Redox Active Thiol Sensors of Oxidative and Nitrosative Stress

Abstract

Significance: The reactivity of the thiol in the side chain of cysteines is exploited by bacterial regulatory proteins that sense and respond to reactive oxygen and nitrogen species. Recent Advances: Charged residues and helix dipoles diminish the pKa of redox active cysteines, resulting in a thiolate that is stabilized by neighboring polar amino acids. The reaction of peroxides with thiolates generates a sulfenic acid (–SOH) intermediate that often gives rise to a reversible disulfide bond. Peroxide-induced intramolecular and intermolecular disulfides and intermolecular mixed disulfides modulate the signaling activity of members of the LysR/OxyR, MarR/OhrR, and RsrA family of transcriptional regulators. Thiol-dependent regulators also help bacteria resist the nitrosative and nitroxidative stress. −SOHs, mixed disulfides, and S-nitrosothiols are some of the post-translational modifications induced by nitrogen oxides in the thiol groups of OxyR and SsrB bacterial regulatory proteins. Sulfenylation, disulfide bond formation, S-thiolation, and S-nitrosylation are reversible modifications amenable to feedback regulation by antioxidant and antinitrosative repair systems. The structural and functional changes engaged in the thiol-dependent sensing of reactive species have been adopted by several regulators to foster bacterial virulence during exposure to products of NADPH phagocyte oxidase and inducible nitric oxide synthase. Critical Issues: Investigations with LysR/OxyR, MarR/OhrR, and RsrA family members have helped in an understanding of the mechanisms by which thiols in regulatory proteins react with reactive species, thereby activating antioxidant and antinitrosative gene expression. Future Directions: To define the determinants that provide selectivity of redox active thiolates for some reactive species but not others is an important challenge for future investigations. Antioxid. Redox Signal. 17, 1201–1214.

Introduction

T he sulfur atom in cysteine is strategically positioned in some regulatory proteins to function as a sensor of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The oxidation states of cysteine vary from −2 to +6, making cysteine the most versatile redox sensor of all amino acids, and organic and inorganic cofactors (46). Thiols and thiolates, and the oxidized sulfenic, sulfinic, or disulfide derivatives often occur in biomolecules. The cysteine oxidation states are kept in equilibrium through enzymatically catalyzed oxidoreduction reactions. Signaling cascades in living organisms take advantage of both the reversibility of cysteine oxidation states and the structural changes associated with oxidized cysteines to coordinate adaptive responses to reactive species engendered by endogenous metabolic processes or exogenous enzymatic or chemical means.

By mediating electron donation, disulfide formation, hydrolysis, metal binding, and redox catalysis and sensing, the sulfur in cysteines can fulfill a wide variety of molecular functions (33, 46). Bacterial redox sensors of various reactive species utilize cysteines because of their ability to bind metals and adopt various oxidation states. Most of the regulators presented in this review rely on the redox-sensing activity of the free thiolate side chain of cysteines. This review also discusses the mechanisms by which redox active cysteines in zinc fingers help bacteria sense ROS. By analogy to other metalloproteins, the cysteines in zinc fingers provide a scaffold where a structural metal cation is assembled. In addition to serving a structural function, redox active cysteines in zinc fingers transmit signals in response to ROS. Free cysteine side chains and cysteines bound in zinc fingers are common strategies of thiol-dependent bacterial redox sensors of ROS and RNS. To learn how cysteines indirectly sense oxidative and nitrosative stress through ligation of redox active metal prosthetic groups, the reader is directed to the accompanying reviews in this forum.

The contribution of thiol-dependent signaling to the resistance of bacteria to oxidative stress was first hinted at in investigations with the frank pathogen Salmonella enterica serovar Typhimurium (20). Many investigations on thiol switches have been carried out in commensal Escherichia coli and saprophytic environmental bacteria of the species Bacillus subtilis and Streptomyces coelicolor [see (1, 77) for recent reviews]. A growing number of Gram-positive and Gram-negative pathogens are now known to apply the signaling properties of thiol switches in order to increase fitness during their associations with vertebrate hosts. The mechanisms by which thiol-dependent redox sensors respond to ROS and RNS can be broadly categorized within families of transcriptional regulators, irrespective of whether they are expressed by commensal, saprophytic, or pathogenic bacteria. Consequently, the mode of action by which active thiols sense reactive species is presented herein within the context of families of transcriptional regulators, and their contributions to bacterial pathogenesis are highlighted. A section in this review also discusses the structural and chemical determinants that impact the sensing activity of cysteines. The last section presents the dominant chemistry driving the interaction of ROS and RNS with redox active cysteines.

Sensing of Oxidative and Nitrosative Stress by Members of the LysR/OxyR Family of Transcriptional Regulators

The first indication that bacteria adapt to oxidative stress was the observation that the exposure of Salmonella to sublethal concentrations of hydrogen peroxide (H₂O₂) elicits hyperresistance to a subsequent challenge with high amounts of this ROS (20). OxyR was identified as a member of the LysR family of transcriptional activators after mapping of an interesting transposon mutant named oxyR2 (21). The critical role that the oxyR locus plays in the adaptation of E. coli to oxidative stress was demonstrated by the fact that OxyR-deficient bacteria are readily killed by H₂O₂ (20, 21). In addition to regulating antioxidant resistance, OxyR helps coordinate the expression of antinitrosative defenses (38). OxyR is less responsive to nitrogen oxides than to H₂O₂, especially if the bacterial cytoplasm is rich in glutathione (GSH) (38, 111). Nonetheless, E. coli oxyR mutants are hypersusceptible to the bacteriostatic effects of nitric oxide (NO) (38). Why is it that a highly specialized H₂O₂ sensor such as OxyR can also be modulated by RNS? Nitroxidative stress may explain this conundrum. Most of the biological chemistry of NO is dominated by oxidative processes (61). Taking into consideration this notion, the OxyR-regulated GSH oxidoreductase, glutaredoxin (Grx), thioredoxin (Trx), catalase, and alkyl hydroperoxide reductase re-establish thiol homeostasis and detoxify a variety of peroxides (9, 45, 71), independently of whether the bacterial cells are exposed to oxidative or nitroxidative species.

OxyR is a redox active regulator that maintains thiol homeostasis

The exposure of Salmonella to sublethal concentrations of H₂O₂ induces the expression of about 30 proteins (20). Hydroperoxidase I, alkyl hydroperoxide reductase, the nonspecific DNA-binding protein Dps, Grx1, GSH oxidoreductase, Trx 2, and the DsbG disulfide isomerase are some of the gene products positively regulated by oxidized OxyR (21, 44, 111). The first two enzymes detoxify H₂O₂, organic hydroperoxides, and peroxynitrite (ONOO⁻), while the iron-binding Dps protein protects DNA from Fe⁺⁺-catalyzed, H₂O₂-dependent Fenton chemistry (9, 37, 83). GSH oxidoreductase, Grx1, DsbG disulfide isomerase, and Trx 2 re-establish thiol/disulfide homeostasis and protect the cell against disulfide stress. Thiol/disulfide exchange systems exert feedback regulation over oxidized OxyR, as suggested by the fact that OxyR is more easily activated by ROS and RNS in E. coli strains deficient in GSH oxidoreductase or Grx1 (38, 111). Moreover, the intramolecular disulfide bond of OxyR is a substrate of thiol/disulfide exchange by Grx1 (111).

Mechanisms of sensing oxidative and nitrosative stress by OxyR redox active thiols

OxyR activates gene transcription after the intramolecular oxidation of strategically situated cysteines (Fig. 1). It was noted that oxidized OxyR drives katG transcription in vitro (90). Although both oxidized and reduced OxyR bind target genes, they do it with different conformations and outcomes. Oxidized, tetrameric OxyR cooperatively binds four adjacent major DNA grooves, whereas reduced OxyR binds two pairs of adjacent DNA grooves separated by a single helical turn (96). Oxidized OxyR binds the RNA polymerase and drives transcription; the reduced protein does neither.

FIG. 1.

Hydrogen peroxide (H₂O₂) sensing by OxyR. Reduced OxyR binds to DNA promoter regions of target genes, thereby preventing transcription (A). Oxidation of the OxyR Cys¹⁹⁹ thiolate group (–S⁻) by H₂O₂ generates a sulfenic acid (–SOH) derivative that attacks the Cys²⁰⁸ thiol side chain. The resulting intramolecular disulfide bond drives the structural changes in OxyR that allow for the recruitment of RNA polymerase and the activation of transcription (B). The Cys¹⁹⁹-Cys²⁰⁸ disulfide bond is reduced by glutaredoxin 1 (Grx1), a member of the OxyR regulon.

Oxidation of the thiolate side chain of OxyR Cys¹⁹⁹ to a −SOH precedes the dramatic structural changes responsible for the activation of OxyR (111). The attack on Cys²⁰⁸ by the Cys¹⁹⁹ sulfenic derivative generates an intramolecular disulfide that drives massive conformational changes in the protein (18, 63, 93, 111). Unexpectedly, mutational analysis indicates that Cys¹⁹⁹ is more important than Cys²⁰⁸ for the activation of OxyR (111). The fact that a C208S substitution renders a partially constitutive active OxyR variant rationalizes the seemingly greater importance of Cys¹⁹⁹ over Cys²⁰⁸ (63). Having a redox potential of −185 mV (111), Cys¹⁹⁹ is maintained in the reduced form in the −280 mV potential of the cytoplasm of resting E. coli. However, these thermodynamic considerations cannot explain the rapid oxidation of OxyR in response to small rises in the intracytoplasmic concentration of H₂O₂. OxyR is oxidized within 30 s after E. coli is treated with 5 μM H₂O₂, a concentration of peroxide that does not appreciably change the ratio of the oxidized glutathione/GSH redox couple (2, 93). OxyR and H₂O₂ react with a second-order rate constant of about 10⁵ M ⁻¹ s⁻¹ (2). Therefore, kinetics rather than thermodynamics drives the fast oxidation of OxyR by H₂O₂. On the other hand, re-reduction of OxyR is a slow process that takes about 5–10 min (2, 93). The delay in the re-reduction of OxyR allows for the transient expression of antioxidant defenses encoded in the regulon.

In addition to undergoing oxidation in response to H₂O₂, OxyR gets S-nitrosylated in E. coli exposed to RNS (38, 55). Similar to the oxidized protein (90), S-nitrosylated OxyR drives katG transcription in vitro (38). In addition to S-nitrosylating OxyR, nitrogen oxides modify the Cys¹⁹⁹ thiol side chain to sulfenylated and S-glutathionylated derivatives (55). All these covalent modifications affect the secondary α-helical structure of OxyR. These RNS-modified OxyR products are transcriptionally active; however, they differ in cooperativity, structure, DNA-binding affinity, and promoter activity. One of the most salient conclusions that can be drawn from these investigations lies in the realization that a redox active regulator does not have to be a simple switch that alternates between “ON” and “OFF” positions, but one whose activity can be fine tuned according to the nature of its post-translational modifications.

Contribution of LysR/OxyR regulators to bacterial pathogenesis

OxyR is dispensable for the pathogenesis of Mycobacterium tuberculosis and Sal. enterica. The M. tuberculosis oxyR gene has several inactivating nonsense and frameshift mutations, and oxyR-deficient Salmonella are completely virulent (25, 95). However, members of the OxyR regulon such as Dps are required for Salmonella virulence (37). dps and other members of the regulon are controlled by several regulators, which may explain the dispensability of OxyR in Mycobacterium and Salmonella. For example, the expression of dps in Salmonella is under the control of the ferric uptake regulator Fur (109), a gene product that is not only under OxyR regulation, but also essential for Salmonella pathogenesis (98, 104). One of the advantages of putting dps under the control of Fur is that its expression becomes responsive to iron levels as well as ROS and RNS (23, 94). Curiously, the OxyR-regulated KatG catalase capable of detoxifying high fluxes of H₂O₂ is dispensable for the survival of Salmonella in macrophages sustaining a respiratory burst (10). Several other catalases in the Salmonella genome may compensate for a lack of KatG (4).

In contrast to Salmonella and M. tuberculosis, OxyR augments the virulence potential of pathogenic microorganisms such as E. coli and Pseudomonas aeruginosa (47, 62). E. coli is an extraordinarily diverse species, encompassing common inhabitants of human and animal intestines as well as enterotoxigenic, enterohemorrhagic, and uropathogenic strains associated with high morbidity and mortality in humans and a variety of domestic animals. OxyR potentiates the ability of E. coli to cause ascending urinary tract infections (47). Curiously, OxyR-dependent antioxidant defenses do not antagonize the ROS produced by the NADPH phagocyte oxidase (47). Instead, OxyR may foster E. coli virulence by enhancing resistance to the reactive species generated during endogenous metabolic processes. According to this idea, alkyl hydroperoxide reductase, a member of the OxyR regulon, is quite effective at detoxifying low micromolar concentrations of H₂O₂, but it is inefficient against the large fluxes of H₂O₂ usually associated with inflammation (87). OxyR also augments the virulence of P. aeruginosa in rodent and insect models of infection (62). Future studies may reveal that other members of the growing LysR/OxyR family of transcriptional regulators do, in fact, foster colonization, invasion, and growth of many pathogenic microorganisms during their associations with vertebrate hosts. Recent evidence indicates that the redox-sensing activity of Cys²³⁵ in AphB, a member of the LysR/OxyR family of transcriptional regulators, is necessary for toxigenic Vibrio cholerae to colonize the anaerobic environment of the mammalian intestine (67).

Sensing of Oxidative Stress by Members of the MarA/OhrR Family of Transcriptional Regulators

The DNA binding domain in the MarA/OhrR family of transcriptional regulators has a characteristic winged helix-turn-helix. A growing number of regulators in the MarA/OhrR family have been noted as containing one or two redox-active cysteines. OhrR homologs have been described in Staphylococcus aureus, Str. coelicolor A3(2), Xanthomonas campestris, and P. aeruginosa (3, 15, 72, 75, 92). A great deal of what we know about OhrR homologs has been unmasked while studying OhrR from the soil saprophyte B. subtilis. Reduced OhrR binds to the operator regions of target genes via a winged helix-turn-helix (Fig. 2), and, thus, represses the transcription of antioxidant defenses (31). The exposure of B. subtilis to organic hydroperoxides increases antioxidant defenses through the oxidation of OhrR (30).

FIG. 2.

Regulation of gene transcription by the peroxide sensor OhrR. The dimeric, reduced OhrR protein represses the transcription of target genes in Bacillus subtilis (A). Organic hydroperoxides oxidize the OhrR Cys¹⁵ −S⁻ to yield a remarkably stable −SOH (B). Sulfenylated OhrR binds to the operator regions of target genes; however, the −SOH is S-thiolated by low-molecular-weight thiols (LMWTs), driving the conformational changes associated with the dissociation of OhrR from DNA (C). The dissociation of oxidized OhrR allows the recruitment of RNA polymerase to the promoters of several antioxidant genes, including thioredoxins (Trxs) and Trx reductase involved in thiol/disulfide exchange reactions. Staphylococcus aureus MgrA and SarZ regulators are homologs of B. subtilis OhrR. Gram-negative Xanthomonas campestris and Pseudomonas aeruginosa also express OhrR-like repressors; however, these regulators use two redox-active cysteines to sense oxidative stress (not shown).

Mechanisms of sensing oxidative stress by MarA/OhrR thiol switches

In Gram-positive bacteria, members of the MarA/OhrR family of transcriptional regulators sense oxidative stress through a lone cysteine residue, whereas their counterparts in Gram-negative bacteria involve two cysteines. A lone cysteine in B. subtilis OhrR, Sta. aureus SarZ, and Sta. aureus MgrA is oxidized by organic hydroperoxides to a remarkably stable −SOH (15, 31, 84). The formation of a −SOH is necessary but not sufficient to derepress transcription. In fact, reduced and sulfenylated B. subtilis OhrR and Sta. aureus SarZ bind with apparently similar affinities to the operator regions of target genes (64, 84). In B. subtilis, sulfenamide generated from the reaction of sulfenylated Cys¹⁵ with the amide group of the adjacent Phe¹⁶ may trigger the conformational changes required for the dissociation of OhrR from the operator regions (64). However, the OhrR sulfenamide derivative is far less abundant than an S-thiolated, mixed disulfide-bonded derivative (64). The −SOH in oxidized OhrR is prone to nucleophilic substitution with endogenous, low-molecular-weight thiols (LMWTs) (64, 84). Both mixed disulfides and sulfenamide are reversible modifications and, hence, the DNA binding activity of OhrR-like proteins can be restored on re-reduction. Together, these data unequivocally identify the redox-active thiol in members of the OhrR family of transcriptional regulators as a genuine signaling switch.

As just mentioned, the OhrR homologs in Gram-negative bacteria have two redox-active cysteines (13, 14, 80). In X. campestris, OhrR Cys²² is oxidized by organic hydroperoxides to −SOH, which rapidly reacts with Cys¹²⁷ to form an intermolecular disulfide in the homodimer (80). As expected, disulfide-linked dimer formation drives the dissociation of X. campestris OhrR from DNA. P. aeruginosa MexR also has two redox-active cysteines (13). Organic hydroperoxides trigger the oxidation of MexR Cys³⁰ and Cys⁶² to form two intermolecular disulfides with Cys⁶²’ and Cys³⁰’ of the homodimer (14). Disulfide formation is the driving force behind the depression of the mexAB-oprN drug efflux operon, and it increases the resistance of P. aeruginosa to antibiotics (14).

Contribution of the redox-sensing activity of the MarA/OhrR family members to bacterial pathogenesis

The contribution of the MarR/OhrR members to bacterial pathogenesis has been investigated in Sta. aureus and P. aeruginosa. A Sta. aureus sarZ mutant is attenuated in both silkworms and a 24-h mouse model of infection, but regains full virulence at later times of the infection (50). The transitory contribution of SarZ to Sta. aureus virulence probably reflects the complex SarZ regulatory network. SarZ regulates the expression of 87 genes in Sta. aureus (6, 16), including the repression of sarS and the induction of genes encoding exoproteins and cell wall biosynthesis. Decreased expression of these genes could contribute to the initial attenuation of sarZ-deficient Sta. aureus. However, by derepressing antioxidant defenses, the lack of sarZ increases the resistance of Sta. aureus to organic hydroperoxides and H₂O₂ (16). Moreover, an sarZ mutant forms denser biofilms, another aspect that may also foster Sta. aureus pathogenesis (92). In comparison to SarZ, MgrA plays a much greater and more durable role in Sta. aureus virulence, as measured by bacterial growth in viscera and the degree of sinovitis and arthritis affecting infected animals (48). The predominant role of MgrA over SarZ in the pathogenesis of Sta. aureus is not surprising, as MgrA regulates sarZ as well as more than 300 other genes (48, 69) such as those encoding α-hemolysixnα-toxin, capsular polysaccharide biosynthesis, coagulase, efflux pumps, leukotoxins, serine proteases, and sortase A.

In analogy to Sta. aureus, the opportunistic pathogen P. aeruginosa expresses two members of the MarR/OhrR family of transcriptional regulators, OhrR and OspR. A P. aeruginosa strain lacking ospR is hypervirulent in an acute pneumonia model of infection, because the derepression of GSH peroxidase enhances resistance to H₂O₂ (60). However, P. aeruginosa OhrR mutants are hyperresistant to organic hydroperoxides, but attenuated in a Caenorhabditis elegans model of infection (3). These findings indicate that the resistance to oxidative stress does not always equate to enhanced virulence. The overproduction of antioxidant defenses may, in turn, elicit reductive stress that leads to detrimental changes in gene expression.

Sensing of Oxidative Stress by Redox Active Thiols That Coordinate Zinc

Most of the characterization of RsrA and the σ^R regulon has been done in the soil saprophyte Str. coelicolor A3(2). The σ^R regulon coordinates the response of Streptomyces to disulfide and oxidative stress associated with diamide, H₂O₂, and the superoxide (O₂ ^•−) generator plumbagin (78), suggesting that members in this regulatory pathway are prone to redox control. The σ^R protein lacks cysteines. In contrast, its cognate antisigma factor RsrA has several cysteines, raising the possibility that redox active thiols in RsrA could mediate the response of the σ^R regulon to oxidative challenges (Fig. 3).

FIG. 3.

Regulation of antioxidant defenses by thiols in zinc-finger-containing antisigma factors. Cys¹¹, Cys⁴¹, Cys⁴⁴, and His³⁷ coordinate a zinc finger in the antisigma factor RsrA of the soil saprophyte Streptomyces coelicolor (A). Zinc-bound RsrA sequesters its cognate σ^R sigma factor to the vicinity of the bacterial membrane (M). Disulfide stress and several reactive oxygen species (ROS) oxidize the triggering Cys¹¹, which forms a disulfide bond with Cys⁴⁴. The disulfide bond releases the zinc cation, and promotes structural changes that drive dissociation of the RsrA-σ^R complex (B). The liberated σ^R recruits the RNA polymerase to the promoters of genes encoding antioxidant defenses. Similar to OhrR, Trx-mediated thiol/disulfide exchange exerts feedback regulation over the system. Mycobacterium tuberculosis RshA is a homolog of the Str. coelicolor RsrA protein.

Regulation of antioxidant defenses by members of the σ^R family of extracytoplasmic function sigma factors

A functional σ^R is necessary for the proper expression of disulfide reductase activity in Str. coelicolor (78). Further analysis revealed that σ^R in fact regulates transcription of the trxAB operon encoding Trx 1 and Trx reductase, and the trxC-encoded Trx 2 (78, 79). A search of the Str. coelicolor genome for conserved σ^R binding sequences identified a total of 27 σ^R targets (79). These include the aforementioned trxC, and genes in the cysteine and molybdepterin biosynthetic pathways. σ^R also regulates the expression of msrA and msrB methionine sulphoxide reductase genes, the mshA-encoded mycothiol biosynthesis gene, and the mca-endoced amidase (81). The expression of these genes is consistent with the fact that the oxidation of RsrA derepresses σ^R-dependent antioxidant defenses. Oxidation of the cell also induces the accumulation of unfolded proteins in cytoplasmic aggregates (73). It is, therefore, not surprising that σ^R directly regulates Clp and Lon ATP-dependent AAA(+) proteases in order to eliminate damaged proteins (51). M. tuberculosis expresses σ^H, a σ^R homolog. Detailed transcriptional analysis indicates that σ^H regulates 31 genes in M. tuberculosis, including genes involved in Trx recycling, a Grx-like protein, ferredoxin, chaperones, and universal stress proteins (53, 70, 85). Therefore, it is becoming clear that actinomycetes use σ^R-like extracytoplasmic function sigma factors which regulate the transcription of classical antioxidant defenses and stress responses to unfolded proteins.

Mechanisms of sensing oxidative stress by the RsrA antisigma factor

The metalloprotein RsrA binds a single zinc cation with an affinity of 10¹⁷ M ⁻¹ (5, 76). The zinc metal is ligated by His³⁷, Cys⁴¹, and Cys⁴⁴ that form a characteristic HX₃CX₂C zinc-containing antisigma (ZAS) motif (49, 76, 110). Cys¹¹ completes the tetrahedral coordination of zinc by RsrA (110). Genetic studies indicate that Cys¹¹, Cys⁴¹, and Cys⁴⁴ are essential for the antisigma activity of RsrA (76). Cys¹¹ is the triggering residue that responds to oxidative stress, most likely by forming a −SOH intermediate that attacks the neighboring thiol Cys⁴⁴ (5, 66, 76). The importance of Cys¹¹ as the triggering residue responsive to oxidative stress is circumstantially supported by the fact that members of the ZAS family of transcriptional regulators that lack a cysteine at this position are indifferent to oxidative stress (66). In addition to Cys¹¹-Cys⁴⁴, a second disulfide forms between Cys⁴¹ and Cys⁶¹ (5). Disulfide formation releases zinc from RsrA (66). Nonetheless, disulfide bond formation, rather than the dissociation of zinc from the protein, increases the α-helical structure of RsrA that releases σ^R and allows it to transcriptionally activate target genes (66).

Similar to its RsrA homolog in Str. coelicolor, the M. tuberculosis RshA antisigma factor is responsive to oxidative stress (89). The binding of RshA to σ^H can also be modulated in response to metabolic changes in the cell. Thr⁹⁴ is phosphorylated by the eukaryotic-like serine/threonine protein kinase PknB (36, 82). By analogy to the oxidized protein, phosphorylated RshA binds less efficiently to σ^H (82). The oxidation of thiols in the RshA zinc finger and phosphorylation of RshA Thr⁹⁴ may facilitate the graded regulation of M. tuberculosis gene transcription in response to shifting redox and metabolic challenges.

RsrA is redox active

The oxidative modifications that mediate redox signaling are often reversible. Strains of Str. coelicolor deficient in mycothiol biosynthesis exhibit increased expression of σ^R, suggesting that decreases in intracellular mycothiol content could be the natural signal that drives the oxidation of RsrA and the consequent activation of the σ^R regulon (81). Both Trx and mycothiol limit RsrA disulfide bond formation, thereby stimulating the binding of this antisigma factor to σ^R (52, 81). On the other hand, σ^R activates the transcription of Trxs and the mycothiol biosynthetic pathway. These findings demonstrate that RsrA is a bona fide redox switch subjected to feedback regulation by members of the regulon.

Role of redox-active antisigma factors in bacterial pathogenesis

σ^H defends the human pathogen M. tuberculosis against both RNS generated from acidified nitrite, and ROS such as organic hydroperoxides, H₂O₂, and O₂ ^•− (24, 28, 70, 85). σ^H is expressed by M. tuberculosis inside macrophages (34). However, this extracytoplasmic function sigma factor does not increase the intracellular fitness of mycobacteria (70), and M. tuberculosis σ^H mutants grow and persist with apparent normality in murine models of infection (53). Nonetheless, σ^H contributes to M. tuberculosis pathogenesis, and its expression affords M. tuberculosis the ability to recruit CD4⁺ and CD8⁺ T lymphocytes to well-formed granulomas in the lungs of infected mice (53). It remains to be investigated whether the immune response triggered by M. tuberculosis is related to the expression of σ^H-dependent antioxidant defenses.

Sensing of Nitrosative Stress by Members of the NarL/SsrB Family of Transcriptional Regulators

The response regulator SsrB is the only member of the NarL/FixL family of transcriptional regulators that has so far been shown as possessing a redox-active cysteine. In contrast to the thiol switches just described, SsrB is a regulator of genes dedicated to bacterial pathogenesis. SsrB and its cognate SsrA sensor kinase form a two-component regulatory system essential for the intracellular replication of Salmonella (22). By analogy to other two-component regulatory systems, the SsrA sensor kinase phosphorylates Asp⁵⁶ in the receiver domain of SsrB, unfolding the DNA binding domain for activation of gene transcription. SsrB binds to a flexible AT-rich palindrome sequence in ancestral and horizontally acquired genes (11). The phosphorylated SsrB protein relieves the gene silencing imposed by polymerized H-NS nucleoid protein on target DNA (105). SsrB partially binds to the predicted σ⁷⁰ −35 region and activates cyclic AMP receptor protein-type class II promoters independently of the αCTD subunit of the RNA polymerase (105).

More than 100 loci are regulated by SsrB, including the 25 kb Salmonella pathogenicity island 2 (SPI2) that encodes a secretion apparatus, chaperones, translocon, the SsrA/SsrB two-component regulatory system, and effector proteins (39, 97, 108). SPI2 effectors translocated into the host cell cytoplasm help remodel Salmonella phagosomes and Salmonella-containing vacuoles. SsrB increases antioxidant and antinitrosative defenses (7, 12, 32, 91, 100, 101, 103); however, it does it in dramatically different ways from other regulators discussed in this review (Fig. 4). OxyR, OhrR, and RshA regulate the expression of classical enzymatic detoxification systems, whereas SsrB lessens oxidative and nitrosative stress of intracellular Salmonella by activating the expression of the SPI2 type III secretion system that decreases contact of phagosomes with incoming NADPH phagocyte oxidase- and inducible NO synthase-containing vesicles. In addition, the SPI2 type III secretion system prevents phagolysosome formation, while promoting the interactions of Salmonella-containing vesicles with nutrient-rich vacuoles in the exocytic pathway (58, 86, 99). As a consequence of the vacuolar remodeling, the SPI2 type III secretion system allows Salmonella to survive and grow in a variety of epithelial cells and mononuclear phagocytes, thereby playing a vital role in the course of systemic infection (74, 88). SsrB and the SPI2 type III secretion system are essential for Salmonella pathogenesis, as Salmonella strains lacking a functional SPI2 type III secretion system are attenuated at least five orders of magnitude (88).

FIG. 4.

Strategies of antioxidant defenses regulated by thiol-based regulators. Regulatory proteins bearing thiol switches induce the expression of classical antioxidant defenses for the detoxification of peroxides or repair intramolecular or intermolecular disulfides (A). P in the glutaredoxin and thioredoxin reactions denotes any protein. OxyR disulfide bond is a substrate of Grx1. Trx reductase catalyses the reduction of a disulfide bond in Trxs using the reducing power of NADPH. The Salmonella pathogenicity island 2 (SPI2) type III secretion system regulated by the SsrB response regulator minimizes the contact of Salmonella phagosomes with vesicles containing NADPH phagocyte oxidase or inducible nitric oxide synthase (iNOS) and lysosomes, while helping establish contact with the trans-Golgi network (TGN) (B). By doing so, the SPI2 type III secretion system lessens the contact of Salmonella with a variety of ROS and reactive nitrogen species (RNS), thereby indirectly fostering antioxidant and antinitrosative defenses.

As it will become clear next, SsrB Cys²⁰³ is modified by RNS (43). A Salmonella strain expressing the SsrB C203S variant exhibits wild-type virulence in an acute model of infection, which is consistent with the fact that this allele is transcriptionally active (11, 43). Nonetheless, a strain of Salmonella bearing the SsrB C203S variant is attenuated in the context of nitrogen oxides produced in an Nramp^R murine model of oral infection (43). These findings suggest that Cys²⁰³ in the C-terminal dimerization domain of the SsrB response regulator is a sensor of RNS produced in the course of a gastrointestinal infection.

Regulation of SsrB function by the redox-active Cys²⁰³

As is the case for other response regulators, phosphorylation of a key aspartate in the receiver domain triggers the binding of SsrB to cognate promoters (11, 27). Recent investigations indicate that in addition to classical regulation by phosphorylation, two other signals influence SsrB's modus operandi. The EIIA^Ntr component of the nitrogen-metabolic phosphotransferase system prevents the detrimental hyperactivation of SPI2 transcription by post-translationally precluding the association of SsrB to target promoters (19). The DNA-binding activity of SsrB is also subject to regulation by RNS (43). Cys²⁰³ in the α4 helix of the dimerization domain is susceptible to S-nitrosylation and oxidation by RNS such as ONOO⁻, and products of the reaction of NO and oxygen (O₂). ONOO⁻ oxidizes the thiol group of Cys²⁰³ to a mixture of sulfenic, sulfinic, and sulfonic oxidative products (43). C203D or C203E substitutions mimicking the negative charge of high-order oxidative products of ONOO⁻-treated SsrB block dimerization and the transcriptional activity of SsrB (11, 43). The side chain of Cys²⁰³ interacts with the hydrophobic residue Leu¹⁹² located in the loop between the DNA-binding H3 helix and the dimerization H4 helix (11). Sulfinylation or sulfonylation of Cys²⁰³ would disrupt this interaction, and, thus, alter the secondary structure required for homodimerization of SsrB, DNA binding, and transcription. Accordingly, the electronegative charge introduced by C203D or C203E substitutions inhibits transcriptional activity (11, 43). S-nitrosylation would not be expected to alter the interactions between Cys²⁰³ and Leu¹⁹². However, S-nitrosylation is not likely to be a terminal modification, but a precursor of further oxidations and/or S-thiolation. The extent to which these modifications prevent the formation of SsrB homodimers and affect gene transcription awaits further investigation.

Determinants That Define the Redox Activity of Cysteines

Cysteines are abundant in proteins, but only a few of them are modified by ROS and RNS. What makes a cysteine become redox active? A comprehensive review by Stamler and collaborators, and elegant recent work by the Ischiropoulos' group coincide to identify electrostatic interactions that affect pKa, hydrophobicity, and allosteric constraints as important determinants for the S-nitrosylation of selected thiols (26, 35, 40). These principles also dictate the reactivity of thiols toward ROS. Thiol-based bacterial redox sensors discussed in this review nicely illustrate the physical and chemical determinants that affect the reaction of redox-active cysteines with peroxides and nitrogen oxides.

Redox mechanism for the formation of thiolate in OxyR

Cys¹⁹⁹ is located in a hydrophobic pocket between two α/β domains surrounded by acidic and basic residues (18). His¹³⁰, Glu¹⁵¹, His¹⁹⁸, Arg²⁰¹, Asp²⁰², and Arg²⁶⁶ may influence the reactivity of OxyR with H₂O₂ (18, 59). Arg²⁶⁶ has been shown to lower the pKa of Cys¹⁹⁹ (18). The positive effect of Arg²⁶⁶ on Cys¹⁹⁹ redox capacity is demonstrated by the fact that a strain of E. coli expressing the oxyR R266A variant is both hypersusceptible to oxidative stress and defective in oxyS expression in response to H₂O₂ (18). Dipoles from neighboring α-helices may also influence the ionization of Cys¹⁹⁹ thiol (18). The Cys¹⁹⁹ thiolate is likely stabilized by hydrophobic interactions with Leu²⁰⁰, Leu²²⁴, and Pro²⁴¹ (18). The reaction of the thiolate with H₂O₂ forms a −SOH that forces Cys¹⁹⁹ out of the hydrophobic pocket. This, in turn, induces flexibility of the region between 205 and 216 residues where Cys²⁰³ resides, thereby increasing the probability of collision between sulfenylated Cys¹⁹⁹ and reduced Cys²⁰⁸ to form a disulfide bond (63).

Sensing by MarR/OhrR family members

The proximity of charged residues within 6 Å of a thiol group increases its potential to be ionized (26). No basic residues are found within a 7-Å distance from the OhrR Cys¹⁵ side chain (42). Instead, a positively charged macrodipole in helix 1 decreases the B. subtilis OhrR Cys¹⁵ pKa value to 5.2 (42). Therefore, this cysteine is kept in the ionized, thiolated form under physiological conditions, ready to react with incoming organic hydroperoxides. The negative charge of the thiolate in helix α1 is stabilized by hydrogen bonding with the side chains of Tyr²⁹ and Tyr⁴⁰ in helix α2 (42). The mutations of Tyr²⁹ and Tyr⁴⁰ have shown that hydrogen bonding to the Cys¹⁵ thiolate is important for B. subtilis OhrR sensing of organic hydroperoxides (42). The residues that form hydrogen bonding with the redox active thiolate and a hydrophobic pocket near it are conserved between B. subtilis OhrR and Sta. aureus SarZ and MgrA repressors (15, 42, 84). Hydrogen bonding is also important for the stabilization of the sulfenic modifications of SarZ Cys¹³ and MgrA Cys¹². S-thiolation disrupts hydrogen bonding, thereby triggering a conformational change that lowers the affinity of these repressors for DNA.

Activation of thiols in the zinc-binding RsrA antisigma factor

Zinc can increase the reactivity of cysteine by lowering the pKa of the coordinating thiol side chains. However, similar to the situation described for the eukaryotic protein Sp1 (56), zinc protects the redox-active cysteines of RsrA from oxidative stress (66). Since zinc actually lowers the reactivity of the triggering thiolate in the finger, other determinants should activate the critical cysteine(s). The electronegative charge provided by glutamate residues flanking the HX₃CX₂C ZAS motif serves as an important redox determinant for the reactivity of RsrA (49). It remains to be investigated to what extent these glutamate residues influence the pKa and reactivity of the Cys¹¹ triggering switch.

Activation of SsrB Cys²⁰³

The fact that Cys²⁰³ reacts with nitrogen oxides and forms a disulfide bond with Cys^203’ of the homodimer (11, 43) indicates that the thiol group of this cysteine is redox active. The determinants that make SsrB Cys²⁰³ redox active have yet to be identified. However, lessons learned with the bacterial regulators just mentioned and well-characterized redox active cysteines in a variety of proteins help make some informed predictions on how the environment surrounding SsrB Cys²⁰³ may dictate the redox activity of this response regulator (Fig. 5). As predicted by the fact that most reactive cysteines are overrepresented in α-helices (26), Cys²⁰³ is located in the SsrB's H4 α-helix. Basic residues at position 2 and acid residues at positions −3 and −4 influence the pKa of thiol groups (35). The analysis of the flanking residues around Cys²⁰³ indicate the presence of both positive and negative residues, raising the possibility that Cys²⁰³ is ionized in the cytoplasm of Salmonella. Of note, the guanidine group of Arg²⁰⁶ is 4.1 Å from the Cys²⁰³ thiol side chain. Moreover, the carboxyl group of Glu¹⁹⁹ is 6.1 Å from Cys²⁰³. This configuration raises the possibility that the Cys²⁰³ side chain is, in fact, in the thiolate form. The hydrophobicity of the H4 helix and Leu¹⁹² located only 4.2 Å away could stabilize the Cys²⁰³ thiolate. These interesting scenarios await further investigation.

FIG. 5.

Model for the activation of the thiol side chain in SsrB Cys²⁰³. The redox active, RNS-modifiable Cys²⁰³ is located in the H4 α-helix dimerization domain of SsrB (11). Analysis of the nuclear magnetic resonance structure of the C-terminus domain of SsrB reveals that the sulfur atom of Cys²⁰³ is 4.1 Å from the positively charged guanidine group of Arg²⁰⁶. The proximity of Arg²⁰⁶ is likely to draw the proton from Cys²⁰³, keeping this cysteine in the ionic, redox-active, thiolated state. The carboxyl group of Glu¹⁹⁹ likely influences the pKa of the Cys²⁰³ thiol group located 6.1 Å away. The Cys²⁰³ −S⁻ could be stabilized through hydrophobic interactions with Leu¹⁹² located 4.2 Å away.

Chemistry Underlying the Modification of Redox-Active Thiols by ROS and RNS

Redox active thiol groups involved in signaling react selectively with ROS and RNS. The resulting oxidative or nitrosative modifications are often reversible. The mechanism by which thiol-dependent bacterial redox sensors react with peroxides is well understood (Fig. 6); however, several models could explain the reactivity of thiol switches with RNS for the formation of S-nitrosothiols.

FIG. 6.

Biological chemistry for the reaction of thiols with peroxides. Thiol groups of reactive cysteines are kept in the thiolated state through interactions with nearby charged residues. The thiolate reacts with H₂O₂ or organic hydroperoxides to form a −SOH. In B. subtilis OhrR, and Sta. aureus MgrA and SarZ, the sulfenylated cysteine can be quite stable. However, in most cases, the −SOH attacks nearby thiols, forming disulfide bonds that promote structural and functional changes in the protein. Disulfide bonds can form between cysteines within a protein (e.g., Escherichia coli OxyR) or cysteines in the homodimer (e.g., X. campestris OhrR). Mixed disulfides can also arise between the sulfenylated protein and LMWTs (e.g., Sta. aureus SarZ). In addition, a −SOH can attack a nearby amide group, giving rise to a sulfenamide (e.g., B. subtilis OhrR). All of these cysteine oxidation states are reversible, therefore providing flexibility as signaling switches. The −SOH intermediate can be oxidized further to sulfinic and sulfonic acids, which, for most part, are irreversible signatures of oxidative stress.

Selectivity of the reaction of thiols and ROS

The rate constants of hydroxyl radical, O₂ ^•−, and H₂O₂ with LMWTs are 10¹⁰, 30–1000, and 20 M ⁻¹ s⁻¹, respectively (106, 107). Although it has high affinity for thiols, the hydroxyl radical reacts nonspecifically with nearly all biomolecules, and, therefore, it is not endowed with the discriminatory power to serve as a signaling molecule. Superoxide dismutases detoxify O₂ ^•− with a rate constant of 2×10⁹ M ⁻¹ s⁻¹ (29), making the O₂ ^•−-dependent oxidation of thiols of little physiological importance. On the other hand, H₂O₂ can react with fast kinetics with certain thiols. For example, the second-order rate constant for the reaction of OxyR Cys¹⁹⁹ and H₂O₂ is about 10⁵ M ⁻¹ s⁻¹ (2), making this regulator an excellent H₂O₂ sensor. As discussed elsewhere in this review, OxyR Cys¹⁹⁹ reacts with RNS, but with apparently lower affinity than ROS. It is currently unknown why OxyR Cys¹⁹⁹ in particular and redox active cysteines in general exhibit preferential affinity toward selected reactive species. To define the determinants that provide selectivity of redox-active thiolates for some reactive species but not others is an important goal for the future.

Oxidation of redox active thiols by H₂O₂

Although the reactivity of thiols with ROS is inversely related to their pKa (107), there are examples where this does not seem to be the case (106). In addition to a low pKa that ionizes the thiol to a thiolate, the environment of the protein is critical for the stability and reactivity of the thiolate. Reactive thiols in MarR, LysR, and zinc-finger family members just discussed have low pKa values and are generally located in hydrophobic pockets. As described in other systems (83), the two-electron oxidation of the redox-active thiolate in the regulators just presented by H₂O₂ or organic hydroperoxides gives rise to a sulfenic group. The oxidation of a thiol to a −SOH is not enough to transmit the signal in most regulatory proteins. The −SOH is frequently oxidized to a disulfide in order to drive the structural changes that modulate protein function. In the case of E. coli OxyR and Str. coelicolor RsrA, intramolecular disulfides are responsible for major structural changes, whereas mixed disulfides are necessary in the case of B. subtilis OhrR. In addition to mediating dramatic changes in secondary structures that transduce the signal, disulfide bonds prevent further oxidation of cysteine −SOHs to high-order oxidative species such as sulfinic and sulfonic groups. Disulfides are reversible modifications that on reduction turn off the signal elicited by H₂O₂, whereas the oxidation of thiols to sulfinic and sulfonic are, for the most part, irreversible and less amenable for signaling. Although the reaction of cysteines with peroxides is facilitated when the sulfur in the side chain is a thiolate, the modification of cysteines by other reactive species does not have to be limited to the ionic form of cysteines. For example, as will be discussed next, radicals can promote modifications of cysteines through a sulfenyl (i.e., thiyl) radical intermediate.

Selective reaction of thiols with peroxides

Not all redox active cysteines react equally with ROS. Cys¹⁵ of B. subtilis OhrR is an excellent case in point. Antioxidant defenses and intrinsic allosteric determinants explain the selective reaction of B. subtilis OhrR with organic hydroperoxides. H₂O₂ can oxidize the thiol of OhrR; however, this reactive species does not appear to be a relevant signal in vivo. The activity of catalases in B. subtilis diminishes the effective concentration of H₂O₂ to levels that are insufficient to initiate the oxidation of the critical cysteine residue in OhrR (31). The reaction of OhrR with organic hydroperoxides is fast. A patch of hydrophobic residues in the vicinity of the redox active cysteine increases the affinity of OhrR for organic hydroperoxides (42). Most eukaryotic organic hydroperoxides are derived from membrane lipids. A radical abstracts an allylic hydrogen in the polyunsaturated hydrocarbon, giving rise to a conjugated diene that is susceptible to the O₂ attack for the generation of lipid hydroperoxides (41). The lack of polyunsaturated hydrocarbons in prokaryotic lipids greatly limits the formation of lipid hydroperoxides in their membranes. Although extremely active when supplied in pure form, it remains to be seen whether organic hydroperoxides are the actual signal for members of the OhrR family of regulators in vivo. The thiol oxidizer diamide also derepresses the OhrR regulon (31), raising the possibility that the cysteines triggered in OhrR may respond to thiol/disulfide exchange reactions. OhrR also appears to be a sensor of hypochlorite stress (17).

Reaction of thiols with RNS

Thiol groups in regulators such as OxyR and SsrB can be modified by RNS. S-nitrosothiols can be generated by several independent mechanisms (Fig. 7). Transnitrosation and radical reactions may account for the nitrosylation of redox-active thiols in the regulators discussed in this review. In the case of OxyR, RNS modify the Cys¹⁹⁹ thiolate to form an S-nitrosothiol. The nitrosonium cation is the most likely candidate that covalently modifies the nucleophilic thiolate of OxyR Cys¹⁹⁹. However, nitrosonium does not exist free in solution. It has consistently been reported that OxyR is more efficiently activated by nitrogen oxides in GSH-deficient E. coli strains, suggesting that transnitrosation reactions from species other than S-nitrosoglutathione are the most likely nitrosating species. Lancaster has proposed that dinitrosyl iron complexes (DNICs) are the most important nitrating agents in the cell (8). DNIC arising from the complexation of NO, iron, and cysteine, or other LMWTs, could be a source of the nitrosonium-like species that S-nitrosylates the OxyR Cys¹⁹⁹ thiolate group.

FIG. 7.

Mechanisms for the formation of S-nitrosothiols in biological systems. The formation of S-nitrosothiols in biological systems involves two dominant biochemistries. Thiolates in redox active cysteines are strong nucleophiles that react with the nitrosonium cation (NO⁺) to form an S-nitrosothiol. Transnitrosation reactions from low-molecular-weight S-nitrosothiols, dinitrosyl iron complexes, and dinitrogen trioxide (N₂O₃) are likely donors of NO⁺. In addition, S-nitrosothiols can be generated through the reaction of thiyl and NO radicals. Nitrogen dioxide (NO₂ ^•) generated in the autoxidation of NO likely mediates the abstraction of an electron from the thiol to form the thiyl radical (S^•).

The Cys²⁰³ of the response regulator SsrB has also been shown to react with nitrogen oxides (43). The DNA binding activity of SsrB is inhibited by high fluxes of NO in the presence of O₂. This indicates that products of the autooxidation of NO mediate the inhibition of binding of SsrB with cognate promoters. Together with DNIC and S-nitrosothiols, dinitrogen trioxide (N₂O₃) is a potent nitrosating agent that serves as a source of nitrosonium groups (54). The production of N₂O₃ is second order for NO, and some investigators have argued that the generation of N₂O₃ is not favored under physiological conditions. Alternatively, nitrogen dioxide produced in the autooxidation of NO could abstract an electron from the thiol group of SsrB Cys²⁰³, generating a thiyl radical that could condensate with the NO radical to form S-nitroso SsrB. As is the case for most biomolecules, the chemical species that mediates S-nitrosylation of SsrB is currently unknown.

Concluding Remarks

Cysteine and methionine have a sulfur atom in their structures. Sulfur in methionine is frequently oxidized to a sulfoxide group. This oxidative modification can be repaired through the enzymatic activity of the methionine sulfoxide reductases. The reactivity of methionines with ROS, the reversibility of the oxidation, and the enrichment of oxidizable methionines in protein surface areas and enzyme active sites have led to the proposal that the sulfur in methionine acts as an endogenous antioxidant which protects biomolecules from the attack of reactive species (65, 68). Despite being redox active, methionine has not yet been found to be a redox-sensing moiety of bacterial transcriptional regulators. Several reasons may explain the predominance of cysteines rather than methionines as common signaling redox determinants. Cysteines and methionines can be found in 10 and 3 different oxidation states in the cell, respectively (46). Thus, cysteines provide a larger repertoire in oxidation states, making it possible to sense diverse ROS and RNS. However, of all possible oxidation states, thiol-dependent bacterial sensors rely quite frequently on thiolates and disulfide bonds as effective ways to transduce a signal. Why would disulfide bonds be a preferred mode of signal transduction? Disulfides are reversible modifications that can be in balance with thiolates by the action of Trx, GSH, and Grx thiol/disulfide exchange systems. Equally important, the dramatic structural rearrangements associated with disulfide bond formation are accompanied by changes in function. A case in point is the fact that the newly formed disulfide between Cys¹⁹⁹ and Cys²⁰⁸ in oxidized OxyR brings together regions separated by 17 Å in the reduced protein (18). This massive structural reorganization allows binding of OxyR to the RNA polymerase with the consequent initiation of transcription.

The regulators discussed in this review reveal variability in the chemistry by which disulfide bonds are generated, and the diverse structural means by which they modulate molecular function (Fig. 8). In addition to the aforementioned intramolecular disulfide bond that drives OxyR transcriptional activity, intermolecular and mixed disulfide bonds affect the function of bacterial regulatory proteins. Intermolecular disulfide bonds in X. campestris OhrR and mixed disulfides in redox active thiolates in lone cysteine OhrR-type regulators are additional ways by which redox-active cysteines transduce signals in response to oxidative and nitrosative stress. The mechanisms underlying the S-thiolation of redox-active cysteines are equally varied in nature. Mixed disulfides in the OhrR-family members have been shown to occur through a −SOH oxidative intermediate. S-thiolation can independently arise through an S-nitrosylated intermediate as described for OxyR (55). In either case, mixed disulfides make possible the structural and functional changes of the oxidized or S-nitrosylated regulatory protein.

FIG. 8.

Changes in protein structure after thiol oxidation. Reduced thiolates in regulatory proteins are susceptible to oxidation by a variety of inorganic or organic hydroperoxides and hypochlorate. Further oxidation of −SOH intermediate to intramolecular, intermolecular, and mixed disulfides induces changes in structure that are followed by switches in molecular function. The −SOH intermediate can also react with adjacent amines to form sulfenamide. In addition, redox active thiols can be modified by peroxynitrite (ONOO⁻) and other RNS for the generation of highly oxidized thiols and S-nitrosothiols. Examples of such regulators are shown in white. Redox-active thiols in regulators such as OxyR can be modified by several reactive species.

Thiol-dependent redox sensing coordinates bacterial responses to changes in physiology and pathogenesis. OxyR, OhrR, RshA, and SsrB add to the antioxidant and antinitrosative defenses of bacteria. In addition, the oxidized thiols in some sensors such as OxyR and RsrA have been shown to be substrates of the antioxidant defenses activated in their regulons. This feedback regulation ensures that redox homeostasis is re-established after the burst of reactive species has subsided. This is an important consideration, as bacterial cells experience fluctuations in oxidative stress during different growth phases and during preferential utilization of certain metabolic pathways (57). In addition, pathogenic microorganisms are exposed to a burst of intense oxidative stress (102). The redox activity of thiol-based sensors increases fitness of saprophytic, commensal, and pathogenic bacteria to fluctuations in oxidative and nitrosative stress in their ever-changing environment.

Since investigations with E. coli and Salmonella discovered the thiol-based H₂O₂ sensor OxyR (20), an increasing number of members within the LysR, MarR, RsrA, and NarL families of transcriptional regulators have been shown as using thiol-based redox sensors that coordinate responses to endogenous and exogenous ROS and RNS. As the range of thiol-based prokaryotic redox sensors broadens, it will be interesting to see the extent that the concepts discussed in this review apply.

Footnotes

Acknowledgments

The author thanks Drs. M. Crawford, J. Helmann, H. Ischiropoulos, J. Jones-Carson, and M. Song for their excellent suggestions. He is also grateful to Dr. C. Henard for discussions and help with the in silico analysis of the SsrB NMR structure. This work was supported by the National Institutes of Health grant AI54959 and the Burroughs Wellcome Fund.

Abbreviations Used

References

Antelmann

, Helmann

. Thiol-based redox switches and gene regulation. Antioxid Redox Signal, 14:1049–1063. 2011.

Aslund

, Zheng

, Beckwith

, Storz

. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc Natl Acad Sci U S A, 96:6161–6165. 1999.

Atichartpongkul

, Fuangthong

, Vattanaviboon

, Mongkolsuk

. Analyses of the regulatory mechanism and physiological roles of Pseudomonas aeruginosa OhrR, a transcription regulator and a sensor of organic hydroperoxides. J Bacteriol, 192:2093–2101. 2010.

Aussel

, Zhao

, Hebrard

, Guilhon

, Viala

, Henri

, Chasson

, Gorvel

, Barras

, Meresse

. Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst. Mol Microbiol, 80:628–640. 2011.

Bae

, Park

, Hahn

, Kim

, Roe

. Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor: zinc release and disulfide bond formation. J Mol Biol, 335:425–435. 2004.

Ballal

, Ray

, Manna

. sarZ, a sarA family gene, is transcriptionally activated by MgrA and is involved in the regulation of genes encoding exoproteins in Staphylococcus aureus. J Bacteriol, 191:1656–1665. 2009.

Berger

, Romero

, Ma

, Wang

, Faubion

, Liao

, Compeer

, Keszei

, Rameh

, Wang

, Boes

, Regueiro

, Reinecker

, Terhorst

. SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages. Nat Immunol, 11:920–927. 2010.

Bosworth

, Toledo

JC.

Jr , Zmijewski

, Li

, Lancaster

JR.

Jr . Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide. Proc Natl Acad Sci U S A, 106:4671–4676. 2009.

Bryk

, Griffin

, Nathan

. Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature, 407:211–215. 2000.

10.

Buchmeier

, Libby

, Xu

, Loewen

, Switala

, Guiney

, Fang

. DNA repair is more important than catalase for Salmonella virulence in mice. J Clin Invest, 95:1047–1053. 1995.

11.

Carroll

, Liao

, Morgan

, Cicirelli

, Li

, Sheng

, Feng

, Kenney

. Structural and functional analysis of the C-terminal DNA binding domain of the Salmonella typhimurium SPI-2 response regulator SsrB. J Biol Chem, 284:12008–12019. 2009.

12.

Chakravortty

, Hansen-Wester

, Hensel

. Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates. J Exp Med, 195:1155–1166. 2002.

13.

Chen

, Hu

, Chen

, Lan

, Li

, Hicks

, Dinner

, He

. The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism. Proc Natl Acad Sci U S A, 105:13586–13591. 2008.

14.

Chen

, Yi

, Zhang

, Ge

, Yang

, He

. Structural insight into the oxidation-sensing mechanism of the antibiotic resistance of regulator MexR. Embo Rep, 11:685–690. 2010.

15.

Chen

, Bae

, Williams

, Duguid

, Rice

, Schneewind

, He

. An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus. Nat Chem Biol, 2:591–595. 2006.

16.

Chen

, Nishida

, Poor

, Cheng

, Bae

, Kuechenmeister

, Dunman

, Missiakas

, He

. A new oxidative sensing and regulation pathway mediated by the MgrA homologue SarZ in Staphylococcus aureus. Mol Microbiol, 71:198–211. 2009.

17.

Chi

, Gronau

, Mader

, Hessling

, Becher

, Antelmann

. S-bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics. Mol Cell Proteomics, 10M111.0095062011.

18.

Choi

, Kim

, Mukhopadhyay

, Cho

, Woo

, Storz

, Ryu

. Structural basis of the redox switch in the OxyR transcription factor. Cell, 105:103–113. 2001.

19.

Choi

, Shin

, Yoon

, Kim

, Lee

, Kim

, Seok

, Ryu

. Salmonella pathogenicity island 2 expression negatively controlled by EIIANtr-SsrB interaction is required for Salmonella virulence. Proc Natl Acad Sci U S A, 107:20506–20511. 2010.

20.

Christman

, Morgan

, Jacobson

, Ames

. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell, 41:753–762. 1985.

21.

Christman

, Storz

, Ames

. OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins. Proc Natl Acad Sci U S A, 86:3484–3488. 1989.

22.

Cirillo

, Valdivia

, Monack

, Falkow

. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol, 30:175–188. 1998.

23.

D'Autreaux

, Touati

, Bersch

, Latour

, Michaud-Soret

. Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron. Proc Natl Acad Sci U S A, 99:16619–16624. 2002.

24.

Darwin

, Ehrt

, Gutierrez-Ramos

, Weich

, Nathan

. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science, 302:1963–1966. 2003.

25.

Deretic

, Philipp

, Dhandayuthapani

, Mudd

, Curcic

, Garbe

, Heym

, Via

, Cole

. Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative-stress regulatory gene: implications for sensitivity to isoniazid. Mol Microbiol, 17:889–900. 1995.

26.

Doulias

, Greene

, Greco

, Tenopoulou

, Seeholzer

, Dunbrack

, Ischiropoulos

. Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation. Proc Natl Acad Sci U S A, 107:16958–16963. 2010.

27.

Feng

, Walthers

, Oropeza

, Kenney

. The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2. Mol Microbiol, 54:823–835. 2004.

28.

Fernandes

, Wu

, Kong

, Puyang

, Garg

, Husson

. A mycobacterial extracytoplasmic sigma factor involved in survival following heat shock and oxidative stress. J Bacteriol, 181:4266–4274. 1999.

29.

Forman

, Fridovich

. Superoxide dismutase: a comparison of rate constants. Arch Biochem Biophys, 158:396–400. 1973.

30.

Fuangthong

, Atichartpongkul

, Mongkolsuk

, Helmann

. OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis. J Bacteriol, 183:4134–4141. 2001.

31.

Fuangthong

, Helmann

. The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative. Proc Natl Acad Sci U S A, 99:6690–6695. 2002.

32.

Gallois

, Klein

, Allen

, Jones

, Nauseef

. Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane. J Immunol, 166:5741–5748. 2001.

33.

Giles

, Giles

, Jacob

. Multiple roles of cysteine in biocatalysis. Biochem Biophys Res Commun, 300:1–4. 2003.

34.

Graham

, Clark-Curtiss

. Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS) Proc Natl Acad Sci U S A, 96:11554–11559. 1999.

35.

Greco

, Hodara

, Parastatidis

, Heijnen

, Dennehy

, Liebler

, Ischiropoulos

. Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells. Proc Natl Acad Sci U S A, 103:7420–7425. 2006.

36.

Greenstein

, MacGurn

, Baer

, Falick

, Cox

, Alber

T.M.

tuberculosis Ser/Thr protein kinase D phosphorylates an anti-anti-sigma factor homolog. PLoS Pathog, 3:e49. 2007.

37.

Halsey

, Vazquez-Torres

, Gravdahl

, Fang

, Libby

. The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence. Infect Immun, 72:1155–1158. 2004.

38.

Hausladen

, Privalle

, Keng

, DeAngelo

, Stamler

. Nitrosative stress: activation of the transcription factor OxyR. Cell, 86:719–729. 1996.

39.

Hensel

. Salmonella pathogenicity island 2. Mol Microbiol, 36:1015–1023. 2000.

40.

Hess

, Matsumoto

, Kim

, Marshall

, Stamler

. Protein S-nitrosylation: purview and parameters. Nat Rev Mol Cell Biol, 6:150–166. 2005.

41.

Hogg

, Kalyanaraman

. Nitric oxide and lipid peroxidation. Biochim Biophys Acta, 1411:378–384. 1999.

42.

Hong

, Fuangthong

, Helmann

, Brennan

. Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family. Mol Cell, 20:131–141. 2005.

43.

Husain

, Jones-Carson

, Song

, McCollister

, Bourret

, Vazquez-Torres

. Redox sensor SsrB Cys²⁰³ enhances Salmonella fitness against nitric oxide generated in the host immune response to oral infection. Proc Natl Acad Sci U S A, 107:14396–14401. 2010.

44.

Imlay

. Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem, 77:755–776. 2008.

45.

Imlay

. Pathways of oxidative damage. Annu Rev Microbiol, 57:395–418. 2003.

46.

Jacob

, Giles

, Sies

. Sulfur and selenium: the role of oxidation state in protein structure and function. Angew Chem Int Ed Engl, 42:4742–4758. 2003.

47.

Johnson

, Clabots

, Rosen

. Effect of inactivation of the global oxidative stress regulator oxyR on the colonization ability of Escherichia coli O1:K1:H7 in a mouse model of ascending urinary tract infection. Infect Immun, 74:461–468. 2006.

48.

Jonsson

, Lindholm

, Luong

, Lee

, Tarkowski

. mgrA regulates staphylococcal virulence important for induction and progression of septic arthritis and sepsis. Microbes Infect, 10:1229–1235. 2008.

49.

Jung

, Cho

, Kim

, Yoo

, Hong

, Roe

. Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response. Nucleic Acids Res, 39:7586–7597. 2011.

50.

Kaito

, Morishita

, Matsumoto

, Kurokawa

, Sekimizu

. Novel DNA binding protein SarZ contributes to virulence in Staphylococcus aureus. Mol Microbiol, 62:1601–1617. 2006.

51.

Kallifidas

, Thomas

, Doughty

, Paget

. The sigmaR regulon of Streptomyces coelicolor A32 reveals a key role in protein quality control during disulphide stress. Microbiology, 156:1661–1672. 2010.

52.

Kang

, Paget

, Seok

, Hahn

, Bae

, Hahn

, Kleanthous

, Buttner

, Roe

. RsrA, an anti-sigma factor regulated by redox change. EMBO J, 18:4292–4298. 1999.

53.

Kaushal

, Schroeder

, Tyagi

, Yoshimatsu

, Scott

, Ko

, Carpenter

, Mehrotra

, Manabe

, Fleischmann

, Bishai

. Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH. Proc Natl Acad Sci U S A, 99:8330–8335. 2002.

54.

Kharitonov

, Sundquist

, Sharma

. Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J Biol Chem, 270:28158–28164. 1995.

55.

Kim

, Merchant

, Nudelman

, Beyer

WF.

Jr , Keng

, DeAngelo

, Hausladen

, Stamler

. OxyR: a molecular code for redox-related signaling. Cell, 109:383–396. 2002.

56.

Knoepfel

, Steinkuhler

, Carri

, Rotilio

. Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1. Biochem Biophys Res Commun, 201:871–877. 1994.

57.

Korshunov

, Imlay

. Two sources of endogenous hydrogen peroxide in Escherichia coli. Mol Microbiol, 75:1389–1401. 2010.

58.

Kuhle

, Abrahams

, Hensel

. Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner. Traffic, 7:716–730. 2006.

59.

Kullik

, Toledano

, Tartaglia

, Storz

. Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation. J Bacteriol, 177:1275–1284. 1995.

60.

Lan

, Murray

, Kazmierczak

, He

. Pseudomonas aeruginosa OspR is an oxidative stress sensing regulator that affects pigment production, antibiotic resistance and dissemination during infection. Mol Microbiol, 75:76–91. 2010.

61.

Lancaster

JR.

Jr . Nitroxidative, nitrosative, and nitrative stress: kinetic predictions of reactive nitrogen species chemistry under biological conditions. Chem Res Toxicol, 19:1160–1174. 2006.

62.

Lau

, Britigan

, Hassett

. Pseudomonas aeruginosa OxyR is required for full virulence in rodent and insect models of infection and for resistance to human neutrophils. Infect Immun, 73:2550–2553. 2005.

63.

Lee

, Lee

, Mukhopadhyay

, Kim

, Lee

, Ahn

, Yu

, Storz

, Ryu

. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat Struct Mol Biol, 11:1179–1185. 2004.

64.

Lee

, Soonsanga

, Helmann

. A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR. Proc Natl Acad Sci U S A, 104:8743–8748. 2007.

65.

Levine

, Mosoni

, Berlett

, Stadtman

. Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci U S A, 93:15036–15040. 1996.

66.

, Bottrill

, Bibb

, Buttner

, Paget

, Kleanthous

. The Role of zinc in the disulphide stress-regulated anti-sigma factor RsrA from Streptomyces coelicolor. J Mol Biol, 333:461–472. 2003.

67.

Liu

, Yang

, Peterfreund

, Tsou

, Selamoglu

, Daldal

, Zhong

, Kan

, Zhu

. Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB. Proc Natl Acad Sci U S A, 108:810–815. 2011.

68.

Luo

, Levine

. Methionine in proteins defends against oxidative stress. FASEB J, 23:464–472. 2009.

69.

Luong

, Dunman

, Murphy

, Projan

, Lee

. Transcription profiling of the mgrA regulon in Staphylococcus aureus. J Bacteriol, 188:1899–1910. 2006.

70.

Manganelli

, Voskuil

, Schoolnik

, Dubnau

, Gomez

, Smith

. Role of the extracytoplasmic-function sigma factor sigma(H) in Mycobacterium tuberculosis global gene expression. Mol Microbiol, 45:365–374. 2002.

71.

McLean

, Bowman

, Poole

. KatG from Salmonella typhimurium is a peroxynitritase. FEBS Lett, 584:1628–1632. 2010.

72.

Mongkolsuk

, Praituan

, Loprasert

, Fuangthong

, Chamnongpol

. Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli. J Bacteriol, 180:2636–2643. 1998.

73.

Nystrom

. Role of oxidative carbonylation in protein quality control and senescence. EMBO J, 24:1311–1317. 2005.

74.

Ochman

, Soncini

, Solomon

, Groisman

. Identification of a pathogenicity island required for Salmonella survival in host cells. Proc Natl Acad Sci U S A, 93:7800–7804. 1996.

75.

Ochsner

, Hassett

, Vasil

. Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa. J Bacteriol, 183:773–778. 2001.

76.

Paget

, Bae

, Hahn

, Li

, Kleanthous

, Roe

, Buttner

. Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch. Mol Microbiol, 39:1036–1047. 2001.

77.

Paget

, Buttner

. Thiol-based regulatory switches. Annu Rev Genet, 37:91–121. 2003.

78.

Paget

, Kang

, Roe

, Buttner

. sigmaR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2) EMBO J, 17:5776–5782. 1998.

79.

Paget

, Molle

, Cohen

, Aharonowitz

, Buttner

. Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon. Mol Microbiol, 42:1007–1020. 2001.

80.

Panmanee

, Vattanaviboon

, Poole

, Mongkolsuk

. Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor and regulator of organic hydroperoxide stress. J Bacteriol, 188:1389–1395. 2006.

81.

Park

, Roe

. Mycothiol regulates and is regulated by a thiol-specific antisigma factor RsrA and sigma(R) in Streptomyces coelicolor. Mol Microbiol, 68:861–870. 2008.

82.

Park

, Kang

, Husson

. Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A, 105:13105–13110. 2008.

83.

Poole

, Karplus

, Claiborne

. Protein sulfenic acids in redox signaling. Annu Rev Pharmacol Toxicol, 44:325–347. 2004.

84.

Poor

, Chen

, Duguid

, Rice

, He

. Crystal structures of the reduced, sulfenic acid, and mixed disulfide forms of SarZ, a redox active global regulator in Staphylococcus aureus. J Biol Chem, 284:23517–23524. 2009.

85.

Raman

, Song

, Puyang

, Bardarov

, Jacobs

WR.

Jr , Husson

. The alternative sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis. J Bacteriol, 183:6119–6125. 2001.

86.

Salcedo

, Holden

. SseG, a virulence protein that targets Salmonella to the Golgi network. EMBO J, 22:5003–5014. 2003.

87.

Seaver

, Imlay

. Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol, 183:7173–7181. 2001.

88.

Shea

, Hensel

, Gleeson

, Holden

. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci U S A, 93:2593–2597. 1996.

89.

Song

, Dove

, Lee

, Husson

. RshA, an anti-sigma factor that regulates the activity of the mycobacterial stress response sigma factor SigH. Mol Microbiol, 50:949–959. 2003.

90.

Storz

, Tartaglia

, Ames

. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science, 248:189–194. 1990.

91.

Suvarnapunya

, Stein

. DNA base excision repair potentiates the protective effect of Salmonella pathogenicity island 2 within macrophages. Microbiology, 151:557–567. 2005.

92.

Tamber

, Cheung

. SarZ promotes the expression of virulence factors and represses biofilm formation by modulating SarA and agr in Staphylococcus aureus. Infect Immun, 77:419–428. 2009.

93.

Tao

. In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli. FEBS Lett, 457:90–92. 1999.

94.

Tardat

, Touati

. Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli::Fur (ferric uptake regulation) and Arc (aerobic respiration control) Mol Microbiol, 5:455–465. 1991.

95.

Taylor

, Inchley

, Gallagher

. The Salmonella typhimurium AhpC polypeptide is not essential for virulence in BALB/c mice but is recognized as an antigen during infection. Infect Immun, 66:3208–3217. 1998.

96.

Toledano

, Kullik

, Trinh

, Baird

, Schneider

, Storz

. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell, 78:897–909. 1994.

97.

Tomljenovic-Berube

, Mulder

, Whiteside

, Brinkman

, Coombes

. Identification of the regulatory logic controlling Salmonella pathoadaptation by the SsrA-SsrB two-component system. PLoS Genet, 6:e1000875. 2010.

98.

Troxell

, Sikes

, Fink

, Vazquez-Torres

, Jones-Carson

, Hassan

. Fur negatively regulates hns and is required for the expression of HilA and virulence in Salmonella enterica serovar Typhimurium. J Bacteriol, 193:497–505. 2011.

99.

Uchiya

, Barbieri

, Funato

, Shah

, Stahl

, Groisman

. A Salmonella virulence protein that inhibits cellular trafficking. EMBO J, 18:3924–3933. 1999.

100.

Vazquez-Torres

, Fang

. Salmonella evasion of the NADPH phagocyte oxidase. Microbes Infect, 3:1313–1320. 2001.

101.

Vazquez-Torres

, Fantuzzi

, Edwards

3rd , Dinarello

, Fang

. Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages. Proc Natl Acad Sci U S A, 98:2561–2565. 2001.

102.

Vazquez-Torres

, Jones-Carson

, Mastroeni

, Ischiropoulos

, Fang

. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J Exp Med, 192:227–236. 2000.

103.

Vazquez-Torres

, Xu

, Jones-Carson

, Holden

, Lucia

, Dinauer

, Mastroeni

, Fang

. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science, 287:1655–1658. 2000.

104.

Velayudhan

, Castor

, Richardson

, Main-Hester

, Fang

. The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair and virulence. Mol Microbiol, 63:1495–1507. 2007.

105.

Walthers

, Li

, Liu

, Anand

, Yan

, Kenney

. Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription. J Biol Chem, 286:1895–1902. 2011.

106.

Winterbourn

, Hampton

. Thiol chemistry and specificity in redox signaling. Free Radic Biol Med, 45:549–561. 2008.

107.

Winterbourn

, Metodiewa

. Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radic Biol Med, 27:322–328. 1999.

108.

Worley

, Ching

, Heffron

. Salmonella SsrB activates a global regulon of horizontally acquired genes. Mol Microbiol, 36:749–761. 2000.

109.

Yoo

, Kim

, Yu

, Kim

, Cha

, Oh

, Eo

, Lee

, Kang

. Requirement of Fur for the full induction of Dps expression in Salmonella enterica serovar Typhimurium. J Microbiol Biotechnol, 17:1452–1459. 2007.

110.

Zdanowski

, Doughty

, Jakimowicz

, O'Hara

, Buttner

, Paget

, Kleanthous

. Assignment of the zinc ligands in RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor. Biochemistry, 45:8294–8300. 2006.

111.

Zheng

, Aslund

, Storz

. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science, 279:1718–1721. 1998.

Redox Active Thiol Sensors of Oxidative and Nitrosative Stress

Abstract

Introduction

Sensing of Oxidative and Nitrosative Stress by Members of the LysR/OxyR Family of Transcriptional Regulators

OxyR is a redox active regulator that maintains thiol homeostasis

Mechanisms of sensing oxidative and nitrosative stress by OxyR redox active thiols

Contribution of LysR/OxyR regulators to bacterial pathogenesis

Sensing of Oxidative Stress by Members of the MarA/OhrR Family of Transcriptional Regulators

Mechanisms of sensing oxidative stress by MarA/OhrR thiol switches

Contribution of the redox-sensing activity of the MarA/OhrR family members to bacterial pathogenesis

Sensing of Oxidative Stress by Redox Active Thiols That Coordinate Zinc

Regulation of antioxidant defenses by members of the σR family of extracytoplasmic function sigma factors

Mechanisms of sensing oxidative stress by the RsrA antisigma factor

RsrA is redox active

Role of redox-active antisigma factors in bacterial pathogenesis

Sensing of Nitrosative Stress by Members of the NarL/SsrB Family of Transcriptional Regulators

Regulation of SsrB function by the redox-active Cys203

Determinants That Define the Redox Activity of Cysteines

Redox mechanism for the formation of thiolate in OxyR

Sensing by MarR/OhrR family members

Activation of thiols in the zinc-binding RsrA antisigma factor

Activation of SsrB Cys203

Chemistry Underlying the Modification of Redox-Active Thiols by ROS and RNS

Selectivity of the reaction of thiols and ROS

Oxidation of redox active thiols by H2O2

Selective reaction of thiols with peroxides

Reaction of thiols with RNS

Concluding Remarks

Footnotes

Acknowledgments

Abbreviations Used

References

Regulation of antioxidant defenses by members of the σ^R family of extracytoplasmic function sigma factors

Regulation of SsrB function by the redox-active Cys²⁰³

Activation of SsrB Cys²⁰³

Oxidation of redox active thiols by H₂O₂