Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

The catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens. KEYWORDS Streptococcus, cysteine oxidation, hydrogen peroxide, posttranslational regulation, redox signaling, transcriptional regulation R eactive oxygen species (ROS), such as superoxide anions (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (HO·), damage almost all biological macromolecules (1)(2)(3). Therefore, organisms have evolved diverse mechanisms to cope with ROS (1)(2)(3)(4). Facultatively anaerobic streptococci, such as the human opportunistic pathogen Streptococcus pneumoniae and the oral commensal bacterium Streptococcus oligofermentans, do not encode H 2 O 2 -scavenging catalase and thus accumulate endogenous H 2 O 2 (5)(6)(7)(8). Streptococci are also well-known for surviving in the presence of high concentrations of H 2 O 2 (6,9,10). Previously, we determined that statically grown S. oligofermentans cultures have an approximately 200-fold higher survival rate than cells anaerobically cultured in 10 mM H 2 O 2 (11). A similar observation has also been reported for S. pneumoniae (8). This suggests that the low levels of H 2 O 2 that accumulate in statically cultured cells may assist streptococci with resisting the oxidant at higher concentrations. However, the biological basis of this low-H 2 O 2 -concentration-induced adaptation remains unknown.
Bacteria usually use cysteine-based redox reactions to sense H 2 O 2 and activate the downstream peroxide detoxification pathways (12)(13)(14). Escherichia coli OxyR was the first identified archetype of thiol-based redox regulators in bacteria; it is activated by intramolecular thiol-disulfide formation resulting from H 2 O 2 oxidation and thereby induces expression of the genes involved in defending against oxidative stress (15). Gram-positive bacteria, on the other hand, utilize the peroxide-responsive repressor PerR to sense H 2 O 2 and derepress the H 2 O 2 resistance genes (11,16,17). PerR, a member of the Fur family of metal-dependent regulators, possesses two metal-binding sites: a regulatory Fe 2ϩ or Mn 2ϩ binding site consisting of histidine and aspartate residues and a structural Zn 2ϩ binding site comprising four cysteine residues (18,19). The Bacillus subtilis PerR is inactivated by H 2 O 2 via metal-catalyzed oxidation (MCO) (20). When binding Fe 2ϩ , PerR is inactivated by Fenton chemistry-generated HO· from H 2 O 2 , which oxidizes the histidine residues. In contrast, the cysteine residues of the B. subtilis PerR that coordinate Zn 2ϩ for structural maintenance are somehow inert to H 2 O 2 (20). Therefore, PerR:Zn,Fe (Fe 2ϩ -bound PerR) but not PerR:Zn,Mn responds to H 2 O 2 (17,19). Makthal et al. (21) also reported that H 2 O 2 inactivates the recombinant Streptococcus pyogenes PerR:Zn,Fe, suggesting that Fe 2ϩ -triggered Fenton chemistry could inactivate the streptococcal PerR as well. However, an in vivo study demonstrated that the S. pyogenes PerR:Zn,Mn also displays a weaker response to H 2 O 2 (22). Previously, we found that the S. oligofermentans PerR is inactivated by H 2 O 2 and derepresses the antioxidative non-heme iron-containing ferritin, dpr, and manganese importer mntABC genes (11). However, even if grown in Mn 2ϩ -supplemented medium, H 2 O 2 still induces the expression of dpr. This implies that the streptococcal PerR can be inactivated by mechanisms other than Fe 2ϩ -triggered Fenton chemistry.
The redox-sensing transcriptional regulators usually respond to H 2 O 2 challenge through cysteine oxidation (12,13,23). Recently, this thiol redox switch-based regulatory mechanism was found to be employed by other transcriptional regulators, such as AgrA in the control of the quorum sensing of Staphylococcus aureus (24) and MntR in the regulation of manganese uptake and the oxidative stress resistance of S. oligofermentans (25). Thiol redox proteomics is a powerful approach for the quantification of oxidative thiol modifications and the identification of physiologically important proteins in oxidative stress resistance (26)(27)(28). Using this approach, a number of novel redox-regulated proteins that contribute to the protection of E. coli from H 2 O 2 stress (29) have been identified. Recently, proteome-wide quantification and characterization of the oxidation-sensitive cysteine residues have determined complex and multilayered oxidative stress responses in pathogenic bacteria, such as Pseudomonas aeruginosa, S. aureus, and S. pneumoniae (8,30). Therefore, cysteine-containing proteins not only serve as H 2 O 2 -damaged targets but also equip bacteria with the capability to resist H 2 O 2 stress.
To elucidate the mechanisms underlying low-H 2 O 2 -concentration-induced resistance to high concentrations of H 2 O 2 in streptococci, we employed physiological, biochemical, genetic, and redox proteomics approaches to investigate the H 2 O 2sensitive cysteine-containing proteins that may be involved in H 2 O 2 adaptation. We determined that cellular H 2 O 2 levels ranging from 40 to 100 M protected S. oligofermentans from insult by higher H 2 O 2 concentrations. Redox proteomics identified cysteine oxidation in the H 2 O 2 -responsive transcriptional regulators PerR and MntR, which regulate antioxidative stress in response to H 2 O 2 , as well as in the thioredoxin system proteins Tpx and Trx, which function in thiol-disulfide homeostasis. Importantly, 40 M H 2 O 2 oxidized the Zn 2ϩ -coordinated cysteine residues and inactivated PerR, thus derepressing its regulons, which function in the thiol redox circuit and metal homeostasis. The high sensitivity of the cysteine residues to H 2 O 2 enables PerR to sense low levels of H 2 O 2 and thus protect the catalase-negative species S. oligofermentans from H 2 O 2 challenge by maneuvering the H 2 O 2 resistance systems. Moreover, the reversible cysteine oxidation resulting from a low H 2 O 2 concentration can also endow the streptococcal PerR with flexibility in H 2 O 2 -responsive regulation.

RESULTS
Preexposure to a low H 2 O 2 concentration enables S. oligofermentans to resist higher H 2 O 2 concentrations. Previously, we found that aerobically cultured S. oligofermentans exhibits significantly higher resistance to H 2 O 2 stress than anaerobic cultures (11), suggesting that the endogenous H 2 O 2 that accumulates in the static culture may protect streptococci from damage in the presence of higher H 2 O 2 concentrations. To validate this presumption, we deleted both the pox and lox genes, which encode pyruvate oxidase and lactate oxidase, respectively, the two major H 2 O 2 producers in S. oligofermentans (5,6). As expected, when exposed to 20 mM H 2 O 2 , only 0.02% survival was found for pox lox mutant cells; in comparison, 30% survival was found for wild-type cells (Table 1).
To verify if the loss of H 2 O 2 resistance in the pox lox mutant was due to the lack of endogenous H 2 O 2 but not the reduction of acetyl phosphate, which is produced by Pox and which contributes to S. pneumoniae H 2 O 2 resistance (31), we determined whether a preexposure to a low concentration of H 2 O 2 could increase the higher H 2 O 2 resistance of the pox lox mutant. The wild-type and pox lox mutant strains were anaerobically grown until the optical density at 600 nm (OD 600 ) was ϳ0.5. One aliquot of the cultures, noted as the prepulse group, was pulsed for 20 min with 40 M H 2 O 2 prior to a 10-min  (32) and compared them with those excreted into the culture. The wild-type (WT) HyPer reporter strain of S. oligofermentans, WT-HyPer (33), was statically grown in 10, 20, 30, and 40 ml of brain heart infusion (BHI) broth in 100-ml flasks, which built an initial O 2 supply gradient. The growth profiles and the H 2 O 2 amounts in the cultures were measured. Figure 1A shows that the best growth and the lowest H 2 O 2 concentration (approximately 400 M) were measured for the 40-ml culture. In contrast, the poorest growth and the highest H 2 O 2 level (approximately 1,400 M) were detected in the 10-ml culture, indicating that larger amounts of H 2 O 2 are produced by Streptococcus with a rich supply of oxygen and thus suppress its growth.
Next, the mid-exponential-phase cells of the WT-HyPer strain from each volume of cultures were visualized under a confocal laser scanning microscope (Leica model TCS SP8), and the HyPer fluorescence intensities were measured as described in Materials and Methods. The Δpox-HyPer mutant, the pox deletion mutant carrying the HyPer gene (33), was included as a control from which H 2 O 2 was absent. Figure 1B and C show that the HyPer fluorescence intensities were inversely proportional to the culture volumes but directly proportional to the H 2 O 2 concentrations in the cultures, with a good linear regression (R 2 ϭ 0.8745) (Fig. 1D). This indicates that the quantity of H 2 O 2 in a culture indicates an equivalent amount within the cells.
Redox proteomics identifies cysteine-oxidized proteins by the low H 2 O 2 concentration that induces self-protection from oxidative stress. To identify the proteins that are sensitive to a low H 2 O 2 concentration and that might be involved in self-protection from oxidative stress, label-free redox proteomics analysis was performed to identify the cysteine-oxidized proteins in 40 M H 2 O 2 -pulsed anaerobically grown S. oligofermentans. Proteins were extracted from H 2 O 2 -treated and -untreated cells, and cysteine thiol group oxidations were analyzed using a combination of differential alkylation and liquid chromatography (LC)-tandem mass spectrometry (MS/ MS) (28, 34) ( Fig. 2A). The representative MS/MS spectra shown in Fig. S1A  Proteins with reversible (S-S or SOH) or irreversible thiol oxidation (SO 2 H or SO 3 H) were identified by comparison with those in H 2 O 2 -untreated cells. The S-S oxidation ratio (in percent) in each sample was calculated by dividing the intensity of the disulfide-linked peptides by the sum of the peptides and considering a cutoff value of a Ն1.5-fold oxidation ratio in H 2 O 2 -treated cells over that in untreated cells to be significant (29). Proteins identified as SOH or SO 2 H or SO 3 H oxidations were those found only in H 2 O 2 -treated samples or with a Ն1.5-fold elevated peptide intensity compared to that for the control. In summary, 40 M H 2 O 2 treatment resulted in thiol group oxidation in 57 cysteine-containing proteins (Data Set S1E). Among these, 25 proteins containing 32 cysteine residues were oxidized into disulfide linkages (S-S), with a Ͼ50-fold increased disulfide ratio in 21 proteins; 3 proteins were reversibly oxidized as SOH; and the remaining 29 proteins were irreversibly oxidized as SO 2 H or SO 3 H. Thus, these 57 proteins are assumed to be involved in self-protection from H 2 O 2 challenge.
As S. oligofermentans cells statically grown in the 40-ml culture survived the 20 mM H 2 O 2 challenge (Table 1), we identified the cysteine-oxidized proteins in this volume of culture. A total of 1,093 proteins were identified by LC-MS/MS analysis, including 108 cysteine-containing proteins (Data Set S2A and B). Calculations indicated that 35 proteins were oxidized, with 26 cysteine residues in 23 proteins being reversibly oxidized into disulfide linkages (S-S) and the remaining 12 proteins being irreversibly oxidized as SO 2 H or SO 3 H (Data Set S2E). However, in cells cultured in 10-ml cultures that accumulated larger amounts of H 2 O 2 , 66 of the 164 cysteine-containing proteins were oxidized, with 33 being oxidized as S-S and 33 being oxidized as SO 2 H or SO 3 H (Data Set S2C to E). Thirty-one proteins that were specifically oxidized in 10-ml cultures belonged to organic acid and organic nitrogen metabolic processes (Data Set S2F), accounting for the growth retardation of S. oligofermentans under oxidative stress.
To link the biological functions of the H 2 O 2 -sensitive proteins, Gene Ontology (GO) analysis was performed by use of the PANTHER bioinformatics platform (http://www .pantherdb.org/) (35). Figure 2B shows that the proteins oxidized by endogenous H 2 O 2 (in 40-ml aerobic cultures) and exogenously provided H 2 O 2 (for 40 M H 2 O 2 -pulsed anaerobic cells) were categorized into similar biological processes, with approximately 61% and 51% of the proteins, respectively, being involved in metabolic processes and 26% and 38% of the proteins, respectively, being involved in cellular processes. Remarkably, almost all the proteins in the glycolysis and nucleotide salvage pathways were oxidized to form disulfide linkages (Table 2; Fig. 2C; Data Sets S1F and S2F). As expected, the antioxidative thiol-reducing proteins thiol peroxidase (Tpx) and thioredoxin (Trx) were 36.5% to 100% oxidized. It is worth noting that the metalloregulator MntR was markedly oxidized at the thiol group of cysteines (Table 2; Data Sets S1E and  Table 2) either might be involved in self-protection or might simply be hypersensitive to oxidative damage.
Redox Western blotting validates the oxidation of cysteine-containing proteins by a low H 2 O 2 concentration, notably, the cysteine oxidation of PerR. To verify the redox proteomics-identified cysteine residues sensitive to low H 2 O 2 concentrations, Tpx and Trx, the well-known antioxidative proteins, were chosen to examine cysteine oxidation by 40 M H 2 O 2 in Tpx-6ϫHis and Trx-6ϫHis strains, which carried a 6ϫHis tag fusion at the C terminus of the Tpx and Trx proteins, respectively. Redox Western blotting was performed as described in Materials and Methods. As shown in Fig Table 2; Data Set S1E). Although redox proteomics identified Trx Cys82 to be complete oxidized ( Fig. 3D; Fig. S1I), redox Western blotting did not detect a differential migration of the Trx protein upon H 2 O 2 oxidation or DTT reduction (  Previously, we demonstrated that the peroxide-responsive repressor PerR and the metalloregulator MntR are involved in the H 2 O 2 resistance of S. oligofermentans (11,25).   2), and the other was reduced with 50 mM DTT for 1 h (lanes 4 and 5). Redox Western blotting was carried out using an 18% SDS-PAGE gel to detect the Tpx-6ϫHis protein using an anti-His tag antibody. Recombinant Tpx-6ϫHis protein, which was partially oxidized and which formed an intramolecular disulfide linkage during purification, was treated with or without 50 mM DTT (lanes 3 and 6) and used as a reduced and an oxidized molecular control, respectively. (B) Using the same approach described in the legend to panel A, a disulfide linkage upon 40 M H 2 O 2 oxidation was identified for thioredoxin (KEGG accession number I872_03205) in the Trx-6ϫHis strain (lanes 1 and 2 versus lanes 5 and 6). In addition, 15 mM 4-acetamido-4=maleimidylstilbene-2,2=-disulfonic acid (AMS), the free thiol-chelating reagent, was used to detect the nondisulfide oxidation of the thiol groups (lanes 3 and 4 and lanes 7 and 8). Cell lysates from the H 2 O 2 -untreated strain (lanes 3 and 4) and the H 2 O 2 -treated Trx-6ϫHis strain (lanes 7 and 8) were reduced with or without 50 mM DTT. The recombinant Trx-6ϫHis protein was first reduced by 50 mM DTT, and then one aliquot was alkylated with AMS and another was left untreated; these were used as reduced and thiol AMS-bound Trx-6ϫHis protein controls, respectively (lanes 9 and 10). Molecular weight markers are shown at the left, and the increased molecular weight of the protein due to bound AMS molecules (500 Da each) is shown at the right. H 2 O 2 -untreated PerR-6ϫHis culture, whereas the upper band appeared mainly in the H 2 O 2 -treated culture and the lower one appeared exclusively in DTT-treated cell lysates (Fig. 4A, lanes 1 to 4, and Fig. 4B). This is reminiscent of the findings for B. subtilis PerR, which migrated more slowly when the structure maintaining Zn 2ϩ was lost due to the oxidation of cysteine residues (20). Therefore, AMS was employed to examine the cysteine redox status of PerR from H 2 O 2 -treated PerR-6ϫHis cells. Figure 4A shows that AMS addition increased the apparent molecular weight of PerR from DTT-reduced cell lysates (upshifted at approximately 0.5 cm, lane 6 versus lane 4) compared to that of PerR from the non-DTT-reduced ones (upshifted at approximately 0.3 cm, lane 5 versus lane 3). This indicates that some but not all of the four Cys residues of the streptococcal PerR are oxidized by pulsing with a low H 2 O 2 concentration.
Redox Western blotting indicated that PerR was also oxidized in statically grown cells (Fig. S2A). To further verify the endogenous cysteine oxidation resulting from H 2 O 2 , 6ϫHis-tagged PerR protein immunoprecipitated from statically grown cells was subjected to differential alkylation and LC-MS/MS analysis (Fig. S2B). Figure 4D displays the representative MS/MS spectra of the peptide fragments carrying four cysteine residues. By counting the peptide fragments carrying oxidative and reductive cysteine residues, we calculated the oxidation ratios of the four cysteine residues to be 76% (Cys100), 50% (Cys103), 83% (Cys139), and 82% (Cys142) (Table S1), whereas His40 and His95, whose oxidations inactivate B. subtilis PerR (20), were oxidized approximately 28% and 53%, respectively, in S. oligofermentans PerR (Table S1). Collectively, both MS/MS identification and redox Western blotting determined that the Zn 2ϩcoordinated cysteine residues of S. oligofermentans PerR are hypersensitive to H 2 O 2 oxidation.
H 2 O 2 oxidation of the cysteine residues abolishes PerR binding to DNA due to Zn 2؉ loss. To further determine whether H 2 O 2 oxidation occurs at the cysteine residues of the streptococcal PerR in vivo, Cys139 and Cys142 were replaced by serine on the shuttle plasmid pDL278-perR-6ϫHis. Wild-type perR and cysteine-mutated perR were each ectopically expressed in a perR deletion strain, and the resultant complementary strains were treated with or without 40 M H 2 O 2 . Redox Western blotting showed that, different from the findings for wild-type PerR, cysteine-mutated PerR retained the same migration in all samples regardless of 40 M H 2 O 2 oxidation or DTT reduction (Fig. 4B). This result demonstrates that H 2 O 2 oxidizes Cys139 and Cys142 of the streptococcal PerR.
It is worth noting that even if cells were collected inside an anaerobic glove box and lysed in the presence of NEM, EDTA, and catalase, part of the PerR protein was still oxidized (Fig. 4A, lane 1), suggesting the PerR cysteine residues are hypersensitive to oxidants. This was further confirmed by redox Western blotting, which detected oxidized PerR protein from the cells pulsed by 40 M H 2 O 2 for only 1 min (Fig. 4C). Noticeably, invariable lower Western blotting signals were detected for PerR from H 2 O 2 -treated cells than for PerR from DTT-reduced cells, and this was determined to be because DTT increased the anti-His tag antibody signal (Fig. S3).
By reference to the B. subtilis PerR and other Cys 4 Zn proteins, such as Hsp33 and RsrA (37,38), oxidation of Zn 2ϩ -coordinated cysteine residues would cause Zn 2ϩ loss and, therefore, slower protein migration because of the conformational changes. We subsequently verified whether H 2 O 2 oxidation causes Zn 2ϩ loss from PerR. Overexpressed glutathione S-transferase (GST)-tagged PerR protein (GST-PerR) was treated or not treated with 5 mM H 2 O 2 and subsequently reduced or not reduced with 50 mM DTT. Nonreducing SDS-PAGE analysis did reveal a slower-migrating band for the 5 mM H 2 O 2 -treated protein than for the DTT-treated protein (Fig. 5A). Inductively coupled plasma mass spectrometry (ICP-MS) also determined 0.09 mol of Zn 2ϩ per mol of H 2 O 2 -treated PerR and 0.79 mol of Zn 2ϩ per mol of DTT-reduced PerR (Fig. 5B), confirming that H 2 O 2 oxidation causes Zn 2ϩ loss from PerR.
Next, we determined whether Zn 2ϩ loss affects DNA binding by PerR. Fifty nanomoles of PerR:Zn,Mn was used for a electrophoretic mobility shift assay (EMSA), based on a calculated K d (dissociation constant) value of approximate 50 nM for binding (Fig. 5C). Figure 5D shows that 50 M H 2 O 2 treatment for 30 min diminished PerR's binding to the to construct the S. oligofermentans PerR-6ϫHis strain. Using the same approach described in the legends to Fig. 3A and B, redox Western blotting detected cysteine oxidation in the 6ϫHis-tagged PerR protein using the anti-His tag antibody. U and L at the gel left indicate upper and lower protein bands, respectively. (B) A serine substitution of either Cys139 or Cys142 was constructed on a shuttle plasmid (pDL278-perR-6ϫHis), and the plasmid was transformed into the perR deletion strain to construct the perR::pDL278-perRC139S-6ϫHis (C139S) and perR::pDL278-perRC142S-6ϫHis (C142S) strains. The perR deletion mutant harboring pDL278-perR-6ϫHis (WT) was included as a control. The three strains were anaerobically cultured and then treated with 40 M H 2 O 2 . Redox Western blotting, as described in the legend to Fig. 3A, was carried out to detect the oxidation of the PerR mutants. (C) The PerR-6ϫHis strain was anaerobically cultured, and cells were treated with 40 M H 2 O 2 for 1 min and 5 min. Using the methods described in the legend to Fig. 3A, the cysteine oxidation of PerR was detected by redox Western blotting. (D) The 6ϫHis-tagged PerR protein was immunoprecipitated from the statically grown PerR-6ϫHis strain as described in Materials and Methods and then resolved on an 18% nonreducing SDS-PAGE gel. The protein band was then subjected to differential alkylation and LC-MS/MS analysis.  Fig. 5E and F). It is worth noting that the EMSA buffer was treated with Chelex 100 to chelate metal ions that might trigger the Fenton reaction. Noticeably, LC-MS/MS did not detect increased histidine residue oxidation in the H 2 O 2 -treated PerR:Zn,Mn protein (Table S2), while histidine oxidations that occurred before H 2 O 2 treatment might have been generated during the in vitro purification. In conclusion, H 2 O 2 oxidizes cysteine residues but not histidine residues and inactivates PerR.
PerR and MntR regulate the cellular redox system and metal homeostasis. Trx and Tpx are involved in cellular redox homeostasis and belong to the S. aureus and Clostridium acetobutylicum PerR regulons (39,40). Additionally, Dpr, a non-heme ironcontaining ferritin, and MntABC, a manganese ABC transporter, are known to play important roles in maintaining cellular metal homeostasis and are under the control of S. oligofermentans PerR and MntR (11,25). To determine whether the four genes mentioned above plus mntR belong to the PerR or MntR regulons, we performed quantitative PCR (qPCR) to quantify the expression of these genes in 40 M H 2 O 2pulsed and nonpulsed anaerobically grown wild-type strain and perR deletion, mntR deletion, and perR mntR double deletion mutants. In comparison with the 40 M H 2 O 2 -induced 3-to 5.8-fold higher levels of expression of tpx, dpr, mntA, and mntR in the wild-type strain, deletion of mntR abolished the H 2 O 2 induction of mntA; however, the H 2 O 2 induction of the four genes almost disappeared in the mutants either with a deletion of perR or with a deletion of both perR and mntR (Table 3). These demonstrate that the H 2 O 2 -induced expressions of tpx, dpr, mntA, and mntR are under the control of PerR, while mntA is also controlled by mntR in response to H 2 O 2 .
Notably, even in the absence of H 2 O 2 , 2.5-to 4.0-fold higher levels of expression of tpx, dpr, and mntA were detected in the ΔperR and ΔperR ΔmntR mutants than in the wild-type strain, suggesting that PerR may directly regulate the tpx, dpr, and mntA genes. However, the conserved PerR binding sequence (TTAATTAGAAGCATTATAAT TAA) was found only in the dpr promoter region; consistently, EMSA indicated PerR binding to the dpr promoter ( Fig. 5C) but not to the promoters of mntABC, tpx, and mntR (Fig. S4). Our previous work found that MntR bound to the mntABC promoter (25), indicating the direct regulation of mntA by MntR. Thus, PerR directly regulates dpr but indirectly regulates mntA, tpx, and mntR via unknown mechanisms. Of note, similar expression levels of the PerR-regulated genes were detected in cells pulsed only with 40 M H 2 O 2 and cells that were pulsed and then further challenged by 10 mM H 2 O 2 ( PerR, MntR, and the regulated cellular redox and metal homeostatic proteins are involved in the self-protection of S. oligofermentans from H 2 O 2 stress. Given that both PerR and MntR contribute to the high H 2 O 2 resistance of S. oligofermentans (11,25), to determine their roles in self-protection against H 2 O 2 stress, the ΔperR and ΔmntR mutants were prepulsed with or without 40 M H 2 O 2 , and then their survival with 10 mM H 2 O 2 challenge was determined as described above.   The recombinant PerR-GST protein was purified in PBS buffer containing 10 mM EDTA and 3 mM GSH, as described in Materials and Methods. Purified PerR-GST was divided into three aliquots: one was not treated, and the remaining two were treated for 30 min with 5 mM H 2 O 2 , with one of these two aliquots subsequently being subjected to 1 h of reduction with 50 mM DTT. Aliquots of the three protein samples were resolved on nonreducing 18% SDS-PAGE gels, and the remaining samples were ultrafiltered and, finally, resuspended in 650 l PBS buffer. The protein concentrations of PerR-GST were determined using a BCA protein assay kit, and the zinc concentration in the protein was measured using ICP-MS. The molar ratios of zinc ion to the PerR-GST monomer were calculated. The averages Ϯ SD from three independent experiments are shown. *, a result significantly different from that for H 2 O 2 -treated PerR-GST protein, as verified by one-way analysis of variance followed by Tukey's post hoc test (P Ͻ 0.05). (C) The PerR-GST protein was digested with 100 U thrombin to remove the GST tag and then eluted into a buffer containing 10 mM EDTA and 1 mM DTT. Next, 1 M PerR protein was preincubated with 1 mM MnCl 2 , and then gradient concentrations of the recombinant PerR:Zn,Mn protein (0 to 200 nM) were tested for binding to the 5=-biotin-labeled dpr promoter fragment, as described in Materials and Methods. (D) One micromole of PerR protein was incubated with 1 mM MnCl 2 and then treated with increasing concentrations of H 2 O 2 at 30°C for 30 min. After incubation, 150-U/ml catalase was added to decompose the residual H 2 O 2 , and 50 nM PerR protein was used for EMSA to test the affinity of binding to the dpr promoter (lanes 3 to 6). To observe whether the H 2 O 2 oxidation-diminished PerR binding could be restored, 10 mM DTT was added to the 200 M H 2 O 2 -treated PerR and the mixture was incubated at 37°C for 1 h (lane 10). E. One micromole of the PerR protein was preincubated with 1 mM MnCl 2 and then treated with 200 M H 2 O 2 at 30°C for different times (lanes 3 to 7). Fifty nM PerR protein was used in the EMSA. Black and gray arrows point to the free DNA probe and protein-DNA complex, respectively. (F) Band densities of the protein-DNA complex in lanes 2 to 7 of panel E were evaluated using ImageJ software, with the density in lane 2 being set as 100%. The density percentages in lanes 3 to 7 were calculated by dividing the band density of the respective lane by that of lane 2. All the experiments were repeated three times, and the averages Ϯ SD from three independent experiments are shown. the two transcriptional repressors and thereby derepressing the antioxidative systems. Notably, a 9.4-fold elevated survival rate in the presence of a higher concentration of H 2 O 2 was observed for the ΔperR mutant than for the ΔmntR mutant ( Table 1), suggesting that Dpr and redox system proteins might play the major role in protecting S. oligofermentans from challenge with a higher H 2 O 2 concentration.
Next, the role of Dpr and redox system proteins in the H 2 O 2 resistance of S. oligofermentans was determined. The tpx, trx, dpr, and mntABC genes were each deleted, and the mutants were compared with the wild-type strain for growth suppression by 40 and 100 M H 2 O 2 . As shown in Fig. 6, 100 M H 2 O 2 slightly suppressed the growth of the wild-type strain, but 40 M H 2 O 2 already retarded the growth of the  tpx, trx, and dpr deletion mutants, with the Δdpr mutant being the most severely inhibited. The ΔmntABC mutant was reported to exhibit reduced resistance to 10 mM H 2 O 2 (11), but its growth was not significantly inhibited by 100 M H 2 O 2 (data not shown). Collectively, the results indicate that the redox regulators PerR and MntR and their regulated cellular redox and metal homeostasis proteins are involved in the self-protection of S. oligofermentans from H 2 O 2 stress.

DISCUSSION
Although a few studies have reported that endogenous H 2 O 2 protects streptococci from challenge with a higher H 2 O 2 concentration (8,22), the mechanism remains unclear. In the present study, through a combination of physiological, biochemical, genetic, and redox proteomic studies, we elucidated the mechanism underlying the low-H 2 O 2 -concentration-induced adaptation of catalase-negative streptococci to a higher H 2 O 2 concentration. Figure 7 depicts that streptococci employ pyruvate oxidase (Pox) and lactate oxidase (Lox) to produce endogenous H 2 O 2 . Two H 2 O 2 -sensing redox regulators, the peroxide-responsive repressor PerR and the metalloregulator MntR, are inactivated by H 2 O 2 oxidation of the cysteine residues. PerR cysteine oxidation results in Zn 2ϩ loss and the subsequent derepression of dpr, mntABC, tpx, and mntR. H 2 O 2 oxidation of MntR leads to disulfide-linked intermolecular polymers and inactivates the regulator, thus derepressing the manganese uptake regulon mntABC (25). In addition to dpr and mntABC, as indicated in our previous work (11), mntR and the thiol peroxidaseencoding gene tpx were identified to be the PerR regulons. Deletion of these functional genes as well as the redox circuit protein Trx increased the sensitivity of S. oligofer- These include redox transcriptional regulators, e.g., the peroxide response repressor PerR and the metalloregulator MntR, a repressor of the Mn 2ϩ uptake regulon mntABC, as well as the redox homeostatic proteins, e.g., thiol peroxidase (Tpx), which catalyzes the reduction of H 2 O 2 , and thioredoxin (Trx), which specifically reduces H 2 O 2 oxidation-generated disulfide linkages. Inactivation of PerR by trace H 2 O 2 derepresses tpx and the genes encoding metal ion homeostatic proteins, like mntR, dpr, and mntABC, whereas oxidation inactivation of MntR derepresses mntABC (25). Dpr chelates free ferrous ion to avoid Fenton chemistry, whereas MntABC imports Mn 2ϩ to decompose the cellular H 2 O 2 . These functional proteins help S. oligofermentans resist the stress associated with a higher H 2 O 2 concentration. Of note, PerR directly represses the dpr gene and controls the tpx, mntABC, and mntR genes indirectly by unknown mechanisms. H 2 O 2 is identified by the green symbols. mentans to a low H 2 O 2 concentration, and correspondingly, deletion of either mntR or perR resulted in the streptococci becoming constitutively resistant to a higher H 2 O 2 concentration. Thus, this work reveals a redox-regulated anti-H 2 O 2 defense network, in which PerR has evolved to sense H 2 O 2 by a Cys-based redox reaction in the manganese-rich cellular environments of the catalase-negative streptococci.
Cysteine residues are the most sensitive to H 2 O 2 oxidation (41), and therefore, reversibly oxidized cysteine thiol modifications, such as SOH and the disulfide bond, usually function in the activation of redox regulatory proteins. Some redox regulators, such as the E. coli chaperone protein Hsp33 (37) and the Streptomyces coelicolor anti-sigma factor RsrA (38) and Fur-like repressor CatR (42), possess a structural Cys 4 Zn. The Zn 2ϩ at the Cys 4 Zn site stabilizes the cysteine residues as thiolate, which may increase the reactivity of cysteine toward electrophilic H 2 O 2 (43). The S. oligofermentans PerR possesses the structural Cys 4 Zn as well. Redox proteomics, redox Western blotting, and LC-MS/MS identification of the immunoprecipitated protein all demonstrated that the cysteine residues of the streptococcal PerR are oxidized by a low H 2 O 2 concentration (Fig. 4). Oxidation of the cysteine residues causes Zn 2ϩ loss and inactivates PerR (Fig. 5), thereby derepressing the antioxidative genes (Table 3). This explains the underlying mechanism of the PerR-mediated H 2 O 2 adaptation of streptococci.
It has been reported that the B. subtilis PerR, an ortholog of the streptococcal PerR (see Fig. S5 in the supplemental material), is inactivated by histidine oxidation, whereas its Zn 2ϩ -coordinated cysteine residues are inert to H 2 O 2 oxidation (20). In contrast, the streptococcal PerR is inactivated by H 2 O 2 oxidation at the Zn 2ϩ -coordinated cysteine residues ( Fig. 4 and 5). Subsequent structural homology modeling of the S. oligofermentans PerR was performed with the SWISS-MODEL server by automatically selecting the S. pyogenes PerR (PDB accession number 4LMY) as a template. Structural comparison with B. subtilis PerR (PDB accession number 2FE3) did show some differences between the two at the C-terminal Cys 4 Zn site (Fig. S5); specifically, Cys103 and Cys142 of the S. oligofermentans PerR are situated close to the N terminus of the S4 ␤-strand and at a short H6 helix, respectively, while Cys96 and Cys139 of the B. subtilis PerR are situated at the C terminus of the S3 ␤-strand and in the center of a long H6 helix, respectively. These differences may render the two PerRs with different H 2 O 2 sensitivities, as a cysteine residue near the N terminus of a helix more likely possesses lower pK a values (43). Cellular metal environments could be another clue to the distinct inactivation mechanisms of the two PerRs. A much higher ratio of Mn/Fe was determined in S. oligofermentans cells (1.02 Ϯ 0.25) than in B. subtilis cells (0.05 Ϯ 0.01), which was paralleled in this study, in accordance with the Mn-centric definition of streptococci (44)(45)(46). Especially, when grown in a medium supplemented with 2.5 M and 100 M Mn 2ϩ , 1.78 Ϯ 0.46 and 8.04 Ϯ 0.42 cellular Mn/Fe ratios were found in S. oligofermentans, respectively (25), indicating an active manganese uptake system in this bacterium. The higher cellular Mn/Fe ratio in streptococci could result in higher percentages of PerR:Zn,Mn than of the PerR:Zn,Fe found in bacilli; thus, cysteine oxidation contributes to H 2 O 2 inactivation of the streptococcal PerR:Zn,Mn proteins ( Fig. 4 and 5; Table S1). Nevertheless, the possibility of Fe 2ϩ -triggered streptococcal PerR inactivation cannot be excluded, as approximately 28% of His40 residues and 53% of His95 residues in PerR were oxidized when S. oligofermentans was grown in BHI broth containing 0.5 M Mn 2ϩ and 15 M Fe 2ϩ (Table S1). Therefore, the dual-H 2 O 2 -sensing mechanisms of the redox regulator PerR could provide protection for the catalase-negative streptococci from oxidative stress in environments with different metal ions.
Metal homeostasis plays a central role in oxidative stress resistance in Gram-positive bacteria (4,44,47,48). In addition to PerR, streptococci also employ MntR, a metalloregulator protein, to control cellular manganese and iron homeostasis (11,16,21,25). Oxidative inactivation of PerR and MntR derepresses the expression of the metal homeostasis-related genes dpr and mntABC (Table 3) (25). Dpr chelates cellular Fe 2ϩ and so prevents the production of highly toxic HO·, and the manganese importer MntABC takes up Mn 2ϩ to decompose cellular H 2 O 2 . MntABC and Dpr have been verified to protect S. oligofermentans from challenge by a high H 2 O 2 concentration in our previous studies (11). Here, Dpr was further verified to resist a sublethal H 2 O 2 concentration.
The thioredoxin (Trx) system, which is comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin, plays a key role in defense against oxidative stress, particularly in the catalase-lacking streptococci (9,(49)(50)(51). Oxidation of the cysteine thiol groups of Trx and Tpx has been found in 40 M H 2 O 2 -pretreated S. oligofermentans cells (Fig. 3), and deletion of two genes increases the H 2 O 2 sensitivity of the streptococcus (Fig. 6). This observation indicates that the Trx system is involved in the H 2 O 2 adaptation of S. oligofermentans, which is presumably under the control of PerR.
In conclusion, this work reports a novel H 2 O 2 adaptation mechanism. Trace amounts of cellular H 2 O 2 cause thiol oxidation of the redox-based regulatory and functional proteins and activate antioxidative systems; meanwhile, they reduce the level of glycolysis, which generates ROS precursors. This H 2 O 2 adaptation mechanism could be an important antioxidative defense strategy of the catalase-void anaerobes.

MATERIALS AND METHODS
Bacterial strains and culture conditions. S. oligofermentans AS 1.3089 (52) and its derivative strains (see Table S3 in the supplemental material) were grown in brain heart infusion (BHI) broth (BD Difco, Franklin Lakes, NJ) statically or anaerobically under 100% N 2 . Escherichia coli DH5␣, used for cloning, was grown in Luria-Bertani (LB) broth at 37°C under shaking. When required, kanamycin (1 mg/ml) and spectinomycin (1 mg/ml) were used for the selection of Streptococcus transformants, while ampicillin (100 g/ml) and spectinomycin (250 g/ml) were used to select E. coli transformants.
Construction of genetically altered strains. All primers used in this study are listed in Table S3. tpx, trx, dpr, and perR deletion strains were constructed using the PCR-ligation method (53). The upstream and downstream DNA fragments of each gene were amplified from the genomic DNA. The purified, BamHI-digested PCR products were ligated with a kanamycin resistance gene fragment from plasmid pALH124 (54) or a spectinomycin resistance gene fragment from pDL278 (55) at compatible sites. For construction of 6ϫHis-tagged strains, the tpx, trx, and perR genes were amplified from the genomic DNA using a pair of primers, with the reverse primer carrying a sequence encoding 6 histidines just before the termination codon. Meanwhile, an ϳ600-bp DNA fragment immediately downstream of the termination codon of each gene was amplified. The purified PCR products were digested with BamHI and ligated with the kanamycin resistance gene fragment. The ligation mixtures were transformed into the S. oligofermentans wild-type strain, except that the ligation mixture for perR deletion was transformed into the ΔmntR strain (11) to construct a ΔperR ΔmntR strain, as described previously (6). For construction of the perR::pDL278-perRC139S-6ϫHis and perR::pDL278-perRC142S-6ϫHis strains, the perR-6ϫHis gene fusion was amplified from the genomic DNA of the strain with 6ϫHis-tagged PerR. After digestion with EcoRI and SalI, the purified product was inserted into the compatible sites on the E. coli-streptococci shuttle vector pDL278 (55) to produce pDL278-perR-6ϫHis. Then, Cys139 and Cys142 were mutated into serine using a site-directed gene mutagenesis kit (Beyotime Biotechnology Co., Shanghai, China). The correct pDL278-perR-6ϫHis, pDL278-perRC139S-6ϫHis, and pDL278-perRC142S-6ϫHis plasmids were transformed into the perR deletion strain to produce strains ectopically expressing wild-type and cysteinemutated perR.
Detection of intracellular hydrogen peroxide by HyPer imaging. Mid-exponential-phase HyPer reporter cells were pelleted, washed twice with phosphate-buffered saline (PBS), resuspended in 100 l of PBS, and exposed to air in the dark for 30 min. Forty microliters of cells was placed on a Polysine microscope slide (25 by 75 by 1 mm; Thermo Scientific, Waltham, MA), covered with a Fisher-brand microscope glass coverslip (diameter, 15 mm; thickness, 0.13 to 0.17 mm; Thermo Scientific), and then visualized under a confocal laser scanning microscope (Leica model TCS SP8; Leica Microsystems, Buffalo Grove, IL, USA). Excitation was provided at 488 nm, with emission being collected from a wavelength range of 500 to 530 nm (32,56). For each sample, at least 5 fluorescent and differential interference contrast (DIC) images were captured. The fluorescence intensities of 25 regions of interest (ROI), with each ROI containing 5 cells, from each sample were measured using Leica Application Suite (LAS) AF software. For images with fluorescence that was too weak, the ROI in the corresponding DIC images was framed, and the fluorescence was measured in the same ROI in the fluorescence image. The average fluorescence intensities of 25 ROIs were calculated and are expressed in arbitrary units (a.u.) per ROI Ϯ standard deviation.
Redox proteomics analysis by differential alkylation and LC-MS/MS. The differential alkylation method (34) was used to identify H 2 O 2 -induced changes in the thiol redox status of the proteins. Briefly, mid-exponential-phase cells in the tested samples were collected by centrifugation and resuspended in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, sodium fluoride, 1 mM EDTA, 0.5 g/ml leupeptin) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). To minimize an artificial oxidation during sample preparation, cell breakage by sonication was performed inside an anaerobic chamber (Thermo Scientific); moreover, 10 mM EDTA and 1 kU/ml catalase were included in the lysis buffer to prevent an Fe 2ϩ -triggered Fenton reaction and decompose H 2 O 2 , respectively. The sonication was implemented on ice in the dark using a UP-400S ultrasonicator (Xinzi Company, Ningbo, China), and cell lysates were centrifuged at 8,000 ϫ g for 15 min, and then the protein concentration in the supernatant was measured using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). The same amounts of protein from all the samples were separated on a nonreducing one-dimensional SDS-PAGE gel, and each gel lane was cut into 6 slices and washed with MS-grade water three times. The proteins in the gel were alkylated for 30 min with 55 mM [ 13 C]iodoacetic acid in 50 mM NH 4 HCO 3 (pH 8.0) in the dark. After removing the iodoacetic acid, 25 mM DTT reduction was performed for 45 min at 55°C, and then the DTT was removed and the proteins were alkylated with 55 mM [ 12 C]iodoacetic acid for 30 min in the dark. Upon in-gel digestion with MS-grade trypsin (Promega, Fitchburg, WI), LC-MS/MS analysis was implemented with an Easy-nLC integrated nano-high-performance liquid chromatography system (Proxeon, Odense, Denmark) and a Q-Extractive mass spectrometer (Thermo Scientific, Waltham, MA), as described previously (28).
MS/MS spectra were searched against the forward and reverse S. oligofermentans protein database, downloaded from UniProt, using the SEQUEST search engine of Proteome Discoverer software (v1.4). The precursor ion mass tolerance was 20 ppm for all mass spectra acquired in an Orbitrap mass analyzer, and the fragment ion mass tolerance was 0.02 Da for all MS/MS spectra. The following search criteria were employed: full tryptic specificity was required; two missed cleavages were allowed; 13 C carboxymethylation (free cysteine residue), 12 C carboxymethylation (disulfide linkage cysteine residue) and sulfenic, sulfinic, and sulfonic acids were variable modifications for cysteine; oxidation was a variable modification for methionine; and the false discovery rate (FDR) was set to 0.01. All the cysteine-modified MS/MS spectra were manually confirmed. The MaxQuant software package was used to obtain the intensity of the cysteine-modified peptides. Duplicate experiments were performed in parallel.
Protein GO category analysis. Homologues of the S. oligofermentans redox-sensitive proteins were searched for in S. pneumoniae and put into the PANTHER bioinformatics platform (http://www.pantherdb .org/) for Gene Ontology (GO) analysis. GO enrichment analysis was implemented on the Gene Ontology Consortium website (http://www.geneontology.org), the binomial test was used for analysis of statistical significance, and a P value of Ͻ0.05 was used as a cutoff.
Redox Western blotting. Cells were collected by centrifugation and resuspended in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin) with addition of 40 mM N-ethylmaleimide (NEM), 1 mM PMSF, 10 mM EDTA, and 1 kU/ml catalase. Cells were sonicated on ice in the dark for 45 min and alkylated in the dark for 20 min, and then the supernatant were collected by centrifugation. Reduced samples were prepared by incubating the lysates with 50 mM DTT for 1 h. For the 4-acetamido-4=-maleimidylstilbene-2,2=-disulfonic acid (AMS) alkylating experiment, cells were resuspended in PBS buffer containing 15 mM AMS, 1 mM PMSF, 10 mM EDTA, and 1 kU/ml catalase, sonicated, and then incubated at 4°C for 2 h in the dark. Half of the samples were reduced with 50 mM DTT for 1 h, and then the DTT was removed and the samples were alkylated with 15 mM AMS at 4°C for 2 h in the dark. The protein concentration of the lysate was determined using a BCA protein assay kit. Protein samples were diluted in nonreducing loading buffer (4ϫ; 0.2 M Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 0.4% bromphenol blue), separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and hybridized with an anti-His tag antibody (Abmart Company, Shanghai, China) at a 4,000-fold dilution. Detection was performed using a chemiluminescent nucleic acid detection module kit (Thermo Scientific).
IP and LC-MS/MS identification of cysteine thiol oxidation of the PerR protein in vivo. 6ϫHistagged PerR protein was purified by immunoprecipitation (IP) using anti-His tag monoclonal antibodymagnetic agarose (MBL International Corporation, Woburn, MA) according to the instructions of the manufacturer. Briefly, the 6ϫHis-tagged-PerR-expressing strain PerR-6ϫHis was statically grown in BHI broth. The mid-exponential-phase cells were collected and washed with PBS three times. Then, the cells were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% NP-40, 1 mM DTT) containing 55 mM [ 13 C]iodoacetic acid, 10 mM EDTA and 1 kU/ml catalase. The cells were sonicated on ice in the dark for 45 min and alkylated in the dark for 20 min, and then the cell lysate was subjected to centrifugation. The obtained supernatant was mixed and incubated with the magnetic beads. After washing 4 times with lysis buffer, the immunoprecipitated 6ϫHis-tagged PerR protein was eluted by boiling in nonreducing SDS sample buffer (4% SDS, 125 mM Tris-HCl, pH 8.0, 20% glycerol) and separated using 18% nonreducing SDS-PAGE. The target PerR protein band with the expected molecular size was cut from the gel, and cysteine residue oxidation was identified by differential alkylation and LC-MS/MS, as described above, except that the reduced and oxidized cysteine residues were alkylated with 55 mM [ 13 C]iodoacetic acid and [ 12 C]iodoacetamide, respectively.
Overexpression of PerR-GST, Tpx-6؋His, and Trx-6؋His proteins. A 450-bp DNA fragment containing the entire perR coding gene was PCR amplified. The purified PCR product was digested with EcoRI/XhoI and ligated into the compatible sites on pGEX4T-1 (GE Healthcare, Boston, MA), and the produced pGEX-PerR was transformed into E. coli BL21(DE3) cells (Novagen, Madison, WI). Correct transformants were grown at 37°C to an OD 600 of 0.4 to 0.6, and 0.1 mM isopropyl-␤-Dthiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis, MO) was added to induce PerR-GST expression at 22°C overnight. Then, the cells were collected by centrifugation and resuspended in phosphate-buffered saline (PBS; 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) containing 1 mM DTT and 10 mM EDTA and then lysed by sonication for 30 min. The cell lysate was centrifuged at 8,000 ϫ g for 30 min, and the supernatant was filtered through a 0.22-nm-pore-size polyvinylidene difluoride membrane (Millipore, Billerica, MA) and then applied to a GSTrap HP column (GE Healthcare, Boston, MA). The proteins were eluted with elution buffer (20 mM Tris-HCl buffer containing 1 mM DTT, 10 mM EDTA, and 10 mM reduced glutathione [GSH], pH 8.0), and the elution fractions were analyzed by electrophoresis on a 12% SDS-PAGE gel. The fractions with the desired protein were pooled and dialyzed against PBS buffer containing 3 mM GSH and 10 mM EDTA three times. Then, the purified proteins were stored in aliquots in 10% glycerol at Ϫ80°C until use.
For the overexpression of the Tpx-6ϫHis and Trx-6ϫHis proteins, 492-and 552-bp DNA fragments containing the entire tpx and trx coding genes, respectively, were PCR amplified with the primer pairs listed in Table S3. The resultant products were integrated into pET-28a (Novagen, Madison, WI) by Gibson assembly (New England Biolabs, Beverly, MA) to produce pET-28a-Tpx and pET-28a-Trx. The correct constructs were transformed into E. coli BL21(DE3) (Novagen, Madison, WI) cells. Correct transformants were grown at 37°C to an OD 600 of 0.6 to 0.8, 0.1 mM IPTG (Sigma-Aldrich, St. Louis, MO) was added, and the cells were incubated at 22°C overnight. Then, the cells were collected by centrifugation, resuspended in binding buffer (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, 1 mM EDTA, 1 mM DTT, pH 7.4), and lysed by sonication for 30 min. The supernatant was filtered and then applied to an Ni 2ϩcharged chelating column (GE Healthcare, Piscataway, NJ) that had previously been equilibrated with binding buffer. Proteins were eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, 1 mM DTT, pH 7.4). The fractions with the desired protein were pooled and dialyzed against buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, and 1 mM EDTA. The purified Tpx-6ϫHis and Trx-6ϫHis proteins were stored in aliquots in 10% glycerol at Ϫ80°C until use.
Nonreducing SDS-PAGE. Five micrograms of PerR-GST protein was treated or not treated with 5 mM H 2 O 2 for 30 min and with or without a subsequent reduction by 50 mM DTT for 1 h. Before electrophoresis, 40 mM NEM was added, and the mixture was kept in the dark for 30 min. The protein samples were diluted in nonreducing SDS loading buffer (4ϫ; 0.2 M Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 0.4% bromphenol blue) and then separated on a 12% SDS-PAGE gel.
Determination of zinc content in PerR-GST using ICP-MS. The PerR-GST protein was treated or not treated with 5 mM H 2 O 2 for 30 min and with or without a subsequent reduction by 50 mM DTT for 1 h and was then transferred into Chelex 100-treated PBS buffer via ultrafiltration. Protein concentrations were measured with a BCA protein assay kit. The protein samples were treated with nitric acid (ultrapure), and then the zinc content was analyzed by inductively coupled plasma mass spectrometry (ICP-MS; DRCII apparatus; PerkinElmer, USA) at Peking University Health Science Center. Beryllium, indium, and uranium standard solutions (NIST certified; PerkinElmer) were used to calibrate the ICP-MS. Experiments were conducted for triplicate samples and repeated at least three times.
Electrophoretic mobility shift assay (EMSA). The target gene promoter fragments were generated by PCR amplification using the biotin-labeled primer pair listed in Table S3. The PerR-GST protein was first dialyzed into PBS buffer containing 10 mM EDTA and 1 mM DTT and then digested with 100 U thrombin to remove the GST tag. One micromole of the PerR protein was preincubated with 1 mM MnCl 2 , and then 0.2 nM a biotin-labeled double-stranded DNA probe and increasing amounts of PerR (0 to 200 nM) were mixed in the binding buffer [10 mM Tris-HCl, pH 8.0, 5% glycerol, 50 mM NaCl, 10 g/ml bovine serum albumin, 2 ng/l poly(dI·dC), 0.5 mM DTT, 1 mM MnCl 2 ]. The reaction proceeded at 30°C for 30 min. To observe the effect of H 2 O 2 on PerR binding, 1 M PerR protein was preincubated with 1 mM MnCl 2 and then treated with various concentrations of H 2 O 2 (0 to 200 M) at 30°C for 30 min or with 200 M H 2 O 2 at 30°C for various times (0 to 30 min), and then catalase was added to a final concentration of 150 U/ml and the mixture was incubated at 37°C for 30 min. To determine whether oxidation was reversible, 10 mM DTT was added to reduce the 200 M H 2 O 2 -treated PerR at 37°C for 1 h. Then, 50 nM H 2 O 2 -oxidized or DTT-reduced PerR protein was tested for binding to a 0.2 nM biotin-labeled dpr promoter fragment. The binding mixtures were electrophoresed on a 6% polyacrylamide gel on ice. The DNA-protein complex was transferred onto a nylon membrane and detected with a chemiluminescent nucleic acid detection module kit (Thermo Scientific).
Determination of H 2 O 2 survival rate. Overnight cultures of the tested strains were diluted 1:30 into fresh BHI broth and incubated strictly anaerobically. When the OD 600 reached 0.4 to 0.5, the cells were separated into three aliquots. One aliquot was treated with 10 mM H 2 O 2 for 10 min, and another was prepulsed with 40 M H 2 O 2 for 20 min before being subjected to 10 mM H 2 O 2 treatment, while an aliquot not treated with 10 mM H 2 O 2 was used as a control. Then, the cells were collected, washed twice with PBS, and resuspended in 200 l BHI broth. Cell chains were separated by sonication for 30 s with an XC-3200D ultrasonic cleaner (Xinchen Company, Nanjing, China), and then 10-fold serial dilutions were performed. Appropriate dilutions were plated on BHI agar plates, and the numbers of CFU were counted after 24 h of incubation in a candle jar at 37°C. The survival percentage was calculated by dividing the number of CFU of the H 2 O 2 -challenged sample by the number of CFU of the corresponding controls. Experiments were executed in triplicate, and each experiment was repeated at least three times independently.
Assay of growth under H 2 O 2 stress. S. oligofermentans wild-type and gene deletion strains were grown anaerobically in BHI broth until the OD 600 reached ϳ0.5, with three replicates of each strain being included. Two replicate cultures were supplemented with 40 and 100 M H 2 O 2 , respectively, leaving one replicate as an H 2 O 2 -untreated control. The growth profiles were measured by counting the number of CFU at the different time intervals. Triplicates for each sample were measured, and the experiments were repeated at least three times.
Determination of excreted hydrogen peroxide in culture. The hydrogen peroxide in the culture suspension was quantified as described previously (11). Briefly, 650 l of culture supernatant was added to 600 l of a solution containing 2.5 mM 4-amino-antipyrine (4-amino-2,3-dimethyl-1-phenyl-3pyrazolin-5-one) (Sigma-Aldrich) and 0.17 M phenol. The reaction proceeded for 4 min at room temperature; horseradish peroxidase (Sigma-Aldrich) was then added to a final concentration of 50 mU/ml in 0.2 M potassium phosphate buffer (pH 7.2). After 4 min of incubation at room temperature, the optical density at 510 nm was measured with a Unico 2100 visible spectrophotometer (Unico, Shanghai, China). A standard curve was generated with known concentrations of chemical H 2 O 2 .
Quantitative PCR. Total RNA was extracted from mid-exponential-phase (OD 600 , ϳ0.4 to 0.5) H 2 O 2 -treated and -untreated S. oligofermentans cells using the TRIzol reagent (Invitrogen, Carlsbad, CA), as recommended by the supplier. After quality confirmation with a 1% agarose gel, the RNA was treated with RNase-free DNase (Promega, Madison, WI) and analyzed by PCR for possible chromosomal DNA contamination. cDNA was generated from 2 g total RNA with random primers using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI), according to the supplier's instructions, and was used for quantitative PCR (qPCR) amplification with the corresponding primers (Table S3). Amplifications were performed with a Mastercycler ep realplex 2 instrument (Eppendorf, Germany). To estimate the copy numbers of the tested genes, a standard curve for each tested gene was generated by quantitative PCR using a 10-fold serially diluted PCR product as the template. The 16S rRNA gene was used as the biomass reference. The number of copies of the tested gene transcript per 100 16S rRNA copies is shown. All measurements were done for triplicate samples, and the experiments were repeated at least three times.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.