Dominant‐negative mutants of prgX: evidence for a role for PrgX dimerization in negative regulation of pheromone‐inducible conjugation

PrgX negatively regulates prgQ transcriptional readthrough in the pheromone‐inducible enterococcal conjugative plasmid pCF10. We isolated and characterized 13 dominant‐negative prgX mutants, all of which mapped in either the N‐ or the C‐terminus of PrgX. In all mutants, the in vivo level of Qa RNA, an antisense RNA to prgQ RNA, was greatly reduced. When oligomerization of PrgX was tested with a phage lambda cI repressor fusion system, the oligomerization domain was found to be between amino acid residues 78 and 280. When histidine‐tagged PrgX (His‐PrgX) was purified by nickel column chromatography from a strain also expressing PrgX, PrgX was co‐purified with His‐PrgX. Although PrgX was expressed at a much higher level than His‐PrgX, an approximately equal amount of PrgX was co‐purified. Pheromone induction greatly decreased the co‐purification of PrgX. Based on these data, we propose that both the N‐ and the C‐terminal domains of PrgX are required for PrgX positive autoregulation and for the repression of prgQ transcription readthrough. In vivo, PrgX exists as a dimer, and dimerization is mediated by the central region of PrgX.