subtilis ICG-001 manufacturer Fnr [7, 8]. By contrast, B. cereus Fnr appeared active in DNA-binding protein in its apo-form (cluster-free form). This has led to the conclusion that unlike its B. subtilis homologue, B. cereus Fnr is active in both its apo-form and its holo-form (bearing a Fe-S cluster) [9]. However, data evidencing that B. cereus Fnr could coordinate a Fe-S cluster under anaerobiosis were lacking. Here, we show that purified B. cereus apoFnr can bind one [4Fe-4S]2+ cluster per monomer upon incubation with iron, cysteine and cysteine desulfurase. Reconstituted Fnr (also referred to as holoFnr) showed enhanced DNA binding activity within the fnr promoter, but

no activity difference with regard to the hbl and nhe promoters. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary

complex. Our results lend novel insight into the molecular control of enterotoxin gene expression in anaerobically-grown B. cereus. Results B. cereus apoFnr binds a labile [4Fe-4S]2+ cluster B. cereus Fnr was expressed as a tag-less polypeptide learn more in aerobically-grown E. coli and purified in three steps as described in Methods. The M r of the purified polypeptide, as estimated by SDS-PAGE under RG-7388 order reducing conditions (with DTT) was 25,000, consistent with the theoretical value of 25,640 deduced from the DNA sequence (Additional file 1). The apparent molecular mass of recombinant Fnr, as determined by analytical gel filtration chromatography and by SDS-PAGE under non-reducing conditions (no DTT or β-mercaptoethanol), was ca. 60,000, indicating that tag less Fnr occurs mainly Adenosine triphosphate as a dimer in solution. As isolated, the Fnr protein was colorless, contains no detectable iron and its UV-visible spectrum did not feature any absorption band other than that at 280 nm (Figure 1). Thus, we have successfully

purified a recombinant tag-less dimeric apo-form of Fnr that is amenable to further investigation in vitro. Figure 1 UV-visible spectroscopy of B. cereus Fnr Fe-S cluster reconstitution. Reconstitution was carried out inside an anaerobic glove box as described in Methods. Time points at which samples were scanned by a UV-visible spectrophotometer are indicated. The ability of apoFnr to bind an iron-sulfur cluster under anaerobiosis was tested in an enzyme-driven reconstitution system involving the cysteine desulfurase (CsdA) from E. coli (see details in Methods). During anaerobic reconstitution, a brown colour developed resulting from a time-dependent increase of a broad absorbance band around 416 nm, typical for [4Fe-4S] containing proteins (Figure 1). After 90-min reconstitution and subsequent gel filtration, the purified brown-colored protein displayed an A 416/A 280 ratio of 0.33 and was found to contain 3.6 ± 0.1 moles of iron atoms per mole of monomer. These data are consistent with the reconstitution of one [4Fe-4S] cluster per Fnr monomer [8, 10].