The VirR Response Regulator from Clostridium perfringens Binds Independently to Two Imperfect Direct Repeats Located Upstream of the pfoA Promoter

Abstract

Regulation of toxin production in the gram-positive anaerobe Clostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virR gene. We have constructed a VirR expression plasmid in Escherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoR genes, which encode alpha-toxin, collagenase, and a putative pfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoA promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing

“…Thus, RNase Y affects virRS gene expression at the transcriptional level, not at the posttranscriptional level. The expression of the VirR direct target gene pfoA, whose promoter sequence contains the VirR protein-binding box (15), also decreased. However, transcript stability was not affected by RNase Y depletion (Fig.…”

“…The two-component system (TCS) VirR/VirS regulates a number of toxin genes (13,14). The response regulator, VirR, is phosphorylated and activated by a sensor kinase, VirS, inducing target gene expression (15,16). The transcription of a small RNA (sRNA), VR-RNA, is activated by VirR/VirS; this sRNA is an effector molecule and directly or indirectly regulates Ͼ100 genes (17).…”

The Pseudomonas aeruginosa AlgZR two-component system coordinates multiple phenotypes

“…Some of the more well-studied regulators in this family include YehT of E. coli (Kraxenberger et al, 2012), AgrA of S. aureus (Koenig et al, 2004; Sidote et al, 2008), FsrA of Enterococcus faecalis (Del Papa and Perego, 2011), VirR of Clostridium perfringens (Cheung and Rood, 2000; Cheung et al, 2004), and PlnC and PlnD of Lactobacilus plantarum (Risoen et al, 1998, 2001; Straume et al, 2009). A common characteristic of these regulators is their mode of binding to DNA (Sidote et al, 2008).…”

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate‐polyacrylamide gels

“…The stains might be particularly useful for monitoring the efficient removal of oligohistidine tags from fusion proteins by protease digestion as well [15]. Furthermore, the stains should provide a convenient means for monitoring protein-protein or protein-DNA interactions by gel mobility shift assay [16][17][18][19][20]. The Pro-Q Sapphire dyes should also be useful in mapping protease susceptibility sites on N-terminally and C-terminally oligohistidine-tagged proteins, as recently described using biotinylated NTA [21].…”