CIS-ACTING, ORIENTATION-DEPENDENT, POSITIVE CONTROL-SYSTEM ACTIVATES PHEROMONE-INDUCIBLE CONJUGATION FUNCTIONS AT DISTANCES GREATER THAN 10 KILOBASES UPSTREAM FROM ITS TARGET IN ENTEROCOCCUS-FAECALIS

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. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.