The properties of the Fe-S cluster indicate that Fnr is essentially present in the apo- form in aerobically grown B. cereus, and may occur BKM120 mw in both apo- and holo- forms in anaerobically-grown bacteria, the ratio between the two forms depending on the redox status of the cells, as detected by the Fnr cluster (Figure 7). The stability of the holo form might also be FK228 datasheet modulated through interactions with DNA, protein partners and (or) low-molecular weight thiols [16–18]. Given the higher DNA binding affinity of the holo form compared with the apo form to its

own promoter, we assume that higher levels of Fnr (apo + holo) are produced under anaerobiosis than under aerobiosis (Figure 7). In addition, on the basis of these and earlier results, we offer evidence that Fnr can (i) activate the expression of genes encoding the enterotoxin-activators resD and plcR and (ii) associate with PlcR and ResD to form a ternary complex under both anaerobiosis and aerobiosis [4, 5, 9, 11]. By producing higher levels of Fnr [5], anaerobically-grown B. cereus cells might produce higher levels of

the tripartite Fnr-ResD-PlcR complex and, as a result, higher levels of Hbl and Nhe. Hence, the interconversion between apo- and holoFnr I-BET151 supplier may well be a key factor in controlling the regulation of enterotoxin gene expression through the Fnr/PlcR/ResD complex. Figure 7 Proposal for the Fnr-dependent regulation of the hbl and nhe enterotoxin genes in B. cereus. (A) apo- and holoFnr-dependent regulation in either the absence

or presence of oxygen. (B), Fnr is thought to be part of a ternary complex involving ResD (black), PlcR (white), Fnr (gray), acting as positive regulator. Conclusions In conclusion, this work brings further evidence that B. cereus Fnr, unlike its counterpart from B. subtilis, is an active transcriptional regulator in both its apo- and holo- forms. This property may enable B. cereus to ensure optimal enterotoxin gene expression in response to changes in oxygen tension such as those encountered during infection of the human host. Cediranib (AZD2171) Methods Bacterial strains and growth conditions Escherichia coli strain TOP10 (Invitrogen) was used as the general cloning host, and strain BL21 CodonPlus(DE3)-RIL (Stratagene) was used to overexpress fnr and resD. E. coli strain BL21λDE3, containing the pRep4 plasmid [19] was used to overexpress plcR[12]. E. coli strains were routinely grown in Luria broth at 37°C. Recombinant expression of fnr, resD and plcR and protein purifications The coding sequence for B. cereus fnr was PCR amplified from F4430/73 genomic DNA using primers PET101F (5′-CACCATGACATTATCTCAAG-3′) and PET101R (5′-CTAATCAATGCTACAAACAGAAGC-3′). The amplicon was cloned as a blunt-end PCR product into pET101/D-TOPO (Invitrogen), yielding pET101fnr. B. cereus Fnr was produced as a recombinant protein in aerobically grown E. coli BL21(pET101fnr).