Discussion This work has shown that the Fnr protein of B. cereus is homodimeric and can bind one [4Fe-4 S] ABT-263 price iron-sulfur cluster per monomer. Our first challenge was to accurately assemble the Fe-S cluster via an enzymatic system since all our attempts to purify holoFnr under anaerobiosis failed. We demonstrated that CsdA from E. coli was capable of assembling the B. cereus Fnr Fe-S cluster. Interestingly, B. cereus synthesizes [13]one pyridoxal 5-phosphate-containing enzyme (NP_834652) [13] that might be involved in Fe-S cluster biogenesis. When anaerobically reconstituted B. cereus Fnr was exposed to O2, we observed

a rapid loss of the Fe-S cluster, demonstrating that Fnr functions as an oxygen sensor via its Fe-S cluster. Importantly, the cluster of the reconstituted B. cereus Fnr appeared extremely unstable, judging from its fast destruction on exposure to air. In this respect, the B. subtilis holoFnr, which is the closest homolog of B. cereus Fnr [14] displayed greater stability [8]. Sequence comparison of the B. cereus and B. subtilis Fnr revealed a significant variation in the amino acid

residues around the three C-terminal cysteine residues (C219-X 2-C222-X4-C227) that serve as ligands for the cluster (Additional file 3) [7]. These observations LCL161 clinical trial imply that the occurrence of certain amino acid residues close to the cluster ligands may affect the stability of the B. cereus holoFnr, thus providing a possible explanation for its high susceptibility to oxygen damage [15]. As a result, B. cereus Fnr might sense subtle changes in the redox status of the cells, a property that would reflect an adaptation of the pathogenic strain to the environment of its

human host. We proposed previously that B. cereus apoFnr binds promoter regions of enterotoxins only through the monomer pathway. In other words, we proposed that apoFnr was Defactinib in vitro active as a DNA-binding protein only under its monomeric form [9]. Here we showed that, when produced in a tag-less form, apoFnr is active as a DNA binding protein under its dimeric form. In addition, Sulfite dehydrogenase we showed that dimeric apoFnr-DNA complexes were stable in contrast to what we observed previously [9]. We conclude that (i) in our previous studies, tags fused at the N-terminus and C-terminus of Fnr introduced steric hindrance that affected its oligomeric structure and/or DNA binding activity and (ii) B. cereus apoFnr may bind DNA both through the dimer and the monomer pathway under aerobiosis unlike its homologues of B. subtilis and E. coli[8]. There are probably many variables affecting the choice for a monomer or dimer recognition pathway in vivo. Among them, there is the redox state of the cell that may impact directly the ratio of monomeric to dimeric apoFnr since we observed that the addition of reductant (DTT) affected the dimerization state of apoFnr in solution.