Mechanisms of Adaptation to Nitrosative Stress in<i>Bacillus subtilis</i>

Rogstam, Annika; Larsson, Jonas; Kjelgaard, Peter; Wachenfeldt, Claes von

doi:10.1128/jb.01782-06

Cited by 65 publications

(61 citation statements)

References 43 publications

(46 reference statements)

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“…In the presence of oxygen, the Hmp enzyme acts as an NO scavenger and is responsible for the direct detoxification of NO to nitrate (35,79). Furthermore, it has been shown that hmp mutants of B. subtilis and S. aureus, as well as of Salmonella enterica and E. coli, are hypersensitive to NO-related killing (59,83,84,89). Thus, the upregulation of Hmp is a central feature of NO stress adaptation in both B. subtilis and S. aureus.…”

Section: Resultsmentioning

confidence: 99%

Nitric Oxide Stress Induces Different Responses but Mediates Comparable Protein Thiol Protection inBacillus subtilisandStaphylococcus aureus

et al. 2008

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The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.Bacillus subtilis and Staphylococcus aureus are gram-positive model bacteria. Whereas B. subtilis is considered to be harmless, S. aureus is a facultative pathogen and the leading cause of nosocomial and community-acquired infections. Both microorganisms have to cope with high amounts of nitric oxide (NO) (up to the M range) generated from coexisting denitrifying bacteria or from the innate immune response of the host, respectively (17,31,55,68). Hence, the ability of B. subtilis and S. aureus to protect themselves against NO might be crucial for survival in their respective natural habitats.The cytotoxic properties of the small, lipophilic, and freely diffusible radical NO are attributed to its high reactivity. Indirect effects of NO are caused by the reaction of the radical with oxygen or superoxide, resulting in the formation of a number of additional reactive nitrogen species, including nitrogen dioxide, peroxynitrite, and dinitrogen trioxide. These nitrogen species differ in reactivity, stability, and biological activity but result in a broad spectrum of antimicrobial activity (25). In general, reactive oxygen and nitrogen species can interact with numerous targets, including thiols, metal centers, tyrosine residues in proteins, nucleotide bases, and lipids (20,69). NO directly affects the activity of enzymes by the reaction with bound free radicals or with metal centers (51, 72, 100). For example, the formation of metal-nitrosyl complexes in respiratory enzymes was shown to inhibit bacterial respiration (8,13,71,88) and the formation of a dinitrosyl-iron complex of a protein essential for branched-chain am...

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Section: Resultsmentioning

confidence: 99%

Nitric Oxide Stress Induces Different Responses but Mediates Comparable Protein Thiol Protection inBacillus subtilisandStaphylococcus aureus

et al. 2008

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“…NO oxidation to nitrite and nitrate is also facilitated by transition metals and intracellular enzymes, e.g. by flavohemoglobin and truncated hemoglobin (25)(26)(27)(28)(29). Thus, the level of nitrite and nitrate in the medium indirectly reflects NOS activity.…”

Section: Resultsmentioning

confidence: 99%

Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner

Gusarov

Starodubtseva

Wang

et al. 2008

Journal of Biological Chemistry

137

142

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Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.

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“…However, it would be expected that this ArsR-like protein plays an important role in regulating norB during infection, when iron is limiting and Fur would not always be bound to its operator. It has recently been shown that NsrR controls the expression of NO detoxification systems in several pathogenic and environmental organisms, and that genes in the NsrR regulon contribute to resistance to nitrosative stress (Rodionov et al, 2005;Overton et al, 2006;Filenko et al, 2007;Gilberthorpe et al, 2007;Rock et al, 2007;Rogstam et al, 2007). NsrR also regulates NO metabolism in N. meningitidis, and meningococcal reduction of NO produced by activated macrophages has been shown to greatly modulate the resulting cytokine and chemokine response, enhance intracellular survival and inhibit macrophage apoptosis (Stevanin et al, 2005Tunbridge et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae

et al. 2008

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The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulatory effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene (norB) needs to be elucidated. To accomplish this, we analysed the functional regions of the norB upstream region. The promoter contains an extended "10 motif (TGNTACAAT) that is required for high-level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the "10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 bp inverted repeat sequence immediately downstream of the extended "10 motif gave high levels of aerobic expression of a norB : : lacZ fusion. Insertional inactivation of gonococcal nsrR, predicted to bind to this inverted repeat sequence, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In Escherichia coli, expression of a gonococcal nsrR gene repressed gonococcal norB; induction of norB occurred in the presence of exogenously added NO. NsrR also regulates aniA and dnrN, as well as its own expression. We also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homologue, a second repressor whose putative binding site overlaps the Fur binding site.

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Mechanisms of Adaptation to Nitrosative Stress inBacillus subtilis

Cited by 65 publications

References 43 publications

Nitric Oxide Stress Induces Different Responses but Mediates Comparable Protein Thiol Protection inBacillus subtilisandStaphylococcus aureus

Nitric Oxide Stress Induces Different Responses but Mediates Comparable Protein Thiol Protection inBacillus subtilisandStaphylococcus aureus

Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner

cis- and trans-acting elements involved in regulation of norB (norZ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae

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