The 16 S rRNA was used as a loading control. Surprisingly, the

ubiGmccBAluxS operon was not regulated in response to cysteine availability in transcriptome despite the presence upstream of ubiG of a T-boxCys element with all the conserved motifs of functional T-boxes (Fig. 5). MccB-type enzymes have both cystathionine γ-lyase and homocysteine γ-lyase activities [8]. To demonstrate a possible repression of this operon by cysteine, we tested the homocysteine γ-lyase activity of MccB by zymogram (Fig. 7) [19]. Using crude extracts of strain 13 grown Akt inhibitor with 0.5 mM cystine or 1 mM homocysteine as sole sulfur source, the homocysteine γ-lyase activity of MccB cannot be detected (Fig. 7, lane 1 and 2). However, it has been previously shown that the master regulator of virulence, VirR via a small regulatory RNA, the VR-RNA, negatively regulates ubiG expression [28, 46]. Thus, we tested the homocysteine γ-lyase activity in the strain 13 inactivated for the virR gene (TS133), the vrr gene encoding the VR-RNA (TS140)

or selleck compound the virX gene (TS186) encoding another regulatory RNA controlling toxin production [25, 27]. We detected by zymogram the homocysteine γ-lyase activity of MccB in crude extracts of these 3 mutants (Fig. 7, lane 3-8). This activity was about 100-fold higher in crude extracts of strains grown in the presence of homocysteine than in the presence of cystine. We then realized a qRT-PCR analysis using oligonucleotides hybridizing with ubiG. With RNAs extracted from TS133 (virR::tet), TS140 (vrr::tet), or TS186 (virX::erm), ubiG expression is respectively 45-, 67- and 250-fold greater in the presence of homocysteine than in the presence of cystine. This confirmed the results obtained with MccB activity and indicated that ubiG transcription drastically increased during cysteine depletion in the tested mutants. The cysteine specific T-box system is very likely involved in the induction of expression of the ubiG operon fantofarone involved in sulfur metabolism and AI-2 production during cysteine limitation. Actually, a T-boxCys is also present upstream of the ubiGmccBluxSmccA operon of C. botulinum and the

ubiGmccBA operon of C. acetobutylicum [9]. However, the regulation of ubiG expression in C. perfringens and C. acetobutylicum seems to differ. In C. acetobutylicum, the T-boxCys is not fully functional and the control of the ubiG operon involves mainly antisense RNAs whose expression is repressed in the presence of methionine via an S-box riboswitch [19]. Figure 7 Modulation of MccB synthesis in the presence of homocysteine or cystine in various mutants. The homocysteine γ-lyase activity of MccB was detected on zymogram. A total of 100 μg of crude extracts were charged on a native polyacrylamide gel (12%). The release of sulfide from homocysteine due to homocysteine γ-lyase activity was detected by the precipitation of insoluble PbS.