the 5′ end of a gene calledprgN,through a previously identified gene,prgX.The DNA in this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid replication genes were identified. Genetic analysis indicated that theprgN, -Y,and-Xgenes are involved in negative control;prgWmay also play a role in negative control, since it appeared to be required for expression ofprgY. prgX,or a closely adjacent DNA sequence, actedin cis.The region of pCF10 containing negative control genes was also shown to function as an autonomous replicon inE. faecalis.