Identification of novel VirR/VirS-regulated genes in Clostridium perfringens

Novel genes that are regulated in Clostridium perfringens by the two-compon ent regulatory system, VirR/VirS, were identified using a differential disp lay method. A plasmid library was constructed from C. perfringens chromosom al DNA, and the plasmids were hybridized with cDNA probes prepared from tot al RNA of wild-type strain 13 and its virR mutant derivative TS133. Three c lones were identified that carry newly identified VirR/VirS-regulated genes , two of which were positively regulated and one of which was negatively re gulated. Genes located on the identified clones were deduced by nucleotide sequencing, and the target genes of the VirR/VirS system were identified wi th a set of Northern hybridizations. A 4.9 kb mRNA transcribing the metB (c ystathionine gamma-synthase), cysK (cysteine synthase) and ygaG (hypothetic al protein) genes was negatively regulated, whereas 1.6 and 6.0 kb transcri pts encoding ptp (protein tyrosine phosphatase) and cpd (2',3'-cyclic nucle otide 2'-phosphodiesterase) respectively, were shown to be positively regul ated by the VirR/VirS system. The other gene, hyp7, whose transcript was po sitively regulated by the VirR/VirS system, was shown to activate the trans cription of the colA (kappa-toxin) and plc (alpha-toxin) genes, but not the pfoA (theta-toxin) gene in C. perfringens. These results suggested that th e global regulatory system VirR/VirS could regulate various genes, other th an toxin genes, both positively and negatively and that the hyp7 gene might encode a novel regulatory factor for toxin production in C. perfringens.