Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins

Abstract

Summary We analysed the region encoding VR‐RNA (VirR‐regulated RNA), which has been reported to be positively regulated by the two‐component VirR/VirS system in Clostridium perfringens. The VR‐RNA promoter identified by primer extension analysis was preceded by a probable VirR‐binding site (CCAGTTNNNCAC), which resembled a repeated sequence motif present in the promoter region of the theta‐toxin (pfoA) gene. A VR‐RNA‐null mutant, constructed by a homologous recombination, exhibited a reduced amount of transcription of the alpha‐ (plc) and kappa‐toxin (colA) genes, which was restored by the complementation of intact VR‐RNA, indicating that the VR‐RNA region plays an important role in the regulation of the plc and colA genes in C. perfringens. It was found that the regulatory effect was observed even when the hyp7 gene encoded on VR‐RNA was deleted or a nonsense mutation was introduced in the hyp7‐coding region. We found that the small 3′‐portion of VR‐RNA was sufficient for the activation of toxin genes, which suggested that VR‐RNA itself could act as an RNA regulatory molecule for the plc and colA genes mediating the regulatory information from the VirR/VirS system in C. perfringens.

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

“…The plc gene encodes alpha-toxin and has been found to be indirectly regulated by the VirS-VirR system via VR-RNA (6,41). Previous in vitro binding experiments showed that VirR does not bind to this promoter region (11), as was observed in this study (Fig.…”

“…Two of these open reading frames, CPE0845 and CPE0920, were found to encode hypothetical proteins, while CPE0846 and hyp7 encode the protease alpha-clostripain (40) and the regulatory RNA molecule, VR-RNA (6, 41), respectively. The expression of both CPE0846 and hyp7 has been demonstrated to be VirR dependent, and it is assumed that the expression of CPE0845 and CPE0920 is regulated by VirR in a similar manner (6,40,41).…”

“…The mutation or inactivation of either the virS or virR gene alters the organism's ability to produce the various toxins and enzymes, with perfringolysin O and alphaclostripain production being totally dependent on a functional VirS-VirR system (19,37,40). In addition to its role in extracellular toxin and enzyme production, the VirS-VirR regulatory network is also involved in the regulation of several housekeeping genes and the hyp7 gene, which encodes a regulatory RNA molecule, VR-RNA (6,41).…”

“…Phosphorylated VirS then acts as a phosphate donor for the phosphorylation of the conserved Asp-57 residue of VirR. Once activated, VirR then modulates the transcription of its target genes either directly or by altering the transcription of other regulatory genes, in particular by the action of VR-RNA (6,7,18,41).…”

“…By performing in vitro binding studies, we showed that VirR binds to each of these repeats independently, i.e., binding to the VirR boxes is not cooperative (11). The identification of the VirR boxes (11), in conjunction with the sequencing of the C. perfringens strain 13 genome (38), has led to the identification of putative VirRbinding sites upstream of other genes (38), two of which have been demonstrated to be regulated by VirR (40,41).…”