The inability of Bacillus licheniformis perR mutant to grow is mainly due to the lack of PerR-mediated fur repression.

PerR, a member of Fur family protein, is a metal-dependent H2O2 sensing transcription factor that regulates genes involved in peroxide stress response. Industrially important bacterium Bacillus licheniformis contains three PerR-like proteins (PerRBL, PerR2, and PerR3) compared to its close relative Bacillus subtilis. Interestingly, unlike other bacteria including B. subtilis, no authentic perR BL null mutant could be established for B. licheniformis. Thus, we constructed a conditional perR BL mutant using a xylose-inducible promoter, and investigated the genes under the control of PerRBL. PerRBL regulon genes include katA, mrgA, ahpC, pfeT, hemA, fur, and perR as observed for PerRBS. However, there is some variation in the expression levels of fur and hemA genes between B. subtilis and B. licheniformis in the derepressed state. Furthermore, katA, mrgA, and ahpC are strongly induced, whereas the others are only weakly or not induced by H2O2 treatment. In contrast to the B. subtilis perR null mutant which frequently gives rise to large colony phenotype mainly due to the loss of katA, the suppressors of B. licheniformis perR mutant, which can form colonies on LB agar, were all catalase-positive. Instead, many of the suppressors showed increased levels of siderophore production, suggesting that the suppressor mutation is linked to the fur gene. Consistent with this, perR fur double mutant could grow on LB agar without Fe supplementation, whereas perR katA double mutant could only grow on LB agar with Fe supplementation. Taken together, our data suggest that in B. licheniformis, despite the similarity in PerRBL and PerRBS regulon genes, perR is an essential gene required for growth and that the inability of perR null mutant to grow is mainly due to elevated expression of Fur.

The difference in in vivo sensitivity between Bacillus licheniformis PerR and Bacillus subtilis PerR is due to the different cellular environments.

PerR, a member of Fur family of metal-dependent regulators, is a major peroxide sensor in many Gram positive bacteria, and controls the expression of genes involved in peroxide resistance. Bacillus licheniformis, a close relative to the well-studied model organism Bacillus subtilis, contains three PerR-like proteins (PerRBL, PerR2 and PerR3) in addition to Fur and Zur. In the present study, we characterized the role of PerRBL in B. licheniformis. In vitro and in vivo studies indicate that PerRBL, like PerRBS, uses either Fe2+ or Mn2+ as a corepressor and only the Fe2+-bound form of PerRBL senses low levels of H2O2 by iron-mediated histidine oxidation. Interestingly, regardless of the difference in H2O2 sensitivity, if any, between PerRBL and PerRBS, B. licheniformis expressing PerRBL or PerRBS could sense lower levels of H2O2 and was more sensitive to H2O2 than B. subtilis expressing PerRBL or PerRBS. This result suggests that the differences in cellular milieu between B. subtilis and B. licheniformis, rather than the intrinsic differences in PerRBS and PerRBLper se, affect the H2O2 sensing ability of PerR inside the cell and the H2O2 resistance of cell. In contrast, B. licheniformis and B. subtilis expressing Staphylococcus aureus PerR (PerRSA), which is more sensitive to H2O2 than PerRBL and PerRBS, were more resistant to H2O2 than those expressing either PerRBL or PerRBS. This result indicates that the sufficient difference in H2O2 susceptibility of PerR proteins can override the difference in cellular environment and affect the resistance of cell to H2O2.

Bacillus licheniformis Contains Two More PerR-Like Proteins in Addition to PerR, Fur, and Zur Orthologues.

The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur, and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur.

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation.

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerRBS protein, represses target genes when bound to either Mn(2+) or Fe(2+) as corepressor, but only the Fe(2+)-bound form responds to H2O2. The orthologous protein in the human pathogen Staphylococcus aureus, PerRSA, plays important roles in H2O2 resistance and virulence. However, PerRSA is reported to only respond to Mn(2+) as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H2O2 sensing. Here we demonstrate that PerRSA uses either Fe(2+) or Mn(2+) as corepressor, and that, like PerRBS, the Fe(2+)-bound form of PerRSA senses physiological levels of H2O2 by iron-mediated histidine oxidation. Moreover, we show that PerRSA is poised to sense very low levels of endogenous H2O2, which normally cannot be sensed by B. subtilis PerRBS. This hypersensitivity of PerRSA accounts for the apparent lack of Fe(2+)-dependent repressor activity and consequent Mn(2+)-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerRSA is directly correlated with virulence, whereas it is inversely correlated with H2O2 resistance, suggesting that PerRSA may be an attractive target for the control of S. aureus pathogenesis.