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.     

dpr and sod in Streptococcus mutans Are Involved in Coexistence with S. sanguinis,and PerR Is Associated with Resistance to H2O2

Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H₂O₂), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H₂O₂ produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H₂O₂. The knockout of dpr and sod significantly increased susceptibility to H₂O₂, while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H₂O₂. Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H₂O₂ compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H₂O₂ in regulating the expression of Dpr.     

Oxygen exposure increases resistance of Desulfovibrio vulgaris Hildenborough to killing by hydrogen peroxide

Inactivation of PerR by oxidative stress and a corresponding increase in expression of the perR regulon genes is part of the oxidative stress defense in a variety of anaerobic bacteria. Diluted anaerobic, nearly sulfide-free cultures of mutant and wild-type Desulfovibrio vulgaris (10⁵–10⁶ colony-forming units/ml) were treated with 0 to 2,500 μM H₂O₂ for only 5 min to prevent readjustment of gene expression. Survivors were then scored by plating. The wild type and perR mutant had 50% survival at 58 and 269 μM H₂O₂, respectively, indicating the latter to be 4.6-fold more resistant to killing by H₂O₂ under these conditions. Significantly increased resistance of the wild type (38-fold; 50% killing at 2188 μM H₂O₂) was observed if cells were pretreated with full air for 30 min, conditions that did not affect cell viability. The resistance of the perR mutant increased less (4.6-fold; 50% killing at 1230 μM H₂O₂), when similarly pretreated. Interestingly, no increased resistance of either was achieved by exposure with 10.6 μM H₂O₂ for 30 min, the highest concentration that could be used without killing the cells. Hence, in environments with low D. vulgaris biomass only the presence of external O₂ effectively activates the perR regulon. As a result, mutant strains lacking one of the perR regulon genes ahpC, dvu0772, rbr1 or rbr2 displayed decreased resistance to H₂O₂ stress only following pretreatment with air.     

PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H₂O₂, whereas Dpr (an Fe²⁺-chelating protein)-dependent H₂O₂ protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H₂O₂-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl₂, S. oligofermentans increased survival after H₂O₂ stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn²⁺-mediated H₂O₂ resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn²⁺) unless it was supplemented with ≥2.5 μM MnCl₂ and caused 82 to 95% reduction of the cellular Mn²⁺ level, while mntABC overexpression increased cellular Mn²⁺ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn²⁺ transporter in S. oligofermentans. mntA mutation reduced the survival of H₂O₂-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H₂O₂-challenged survival 12-fold, indicating that MntABC-mediated Mn²⁺ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H₂O₂ pulsing upregulated mntABC, while H₂O₂-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H₂O₂ can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H₂O₂ stress defenses and may be distributed across all oral streptococci lacking catalase.     

Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp. strain PCC 6803

We utilized a full genome cDNA microarray to identify the genes that comprise the peroxide stimulon in the cyanobacterium Synechocystis sp. strain PCC 6803. We determined that a gene (slr1738) encoding a protein similar to PerR in Bacillus subtilis was induced by peroxide. We constructed a PerR knockout strain and used it to help identify components of the PerR regulon, and we found that the regulatory properties were consistent with the hypothesis that PerR functions as a repressor. This effort was guided by finding putative PerR boxes in positions upstream of specific genes and by careful statistical analysis. PerR and sll1621 (ahpC), which codes for a peroxiredoxin, share a divergent promoter that is regulated by PerR. We found that isiA, encoding a Chl protein that is induced under low-iron conditions, was strongly induced by a short-term peroxide stress. Other genes that were strongly induced by peroxide included sigD, sigB, and genes encoding peroxiredoxins and Dsb-like proteins that have not been studied yet in this strain. A gene (slr1894) that encoded a protein similar to MrgA in B. subtilis was upregulated by peroxide, and a strain containing an mrgA knockout mutation was highly sensitive to peroxide. A number of genes were downregulated, including key genes in the chlorophyll biosynthesis pathway and numerous regulatory genes, including those encoding histidine kinases. We used PerR mutants and a thioredoxin mutant (TrxA1) to study differential expression in response to peroxide and determined that neither PerR nor TrxA1 is essential for the peroxide protective response.     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.     

Role of Alkyl Hydroperoxide Reductase (AhpC) in the Biofilm Formation of Campylobacter jejuni

Biofilm formation of Campylobacter jejuni, a major cause of human gastroenteritis, contributes to the survival of this pathogenic bacterium in different environmental niches; however, molecular mechanisms for its biofilm formation have not been fully understood yet. In this study, the role of oxidative stress resistance in biofilm formation was investigated using mutants defective in catalase (KatA), superoxide dismutase (SodB), and alkyl hydroperoxide reductase (AhpC). Biofilm formation was substantially increased in an ahpC mutant compared to the wild type, and katA and sodB mutants. In contrast to the augmented biofilm formation of the ahpC mutant, a strain overexpressing ahpC exhibited reduced biofilm formation. A perR mutant and a CosR-overexpression strain, both of which upregulate ahpC, also displayed decreased biofilms. However, the introduction of the ahpC mutation to the perR mutant and the CosR-overexpression strain substantially enhanced biofilm formation. The ahpC mutant accumulated more total reactive oxygen species and lipid hydroperoxides than the wild type, and the treatment of the ahpC mutant with antioxidants reduced biofilm formation to the wild-type level. Confocal microscopy analysis showed more microcolonies were developed in the ahpC mutant than the wild type. These results successfully demonstrate that AhpC plays an important role in the biofilm formation of C. jejuni.     

Role of mrgA in peroxide and light stress in the cyanobacterium Synechocystis sp. PCC 6803

In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the mrgA gene is part of the PerR regulon that is upregulated during peroxide stress. We determined that an Δ mrgA mutant was highly sensitive to low peroxide levels and that the mutant upregulated a gene cluster ( sll1722-26 ) that encoded enzymes involved with exopolymeric substance (EPS) production. We made mutants in this EPS cluster in both a wild type and Δ mrgA background and studied the responses to oxidative stress by measuring cell damage with LIVE/DEAD stain. We show that Synechocystis sp. PCC 6803 becomes highly sensitive to oxidative stress when either mrgA or the sll1722-26 EPS components are deleted. The results suggest that the deletion of the EPS cluster makes a cell highly susceptible to cell damage, under moderate oxidative stress conditions. Mutations in either mrgA or the EPS cluster also result in cells that are more light and peroxide sensitive, and produce significantly less EPS material than in wild type. In this study, we show that in the absence of MrgA, which is known to be involved in the storage or mobilization of iron, cells can be more easily damaged by exogenous oxidative and light stress.