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In this work, we report that flavohemoglobin contributes to the azole susceptibility of Staphylococcus aureus. We first observed that deletion of the flavohemoglobin gene leads to an increase in the viability of imidazole-treated S. aureus cells and that reversion to the wild-type phenotype occurs upon expression of flavohemoglobin from a multicopy plasmid. Further spectroscopic analyses showed that miconazole, the most efficient azole antibiotic against S. aureus, ligates to heme of both oxidized and reduced flavohemoglobin. The binding of miconazole to oxidized flavohemoglobin, with an association constant of 1.7 x [10.sup.6] [M.sup.-1], typical of a tight, specific binding equilibrium, results in augmentation of the superoxide production by the enzyme. These results are corroborated by in vivo studies showing that imidazole-treated S. aureus cells expressing flavohemoglobin contain a larger amount of reactive oxygen species. Moreover, it was observed that the survival of miconazole-treated S. aureus internalized by murine macrophages is higher for cells lacking flavohemoglobin. Altogether, the present data revealed that in S. aureus, flavohemoglobin enhances the antimicrobial activity of imidazoles via an increase of intracellular oxidative stress. doi: 10.1128/JB.01378-09

In this report we show that inactivation of the putative nitroreductase SAOUHSC_00833 (ntrA) increases the sensitivity of Staphylococcus aureus to S-nitrosoglutathione (GSNO) and augments its resistance to nitrofurans. S. aureus NtrA is a bifunctional enzyme that exhibits nitroreductase and GSNO reductase activity. A phylogenetic analysis suggests that NtrA is a member of a novel family of nitroreductases that seems to play a dual role in vivo, promoting nitrofuran activation and protecting the cell against transnitrosylation.

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or [H.sub.2][O.sub.2] required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and [H.sub.2][O.sub.2] can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Desulfovibrio vulgaris Hildenborough is a good model organism to study hydrogen metabolism in sulfate-reducing bacteria. Hydrogen is a key compound for these organisms, since it is one of their major energy sources in natural habitats and also an intermediate in the energy metabolism. The D. vulgaris Hildenborough genome codes for six different hydrogenases, but only three of them, the periplasmic-facing [FeFe], [[FeNi].sub.1], and [FeNiSe] hydrogenases, are usually detected. In this work, we studied the synthesis of each of these enzymes in response to different electron donors and acceptors for growth as well as in response to the availability of Ni and Se. The formation of the three hydrogenases was not very strongly affected by the electron donors or acceptors used, but the highest levels were observed after growth with hydrogen as electron donor and lowest with thiosulfate as electron acceptor. The major effect observed was with inclusion of Se in the growth medium, which led to a strong repression of the [FeFe] and [[NiFe].sub.1], hydrogenases and a strong increase in the [NiFeSe] hydrogenase that is not detected in the absence of Se. Ni also led to increased formation of the [NiFe].sub.1] hydrogenase, except for growth with [H.sub.2], where its synthesis is very high even without Ni added to the medium. Growth with [H.sub.2] results in a strong increase in the soluble forms of the [[NiFe].sub.1] and [NiFeSe] hydrogenases. This study is an important contribution to understanding why D. vulgaris Hildenborough has three periplasmic hydrogenases. It supports their similar physiological role in [H.sub.2] oxidation and reveals that element availability has a strong influence in their relative expression.

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxi-doreductases (ROOl and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hep and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection. [ABSTRACT FROM AUTHOR]