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

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.     

OxyR介导的细菌氧化胁迫应答调控机制研究进展

OxyR属于LysR型转录因子家族的氧化胁迫调控蛋白,是细菌抵抗氧化胁迫压力的重要调控因子。OxyR能够通过调控过氧化氢酶和过氧化物酶等抗氧化基因的表达清除H₂O₂、参与铁代谢控制胞内过氧化物的产生以及修复生物大分子氧化损伤,从而抵抗氧化胁迫。OxyR的基因表达调控功能依赖于其还原态和氧化态之间的转变,改变调控蛋白对下游基因调控区的亲和能力。氧化态OxyR识别启动子区的结合序列,激活或抑制过氧化氢酶等基因的表达。还原态和氧化态的转换依赖于在氧化状态下分子间二硫键的形成。本文综述了近年来细菌OxyR调控基因表达的最新研究进展,有助于深入理解OxyR在细菌抵抗氧化胁迫的作用方式,为相关致病菌的防治奠定分子基础。     

Biochemical basis of sulphenomics: how protein sulphenic acids may be stabilized by the protein microenvironment

Among protein residues, cysteines are one of the prominent candidates to ROS‐mediated and RNS‐mediated post‐translational modifications, and hydrogen peroxide (H₂O₂) is the main ROS candidate for inducing cysteine oxidation. The reaction with H₂O₂ is not common to all cysteine residues, being their reactivity an utmost prerequisite for the sensitivity towards H₂O₂. Indeed, only deprotonated Cys (i.e. thiolate form, ? S^?) can react with H₂O₂ leading to sulphenic acid formation (? SOH), which is considered as a major/central player of ROS sensing pathways. However, cysteine sulphenic acids are generally unstable because they can be further oxidized to irreversible forms (sulphinic and sulphonic acids, ? SO₂H and ? SO₃H, respectively), or alternatively, they can proceed towards further modifications including disulphide bond formation (? SS? ), S‐glutathionylation (? SSG) and sulphenamide formation (? SN?). To understand why and how cysteine residues undergo primary oxidation to sulphenic acid, and to explore the stability of cysteine sulphenic acids, a combination of biochemical, structural and computational studies are required. Here, we will discuss the current knowledge of the structural determinants for cysteine reactivity and sulphenic acid stability within protein microenvironments.     

Challenging Xanthomonas campestris with low levels of arsenic mediates cross-protection against oxidant killing

Xanthomonas encounters highly toxic reactive oxygen species (ROS) from many sources, such as those generated by plants against invading bacteria, other soil bacteria and from aerobic respiration. Thus, conditions that alter intracellular ROS levels such as exposure to toxic metalloids would have profound effects on bacterial physiology. Here, we report that exposure of Xanthomonas campestris pv. phaseoli (Xp) to low levels of arsenic induces physiological cross-protection against killing by H(2)O(2) and organic hydroperoxide but not a superoxide generator. Cross-protection against H(2)O(2) and organic hydroperoxide toxicity was due to increased expression of genes encoding major peroxide-metabolizing enzymes such as alkyl hydroperoxide reductase (AhpC), catalase (KatA) and organic hydroperoxide resistance protein (Ohr). Arsenic-induced protection against H(2)O(2) and organic hydroperoxide requires the peroxide stress response regulators, OxyR and OhrR, respectively. Moreover, analyses of double mutants of the major H(2)O(2) and organic hyproperoxide-scavenging enzymes, Xp ahpC katA and Xp ahpC ohr, respectively, suggested the existence of unidentified OxyR- and OhrR-regulated genes that are involved in arsenic-induced resistance to H(2)O(2) and organic hyproperoxide killing in Xp. These arsenic-induced physiological alterations could play an important role in bacterial survival both in the soil environment and during plant-pathogen interactions.     

Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Hydrogen peroxide (H₂O₂) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H₂O₂ requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H₂O₂ in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H₂O₂, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H₂O₂. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H₂O₂-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H₂O₂ in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H₂O₂ can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H₂O₂, they cannot be used to monitor transient changes in H₂O₂ concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H₂O₂ and can be targeted to various compartments of the cell, but they are not selective for H₂O₂ because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H₂O₂-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.     

The YaaA protein of the Escherichia coli OxyR regulon lessens hydrogen peroxide toxicity by diminishing the amount of intracellular unincorporated iron

Hydrogen peroxide (H₂O₂) is commonly formed in microbial habitats by either chemical oxidation processes or host defense responses. H₂O₂ can penetrate membranes and damage key intracellular biomolecules, including DNA and iron-dependent enzymes. Bacteria defend themselves against this H₂O₂ by inducing a regulon that engages multiple defensive strategies. A previous microarray study suggested that yaaA, an uncharacterized gene found in many bacteria, was induced by H₂O₂ in Escherichia coli as part of its OxyR regulon. Here we confirm that yaaA is a key element of the stress response to H₂O₂. In a catalase/peroxidase-deficient (Hpx⁻) background, yaaA deletion mutants grew poorly, filamented extensively, and lost substantial viability when they were cultured in aerobic LB medium. The results from a thyA forward mutagenesis assay and the growth defect of the yaaA deletion in a recombination-deficient (recA56) background indicated that yaaA mutants accumulated high levels of DNA damage. The growth defect of yaaA mutants could be suppressed by either the addition of iron chelators or mutations that slowed iron import, indicating that the DNA damage was caused by the Fenton reaction. Spin-trapping experiments confirmed that Hpx⁻ yaaA cells had a higher hydroxyl radical (HO^•) level. Electron paramagnetic resonance spectroscopy analysis showed that the proximate cause was an unusually high level of intracellular unincorporated iron. These results demonstrate that during periods of H₂O₂ stress the induction of YaaA is a critical device to suppress intracellular iron levels; it thereby attenuates the Fenton reaction and the DNA damage that would otherwise result. The molecular mechanism of YaaA action remains unknown.     

A genetically encoded sensor for H2O2 with expanded dynamic range

Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H₂O₂] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H₂O₂ due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator’s dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H₂O₂] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.     

Tolerance to oxidative stress is required for maximal xylem colonization by the xylem‐limited bacterial phytopathogen,Xylella fastidiosa

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem‐limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H₂O₂) relative to the wild‐type. In addition, during early stages of grapevine infection, the survival rate was 1000‐fold lower for the oxyR mutant than for the wild‐type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell–cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.     

Construction and Physiological Analysis of a Xanthomonas Mutant To Examine the Role of the oxyR Gene in Oxidant-Induced Protection against Peroxide Killing

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H₂O₂ and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.     

Crystal Structure of Peroxide Stress Regulator from Streptococcus pyogenes Provides Functional Insights into the Mechanism of Oxidative Stress Sensing

Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.     

A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria

In this paper we propose and provide evidence for a mechanism, oxidative nitrogen scavenging (ONS), whereby seedlings of some grass species may extract nitrogen from symbiotic diazotrophic bacteria through oxidation by plant-secreted reactive oxygen species (ROS). Experiments on this proposed mechanism employ tall fescue (Festuca arundinaceae) seedlings to elucidate features of the oxidative mechanism. We employed 15N₂ gas assimilation experiments to demonstrate nitrogen fixation, direct microscopic visualization of bacteria on seedling surfaces to visualize the bacterial oxidation process, reactive oxygen probes to test for the presence of H₂O₂ and cultural experiments to assess conditions under which H₂O₂ is secreted by seedlings. We also made surveys of the seedlings of several grass species to assess the distribution of the phenomenon of microbial oxidation in the Poaceae. Key elements of the proposed mechanism for nitrogen acquisition in seedlings include: 1) diazotrophic bacteria are vectored on or within seeds; 2) at seed germination bacteria colonize seedling roots and shoots; 3) seedling tissues secrete ROS onto bacteria; 4) bacterial cell walls, membranes, nucleic acids, proteins and other biological molecules are oxidized; 5) nitrates and/or smaller fragments of organic nitrogen-containing molecules resulting from oxidation may be absorbed by seedling tissues and larger peptide fragments may be further processed by secreted or cell wall plant proteases until they are small enough for transport into cells. Hydrogen peroxide secretion from seedling roots and bacterial oxidation was observed in several species in subfamily Pooideae where seeds possessed adherent paleas and lemmas, but was not seen in grasses that lacked this feature or long-cultivated crop species.     

DRA0336, another OxyR homolog,involved in the antioxidation mechanisms in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

A novel OxyR (DR0615) with one conserved cysteine that senses hydrogen peroxide in Deinococcus radiodurans had been identified in our previous work. Comparative genomics revealed that D. radiodurans possesses another OxyR homolog, OxyR₂ (DRA0336). In this study, we constructed the deletion mutant of oxyR ₂ and the double mutant of both the OxyR homologs to investigate the role of OxyR in response to oxidative stress in D. Radiodurans. Deletion of oxyR ₂ resulted in an obviously increased sensitivity to hydrogen peroxide, and the double mutant for oxyR and oxyR ₂ was significantly more sensitive than any of the two single mutants. The total catalase activity of the double mutant was lower than that of any of the single mutants, and reactive oxygen species (ROS) accumulated to a greater extent. DNA microarray analysis further suggested that oxyR ₂ was involved in antioxidation mechanisms. Site-direct mutagenesis and complementation analysis revealed that C₂₂₈ in OxyR₂ was essential. This is the first report of the presence of two OxyR in one organism. These results suggest that D. radiodurans OxyR and OxyR₂ function together to protect the cell against oxidative stress.     

Identification of altered function alleles that affect Bacillus subtilis PerR metal ion selectivity

Bacillus subtilis PerR is a Fur family repressor that senses hydrogen peroxide by metal-catalyzed oxidation. PerR contains a structural Zn(II) ion (Site 1) and a regulatory metal binding site (Site 2) that, upon association with either Mn(II) or Fe(II), allosterically activates DNA binding. In addition, a third less conserved metal binding site (Site 3) is present near the dimer interface in several crystal structures of homologous Fur family proteins. Here, we show that PerR proteins with substitutions of putative Site 3 residues (Y92A, E114A and H128A) are functional as repressors, but are unexpectedly compromised in their ability to sense H(2)O(2). Consistently, these mutants utilize Mn(II) but not Fe(II) as a co-repressor in vivo. Metal titrations failed to identify a third binding site in PerR, and inspection of the PerR structure suggests that these residues instead constitute a hydrogen binding network that modulates the architecture, and consequently the metal selectivity, of Site 2. PerR H128A binds DNA with high affinity, but has a significantly reduced affinity for Fe(II), and to a lesser extent for Mn(II). The ability of PerR H128A to bind Fe(II) in vivo and to thereby respond efficiently to H(2)O(2) was restored in a fur mutant strain with elevated cytosolic iron concentration.     

Reactive oxygen species in the killing of Pseudomonas aeruginosa by human leukocytes

Pseudomonas aeruginosa (PA) infects hosts with compromised host defenses. An important defense mechanism is the generation of reactive oxygen species (ROS) by white blood cells (WBCs). What roles do ROS play in host defense against PA? Human WBCs killed PA in vitro, and they generated a respiratory burst as measured by the production of H₂O₂. ROS efficiently killed PA; in acellular assays, less than 10mm of H₂O₂ or OCl^- eliminated all bacteria in 90 min. However, WBCs with suppressed production of ROS (caused by hypoxia) killed PA normally. In addition, none of the antioxidants vitamin C, N-acetylcysteine, superoxide dismutase, or catalase affected PA killing by WBCs. Thus, PA stimulates WBCs to produce ROS, which can kill the bacteria, but disturbances of WBC ROS production do not interfere with the killing of PA. WBCs have robust, redundant mechanisms for PA elimination.     

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (E_GSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in E_GSH and H₂O₂ levels with the genetically encoded biosensors Grx1-roGFP2 (for E_GSH) and roGFP2-Orp1 (for H₂O₂) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Methyl viologen-induced photo-oxidative stress increases hydrogen peroxide and oxidation of glutathione in chloroplasts, cytosol, and mitochondria, as well as autonomous oxidation in mitochondria.