Response ofBacillus subtilisto Nitric Oxide and the Nitrosating Agent Sodium Nitroprusside

Abstract

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Nitric oxide (NO) is a lipophilic, freely diffusible radical that can inhibit enzymes, damage DNA, initiate lipid peroxidation, and exacerbate peroxide-induced damage (49,53,66). NO chemistry can be divided into those reactions that occur between NO and biomolecules (direct effects) and those reactions that can only occur subsequent to NO reacting with oxygen or superoxide to form reactive nitrogen oxide species (RNOS) (indirect effects). Direct effects of NO include the formation of metal-nitrosyl complexes (63) and reactions with lipid-derived (50) and other high-energy radicals (34). For example, NO coordinates free or enzyme-bound Fe(II) to form Fe-NO as described for cytochrome P450 (36, 43, 64). Fe nitrosylation leads to altered activity of at least two bacterial metalloregulatory proteins: Fur and Fnr (13,14). Indirect effects occur after NO reacts with either oxygen to generate N 2 O 3 or superoxide to generate the highly reactive oxidant peroxynitrite (OONO Ϫ ). Peroxynitrite rapidly decomposes to form nitrate (NO 3 Ϫ ), hydroxyl radical ( ⅐ OH), and nitrogen dioxide radical ( ⅐ NO 2 ). Some RNOS have the propensity to react with thiol groups and amines to form S-nitrosothiols and nitrosamines.Bacteria encounter NO from a variety of sources. Macrophages of the mammalian immune system generate NO with an inducible NO synthase as part of their arsenal employed against microbial pathogens (38,57). NO synthases have also been identified in some bacteria, including Bacillus subtilis (1, 2), although their function in many cases remains elusive.Denitrifying bacteria produce NO as an intermediate in the reduction pathway from nitrate to dinitrogen (33). B. subtilis is not capable of denitrification, yet it coexists with denitrifying bacteria in subsurface environments. It is therefore likely that B. subtilis has developed a targeted response to exogenously and perhaps endogenously produced NO.Several bacterial enzymes can alleviate NO stress. The Escherichia coli flavohemoglobin Hmp has NO reductase activity under anaerobic conditions (31) and NO dioxygenase (22) or denitrosylase activity (25) under aerobic conditions; however, only the aerobic Hmp activities appear to confer NO stress resistance (20). In addition, both E. coli and Salmonella enterica hmp mutants are hypersensitive to NO (41,56,57). Nakano showed that B. subtilis hmp is regulated by ResDE, a two-comp...

Peroxide‐Sensing Transcriptional Regulators in Bacteria

“…Exposure of B. subtilis to either an NO bolus or the NO generator Na-nitroprusside results in PerR-mediated induction of the PerR regulon during growth under aerobic and anaerobic conditions (77). The observation that anaerobic growth in the presence of Mn 2ϩ reduces NO-induced expression of the PerR regulon while high iron levels enhance it suggests that induction is mediated by PerR-Zn 2ϩ -Fe 2ϩ .…”

Catalase (KatA) and Alkyl Hydroperoxide Reductase (AhpC) Have Compensatory Roles in Peroxide Stress Resistance and Are Required for Survival, Persistence, and Nasal Colonization inStaphylococcus aureus

“…In B. subtilis, the fur mutant has a growth defect in the presence of SNP (39). Moore et al (39) suggested that this is due to the formation of Fe-nitrosyl complexes, which leads to the release of nitroxyl anion and formation of nitrous oxide (45). The ahpC katA mutant of S. aureus was more sensitive to SNP than the wild type but less than the fur mutant.…”

Biochemical Characterization of the Structural Zn2+ Site in the Bacillus subtilis Peroxide Sensor PerR

“…If similar values pertain to B. subtilis, this would explain why, in the presence of both iron and manganese in the growth medium, PerR is predominantly in the peroxide-sensing PerR:Zn,Fe form. Apparently in cells grown in manganese-supplemented minimal medium (with no added iron), the intracellular level of iron is lowered sufficiently to allow the PerR:Zn,Mn form of the repressor to predominate (2,25). Yet because of the relatively higher affinity for Fe 2ϩ , a small increase in intracellular free Fe 2ϩ could shift the repressor from the PerR:Zn,Mn to the Per-R:Zn,Fe form.…”

“…In medium containing iron or iron and manganese, induction is efficient with complete derepression elicited by Ͻ100 M H 2 O 2 or by exposure to NO-generating agents. In contrast, in low iron minimal medium supplemented with manganese, the resulting PerR:Zn,Mn form of the repressor is relatively insensitive to peroxide-and NO-mediated inactivation (2,10,25).…”