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.
Department of Microbiology, 1460 Mayo Memorial Bldg., University of Minnesota, Minneapolis 55455, USA.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003227:Conjugation, Genetic / Q000187:drug effects; D019281:Dimerization; D013293:Enterococcus faecalis / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005799:Genes, Dominant; D008969:Molecular Sequence Data; D009154:Mutation; D010675:Pheromones / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D010675:Pheromones; D012333:RNA, Messenger