7% β-Gal activity was observed using 100 μM 2,2′-dipyridyl, an iron chelator) compared with high-iron conditions Ku-0059436 supplier (50 μM FeCl3 and 50 μM haem, 34% and 26% β-Gal activity, respectively) (Fig. 4). The results suggested that repression of mbfA by IrrAt does not require iron or haem as a cofactor. To further identify amino acid residues that are important for the iron sensing of IrrAt in mediating the derepression of mbfA, the iron responsiveness of the mutant IrrAt proteins (pIRR38, pIRR45, pIRR65,

pIRR86, pIRR92, pIRR93, pIRR94, pIRR105, pIRR127, pIRRHHH and pIRRHHH86) in the iron regulation of mbfA-lacZ was compared with wild-type IrrAt (pIRR) (Fig. 4). A single mutation in IrrAt at H38, D86, H92, H93 or D105 led to a hyper-repressed phenotype in which the expression of mbfA-lacZ was low and was not derepressed in response to iron and haem (Fig. 4). These residues appeared to play a role in the iron responsiveness of IrrAt. Although single mutations at H45, H65 and H127 reduced the repressor activity of IrrAt, the mutant proteins still retained iron responsiveness (Fig. 4). In contrast, the H94 mutant protein showed a greater reduction in repressor activity, and its iron responsiveness was lost (Fig. 4). The results suggested that H45, H65, H94 and H127 may play a role in the DNA-binding selleck compound ability

of IrrAt. Moreover, H94 was involved in iron sensing by IrrAt. The iron responsiveness was also lost in the HHH mutant protein, which likely resulted from the mutation at H94 (Fig. 4). The HHH86 mutant protein showed a hyper-repressed phenotype (Fig. 4). In conclusion, site-directed mutagenesis analysis revealed that residues H45, H65, H94 and H127 and the HHH motif are important for the repressor function of IrrAt. Mutations at these key residues may cause changes in protein conformation, preventing the protein from functioning properly. Single mutations at H38, D86, H92, H93 and D105 led to a hyper-repressed phenotype

(Fig. 4), implying that these residues CYTH4 may be directly or indirectly involved in the iron or haem binding and the iron-responsive regulatory function of IrrAt. Interestingly, only mutation at D86 was able to restore the repressor function of IrrAt that lacked the HHH motif (Fig. 2b). Residue D86 of IrrAt is equivalent to E80 of FurPa (the structural zinc-binding site) and to E90 of FurHp (regulatory site S2) (Fig. 1). It is possible that the conformation of the HHH mutant protein may undergo further structural modifications due to the mutation of D86 such that the HHH86 mutant protein is readily able to interact with DNA and may lock the protein in its DNA-binding conformation, resulting in loss of iron responsiveness. Residue H94 of IrrAt is a part of the conserved HHH motif (Fig. 1), which is a domain involved in haem sensing and in the IrrBj and IrrRl regulatory switch through different mechanisms (Qi et al.