NemR Is a Bleach-sensing Transcription Factor*

Background: Little is known about how bacteria sense or respond to reactive chlorine species, such as bleach. Results: NemR is a redox-regulated transcription factor which senses bleach. Conclusion: NemR controls expression of genes encoding electrophile detoxification enzymes, which increase bleach resistance. Significance: We demonstrate a bleach-sensing bacterial response system and a new mechanism contributing to bacterial bleach survival. Hypochlorous acid (HOCl), the active component of household bleach, also functions as a powerful antimicrobial during the innate immune response. Despite its widespread use, surprisingly little is known about how cells sense or respond to HOCl. We now demonstrate that Escherichia coli NemR is a redox-regulated transcriptional repressor, which uses the oxidation status of HOCl-sensitive cysteine residues to respond to bleach and related reactive chlorine species. NemR controls bleach-mediated expression of two enzymes required for detoxification of reactive electrophiles: glyoxalase I and N-ethylmaleimide reductase. Both enzymes contribute to bacterial bleach survival. These results provide evidence that bleach resistance relies on the capacity of organisms to specifically sense reactive chlorine species and respond with the up-regulation of enzymes dedicated to detoxification of methylglyoxal and other reactive electrophiles.

Hypochlorous acid (HOCl) 5 is a powerful oxidant and one of the most commonly used disinfectants in the world (1). It is also naturally generated during the microbicidal oxidative burst of neutrophils and appears to play a key role in controlling bacterial colonization of mucosal epithelia (2,3). Despite this physiological importance, little is known about how bacteria sense or defend themselves against reactive chlorine species (RCS), which include HOCl, chloramines, and other related compounds that are able to chlorinate and oxidize biomolecules (4). Very few transcription factors sensitive to HOCl have been reported to date. YjiE has recently been shown to regulate a variety of genes in Escherichia coli in response to HOCl and to play a role in HOCl survival (5). OhrR of Bacillus subtilis, previously thought to be specific for sensing organic hydroperoxides, has been reported to be involved in HOCl resistance as well (6). Relatively little is known, however, about how these regulators and the genes they control contribute to HOCl survival. This is in contrast to the very large body of research that has been conducted to investigate how bacteria sense and defend themselves against other, less reactive bactericidal oxidants (4, 7) such as hydrogen peroxide (H 2 O 2 ) or superoxide (O 2 Ϫ ) (for recent review, see Refs. 8 -11).
Given the importance of HOCl in health and disease, we therefore decided to perform a more detailed investigation of how bacteria sense and respond to bleach. Here, we report our discovery that the widely conserved TetR family repressor NemR functions as a HOCl-responsive transcription factor in E. coli in addition to its previously described role in responding to cysteine-modifying electrophiles (12). In vitro and in vivo studies reveal that HOCl sensitivity is conferred upon NemR by cysteine residues that are highly sensitive toward oxidation by HOCl and related physiologically important RCS, such as N-chlorotaurine, an antimicrobial compound formed when HOCl reacts with taurine (2-aminoethanesulfonic acid) during the oxidative burst of neutrophils (13). RCS oxidation of cysteine residues, which is a fully reversible process in vitro and likely also in vivo, leads to a decrease in NemR DNA binding affinity, causing the derepression of downstream target gene expression. We demonstrate that bleach-mediated up-regulation of the NemR-controlled genes gloA and nemA increases bacterial bleach survival, providing the first evidence that resistance toward bleach relies on the ability of bacteria to detoxify methylglyoxal and reactive electrophiles.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions-All strains and plasmids used in this study are listed in Table 1. E. coli was grown at 37°C in lysogenic broth (LB) or MOPS minimal medium (Teknova, Inc.) containing 0.2% glucose, 1.32 mM K 2 HPO 4 , and 10 M thiamine. Unless specified otherwise, all chemicals were from Fisher or Sigma-Aldrich. N-Chlorotaurine and methylglyoxal were synthesized before each use (14,15).

Microarray Analysis and Data
Processing-E. coli MG1655 was grown in MOPS glucose medium at 37°C with aeration to A 600 ϭ 0.4 -0.5. HOCl was added to a final concentration of 400 M. 5-ml samples were collected in liquid nitrogen immediately before, 5 min after, and 10 min after HOCl addition, and total RNA was prepared using the Rneasy Midi kit (Qiagen). cDNA synthesis, array hybridization to Affymetrix GeneChip E. coli genome 2.0 arrays, and imaging were performed at the Affymetrix and Microarray Core facility at the University of Michigan, Ann Arbor. Quality of raw images and expression values were analyzed using the expresso function and affy package of Bioconductor (16). Three biological replicates were conducted. The TM4 MultiExperiment Viewer (17) was used to identify patterns of gene expression and differentially expressed genes. A total of 5367 MG1655 probes were separated into 25 clusters with 100 iterations. The pattern of each cluster was evaluated based on log2-transformed ratios of treated versus untreated samples. Probes in each cluster have expression pat-terns similar to each other and dissimilar to those in other clusters.

Construction of Chromosomal nemR Variants-The nemR
alleles from pNEMR2, pNEMR32, pNEMR43, and pNEMR44 were amplified using primers [20] and [21]. The cat ϩ cassette was amplified from plasmid pKD3 (19) using primers [22] and [23]. 200 fmol of nemR PCR product and 200 fmol of cat ϩ PCR product were mixed and used as template for amplification with primers [20] and [23]. The resulting nemR-cat ϩ PCR products were gel-purified, transformed into strain MJG244, and selected for chloramphenicol resistance. The cat ϩ cassettes in these strains were then resolved (19).
Methylglyoxal Survival Assays-E. coli MG1655 and isogenic mutant strains were grown at 37°C with shaking (200 rpm) in 25 ml of MOPS glucose medium to A 600 ϭ 0.4 -0.6, then methylglyoxal was added to 150 M, and cells were harvested and spot-titered as above. To test the ability of HOCl to cross-protect against methylglyoxal, 0.4 mM HOCl was added, and cells were incubated for 10 min before the addition of 0.5 mM Na 2 S 2 O 3 and methylglyoxal.
Quantification of Intracellular Free Methylglyoxal-Methylglyoxal was measured using a previously described HPLC method (24).
Metal Analysis-The metal content of purified NemR was analyzed using inductively coupled plasma-high resolution mass spectrometry at the Keck Elemental Geochemistry Laboratory, Department of Geological Sciences, University of Michigan.
In Vitro Oxidation and Gel Mobility Shift Assays of NemR Proteins-Purified NemR proteins were incubated for 15 min at 37°C in 50 mM sodium phosphate (pH 8), 150 mM NaCl with the indicated molar ratio of oxidants, then exchanged into gel shift buffer (10 mM Tris-HCl (pH 7.8 at 4°C), 150 mM NaCl, 3 mM magnesium acetate, 10% glycerol) using a P-30 gel chromatography column. To assay reversibility of oxidation, N-chlorotaurine-treated samples were incubated with 1 mM DTT for 15 min at 37°C. Gel mobility shift assays were a modification of the procedure of Umezawa et al. using a 222-bp fragment of E. coli MG1655 genomic DNA containing the nemR promoter region (P nemR ), amplified with primers [36] and [37] (12). NemR variants were incubated in gel shift buffer with P nemR for 30 min at 37°C. Fragments were separated by electrophoresis on 10% TBE-polyacrylamide gels (Bio-Rad), stained with ethidium bromide, visualized by UV fluorescence, and quantified using ImageJ 1.44o (25).
In Vivo Thiol Trapping of NemR-E. coli BL21 containing plasmids expressing His-tagged NemR variants were grown at 37°C in MOPS glucose medium to A 600 ϭ 0.4 -0.5. NemR expression was induced with 10 M isopropyl 1-thio-␤-D-galactopyranoside for 30 min, after which cells were treated with 1 mM HOCl. At the indicated time points, 1-ml cell aliquots were harvested by centrifugation and resuspended in 100% acetone. After 2 h of incubation at Ϫ20°C, precipitated proteins were pelleted by centrifugation and resuspended in 6 M urea, 200 mM Tris-HCl (pH 8.5), 10 mM EDTA, 0.5% w/v SDS, and supplemented with 0.8 M iodoacetamide. Samples were separated by nonreducing SDS-PAGE and visualized by Western blotting using an anti-His tag antibody (Abcam).

Identification of Bleach-responsive Regulators in E. coli-To
identify regulators that might contribute to bacterial bleach defense, we examined gene expression in E. coli before and after a sublethal dose of HOCl (0.4 mM) using a transcriptional microarray, the full results of which are shown in supplemental Table S1. Among the genes significantly up-regulated by HOCl treatment were two electrophile detoxification genes (nemA and gloA) known to be under the control of NemR, a transcriptional repressor of the TetR family (Fig. 1A, inset). NemR, which was also found to be significantly up-regulated in response to HOCl treatment in our microarray analysis, has previously been reported to be sensitive to the electrophilic cysteine alkylation agent N-ethylmaleimide (NEM) (12). NEM is a synthetic reagent that irreversibly alkylates cysteine thiols. In contrast, HOCl is a physiological oxidant that causes the rapid and reversible oxidation of cysteine thiols to sulfenic acids and disulfide bonds (27). We therefore decided to pursue the study of NemR as a potentially bleach-responsive transcriptional regulator.
NemR Is an HOCl-sensing Transcription Factor-Quantitative RT-PCR confirmed our microarray data and revealed approximately 70-fold up-regulation of nemR and 100-fold and 10-fold up-regulation of the downstream genes nemA and gloA, respectively, in response to sublethal HOCl treatment (Fig. 1A). Expression of both nemA and gloA was constitutively induced and almost completely nonresponsive to HOCl in a ⌬nemR strain (Fig. 1A), indicating that bleach-mediated expression of both genes was almost entirely dependent on NemR. Although Umezawa et al. (12) identified weak NemR binding sites upstream of the pck and yeaU genes of E. coli, neither of these genes showed NemR-dependent up-regulation upon HOCl treatment (data not shown), suggesting that nemA and gloA are the primary regulatory targets of NemR.
To determine the in vivo oxidant specificity of the NemR response, we treated wild-type E. coli cells with various electrophiles and with reactive oxygen, nitrogen, and chlorine species, utilizing concentrations that induced transient growth arrest under our growth conditions without killing the cells (data not shown). These studies revealed that, in addition to responding to NEM, NemR is sensitive to treatment with either HOCl or N-chlorotaurine, a prominent secondary oxidation product of HOCl in vivo (13). It also responded weakly to the disulfidegenerating electrophile diamide (diazenedicarboxylic acid bis(N,N-dimethylamide)) ( Fig. 1B) (28). In contrast, no significant NemR-mediated gene expression was observed in response to H 2 O 2 , the O 2 Ϫ generator methyl viologen, the nitric oxide source diethylamine nitric oxide (DEANO), or, notably, methylglyoxal, the substrate of the NemR downstream target GloA (glyoxalase I) (29) (Fig. 1B). These data demonstrate that induction of the nemR regulon is sensitive to HOCl and related RCS.
Phenotypic studies revealed that deletion of gloA and, to a lesser extent, nemA, increased the HOCl sensitivity of E. coli cells. In contrast, deletion of nemR had no significant effect on bleach survival ( Fig. 2A), indicating that constitutive overexpression of nemA and gloA did not provide additional protection against HOCl. As expected, mutants lacking gloA were more sensitive to methylglyoxal treatment than wild-type E. coli (Fig. 2B) (29). A ⌬nemR strain was considerably more resistant to methylglyoxal than wild type presumably due to the constitutive derepression of gloA. Similarly, pretreatment of E. coli with 0.4 mM HOCl protected against methylglyoxal stress (Fig. 2C), as would be expected due to NemR-dependent up-regulation of gloA. ⌬nemA mutants showed no defect in survival of methylglyoxal stress, indicating that NemA is more important for HOCl stress tolerance than for resistance to exogenous methylglyoxal (Fig. 2, A and B). Consistent with these results, HPLC analysis of HOCl-treated E. coli revealed substantial accumulation of intracellular methylglyoxal after HOCl treatment (Fig. 2D). These results suggest that NemR is a bacterial repressor which is sensitive to HOCl and related RCS and whose gene products contribute to increased bleach resistance in E. coli by detoxifying reactive electrophiles produced during RCS stress.
Cysteine 106 Is Conserved among NemR Homologs-The simplest hypothesis explaining these results was that HOCl-mediated modification of NemR leads to the derepression of the nemR operon, causing the up-regulation of genes that increase bacterial resistance to these oxidants. To first investigate whether the NemR bleach sensing mechanism might involve oxidation of redox-sensitive metals (11), we conducted inductively coupled plasma mass spectrometry of the purified NemR protein. However, we were unable to detect any metals associated with NemR (data not shown), indicating that HOCl sensing in NemR is likely mediated by other means. Because some transcription factors, such as the peroxide-sensing OxyR, have been shown to rely on the reactivity of functionally or structurally important cysteine thiols to sense oxidants (9), we aligned NemR homologs from 166 bacterial genera (BLAST e-value Ͻ 0.00001) to identify potentially evolutionarily important cysteines. Of the six cysteines in E. coli NemR, only one, Cys-106, is highly conserved (Fig. 3A, E. coli numbering in red). Modeling of the E. coli NemR sequence onto the molecular structure of its Acinetobacter homolog (Protein Data Bank ID code 3KNW, 41% identical, 61% similar to E. coli NemR) (21)  predicted that Cys-106 is surface-exposed and faces the predicted DNA binding site (Fig. 3B, green helices). Cys-21 is located directly in the DNA binding helix whereas Cys-116 and Cys-153 are located in the predicted dimerization interface of NemR. None of these other cysteines is, however, particularly well conserved among NemR homologs (Fig. 3A).
Cys-106 Is Sufficient for NemR RCS-sensing Mechanism-To investigate the role of these cysteines in the ability of NemR to respond to HOCl and related RCS, we constructed strains containing different cysteine mutant alleles of nemR at the native locus in the E. coli chromosome. As a read-out, we monitored their ability to respond to HOCl treatment with the overexpression of nemR using RT-PCR.
NemR 6C-S , in which all six cysteines were mutated to serine, did not respond at all to HOCl treatment (Fig. 4, red trace), strongly indicating that HOCl sensing was cysteine-dependent. Repression of the nemR operon was maintained in these strains, albeit slightly relieved compared with to wild-type strains, indicating that this mutant protein largely maintains its DNA binding capacity despite the lack of all cysteines. A similar relief in basal repression was observed in strains expressing NemR 5C-S(C106) , which lacked all but the absolutely conserved Cys-106. In con-trast to the strain expressing the no-cysteine variant, however, this strain responded to HOCl in a wild-type-like manner (Fig.  4, compare black and green traces). These results suggested that the conserved Cys-106 is sufficient for mediating a wild-typelike HOCl response in NemR.
Notably, cells expressing NemR C106S , a variant that lacks the conserved Cys-106 but contains all five other nonconserved  cysteines, were also able to mount a HOCl response (Fig. 4, blue  trace). However, gene expression rapidly declined in these bacteria, suggesting that Cys-106 might be crucial for maintaining a sustained HOCl response. This result was not completely unexpected given that some of the nonconserved cysteines are predicted to be in structurally or functionally critical locations of NemR and their oxidation might lead to derepression as well. Nevertheless, the fact that Cys-106 is absolutely conserved among members of the NemR family and is sufficient to confer the observed bleach response suggests that Cys-106 plays the central role in the NemR RCS-sensing mechanism.
NemR DNA Binding Activity Is RCS-sensitive in Vitro-To investigate the effects of RCS treatment on the DNA binding activity of NemR in vitro, we purified wild-type NemR and cysteine-mutated NemR variants and conducted in vitro gel shift assays using a DNA fragment containing the promoter region of nemR (12). We found that reduced wild-type NemR binds strongly to its own promoter region (Fig. 5A, top left). Incuba- tion of NemR with a 1:1 molar ratio of N-chlorotaurine, used as an HOCl substitute in in vitro experiments because it causes many fewer nonspecific oxidation artifacts than HOCl (30), substantially decreased the NemR DNA binding affinity (Fig.  5A, top right). Incubation of NemR with a 1:1 molar ratio of H 2 O 2 had much less effect on its DNA binding (Fig. 5A, bottom  left), consistent with the failure of H 2 O 2 to induce nemR expression in vivo (Fig. 1B). These results supported our in vivo studies and suggested that RCS oxidation of NemR causes its dissociation from DNA, therefore leading to the observed derepression of NemR target gene expression. Most importantly, incubation of oxidized inactive NemR with excess amounts of the thiolspecific reducing agent DTT fully restored DNA binding (Fig.  5A, bottom right), indicating that, unlike the irreversible inactivation of NemR by NEM alkylation (12), oxidative inactivation of NemR is a fully reversible process in vitro.
In vitro DNA binding analysis of the three NemR cysteine variants yielded results that were also fully consistent with our in vivo findings. The mutant variant NemR 5C-S(C106) , which contains only the conserved Cys-106, revealed a decrease in in vitro DNA binding affinity in response to N-chlorotaurine treatment that was very similar in relative extent to the decrease observed in N-chlorotaurine-treated wild-type NemR (Fig. 5B). The presence of all cysteines except Cys-106 also resulted in in vitro RCS sensitivity (Fig. 5C), supporting our in vivo results suggesting that one or more of the remaining nonconserved cysteines of E. coli NemR are RCS-sensitive as well (Fig. 4). In contrast, the cysteine-free NemR 6C-S protein remained bound to DNA independent of its pretreatment, indicating that absence of all six cysteines did not affect the NemR DNA binding but eliminated its ability to sense reactive chlorine species (Fig. 5D).
Monitoring the in Vitro and in Vivo Thiol Oxidation Status of NemR-To further examine the role that cysteine residues play in the RCS response of NemR, we examined the thiol oxidation status of our purified NemR wild-type and the three cysteine variants on nonreducing SDS-PAGE before and after treatment with N-chlorotaurine. In the absence of oxidants, all purified NemR variants migrated predominantly as monomeric proteins, although some disulfide-bonded species were already observed in untreated wild-type NemR (Fig. 6A). Upon treatment with a 1:1 molar ratio of N-chlorotaurine, however, both wild-type NemR and NemR C106S , which contains all but the conserved Cys-106, formed a variety of higher molecular mass species, which disappeared as soon as reducing conditions were reestablished (Fig. 6A). These results suggested that NemR forms intermolecular disulfide bonds with other NemR molecules upon treatment with RCS in vitro. As expected, NemR 6C-S , which does not contain any cysteine, remained fully monomeric (albeit with a slightly different migration behavior than cysteine-containing variants), further excluding the involvement of any noncysteine modifications in the HOClsensing mechanism of NemR. Importantly, HOCl-treated NemR 5C-S(C106) , which contains only the highly conserved Cys-106 and whose in vivo and in vitro response to HOCl mimics that of wild-type NemR, remained largely monomeric upon treatment with N-chlorotaurine (Fig. 6A). These results suggested that intermolecular disulfide bond formation is not required for NemR functional regulation in vitro. Indeed, Ellman's assay, which we used to monitor the oxidation status of the single Cys-106 in NemR 5C-S(C106) , revealed that of the 80% of reduced molecules in the untreated NemR 5C-S(C106) preparation, all but 8% were oxidized by N-chlorotaurine. Most notably, incubation of oxidized NemR 5C-S(C106) with 5 mM DTT for 30 min reduced almost 60% of all molecules, excluding the presence of irreversible overoxidation products (such as sulfinic or sulfonic acids). The simplest hypothesis to explain this result was that, in the absence of other cysteines, Cys-106 might undergo reversible sulfenic acid formation, the immediate consequence of RCS-mediated chlorination of thiol groups (4). Both reduced thiols and sulfenic acids can be detected using the probe NBD-Cl, which forms adducts with absorption maxima of 420 nm for thiols and 350 nm for sulfenic acid (31). We recorded the UV spectrum of N-chlorotaurine-oxidized NemR 5C-S(C106) in the presence of NBD-Cl but were unable to detect any significant sulfenic acid adducts in our sample (data not shown). Depending on the microenvironment of the cysteine, however, the nucleophilicity of the sulfur in sulfenic acid may not be sufficient to react with the electrophilic NBD-Cl reagent. We therefore decided to conduct a chymotryptic digest of reduced and N-chlorotaurine-oxidized NemR 5C-S(C106) and perform mass spectrometric analysis of the peptides. In contrast to the reduced NemR 5C-S(C106) sample, for which 100% coverage was obtained, we were unable to identify the Cys-106-containing peptide in the oxidized sample despite using an error-tolerant search with the latest version of Unimod (32). Future studies are clearly needed to identify the nature of the reversible thiol oxidation product in oxidized NemR.
In vivo thiol trapping experiments fully supported our in vitro findings and revealed the formation of intermolecular disulfide bonds in HOCl-treated cells expressing either wildtype NemR or the NemR variants lacking Cys-21, Cys-106, and Cys-149 (Fig. 6B). Variants lacking Cys-98 or Cys-153 formed substantially fewer higher molecular mass species, whereas the variant lacking Cys-116 formed none. These results indicate that Cys-116, located within the predicted dimer interface of NemR (Fig. 3B), plays the primary role in the observed intermolecular disulfide formation after HOCl stress in vivo. Neither the redox-active NemR 5C-S(C106) variant nor the inactive NemR 6C-S variant formed any disulfide bonded species in vivo. These results confirm that intermolecular disulfide bond formation is not required for a sustained NemR-mediated RCS response in E. coli and suggest that reversible oxidative modification of the absolutely conserved Cys-106 plays the central role in the sensing of and response to RCS.

DISCUSSION
Our study demonstrates that NemR is a transcriptional regulator that uses RCS-sensitive cysteine residues to rapidly induce expression of specific reductases involved in the detoxification of reactive electrophiles. Our data suggest a model in which NemR uses the reversible oxidation of its highly conserved Cys-106 as a trigger for conformational rearrangements that cause its dissociation from the DNA and subsequently the activation of nemA and gloA expression. Because in vitro exposure of wild-type NemR to an equimolar amount of N-chlorotaurine led not only to the oxidation of Cys-106, but also to the formation of a variety of intermolecularly disulfide-bonded oligomers (Fig. 6), it is likely that RCS-mediated chlorination of Cys-106 not only leads to oxidation of Cys-106 but also to an oxidative cascade, chlorinating and oxidizing other cysteine residues in NemR (4). In vivo analysis demonstrated that Cys-116, predicted to be near the dimer interface (Fig. 3B), is involved in the formation of intermolecular disulfide bonds (Fig. 6C) and may be responsible for the inactivation of NemR by RCS in the absence of Cys-106 (Figs. 4 and 5). However, the NemR 5C-S(C106) mutant, which lacks Cys-116 (as well as all other cysteines apart from Cys-106), is able to respond to RCS in a wild-type manner both in vivo and in vitro, indicating that Cys-106, the only conserved cysteine in NemR, is sufficient for wild-type-like HOCl sensing in vivo. The additional nonconserved cysteine residues appear to be able to partially substitute for its absence. This result is reminiscent of the peroxide-sensing transcription factor OxyR, which uses reversible peroxidemediated sufenic acid formation at its absolutely conserved Cys-199 for sensing but is able to undergo disulfide bond formation with the less conserved Cys-208 (9). At this point, we do not know the nature of the reversible thiol modification that occurs at NemR Cys-106 and which leads to the loss in DNA binding affinity. Based on our preliminary mass spectrometric results, the modification appears to be distinct from the reported sulfenamide formation or S-bacillithiolation that occurs at Cys-15 of B. subtilis repressor OhrR. Like NemR, OhrR is inactivated by HOCl, leading to the derepression of its target gene, peroxiredoxin OhrA (33,34). YjiE, another HOClsensing transcription factor in E. coli, contains no conserved cysteines (5), and the mechanism by which YjiE senses HOCl remains unknown. The role of the NemR nonconserved cysteines, and the properties of NemR that confer its specificity for certain electrophiles and RCS are currently under investigation.
Our studies revealed that NemA and particularly GloA are critical to bacterial bleach survival. NemA is a flavin-dependent reductase, which is active on a variety of electrophilic compounds in vitro, including NEM. However, its physiological substrate is so far unknown (35). The function of GloA is much better understood. GloA is broadly conserved among prokaryotic and eukaryotic species as the primary methylglyoxal-detoxifying enzyme (29). Methylglyoxal is a reactive ketoaldehyde capable of damaging proteins and nucleic acids and is formed by dephosphorylation of the glycolytic intermediate dihydroxyacetone phosphate (36). Numerous oxidative stress conditions have been associated with the accumulation of methylglyoxal and other reactive electrophiles in both bacteria and eukaryotes; however, the precise relationship between oxidative stress and methylglyoxal accumulation remains poorly understood (37). Our results demonstrate that increased methylglyoxal production is a direct consequence of HOCl stress and suggest that detoxification of reactive electrophiles is a critical component of bacterial RCS defense. The pathway(s) responsible for HOCl-induced methylglyoxal production are currently being investigated in our laboratory. The fact that NemR does not respond directly to methylglyoxal implies that E. coli uses NemR to mount a proactive response, anticipating an increase in methylglyoxal levels in response to RCS. Previous work on NemR has shown that it responds to cysteine-modifying electrophiles, including NEM, showdomycin, and, more weakly, iodoacetamide (12). In combination with the results presented here, this suggests that NemR is an RCS-and electrophile-sensing regulator important for defense against a subset of stresses that result in toxic modification of cellular thiols. NemR homologs are broadly conserved among both Gram-negative and Gram-positive bacteria. The E. coli-like nemR operon structure is conserved among Enterobacteriacea (38), suggesting that the NemR-controlled gloA and nemA expression response is conserved throughout this group. In other clades, genes encoding NemR homologs are commonly found in loci containing genes potentially involved in response to oxidative or electrophilic stress, including glutathione S-transferases, oxidoreductases, and alcohol dehydrogenases. Supporting the idea that sensing of RCS may be a common function among NemR homologs, the Rhodococcus rhodochrous NemR homolog DhaR (31% identical, 51% similar to E. coli NemR) has been shown to regulate breakdown of a variety of chlorinated compounds, including 1-chlorobutane and 1,3-dichloropropene, although the detailed mechanism and specificity of DhaR have not been investigated (39).
In conclusion, our studies identified NemR as a broadly conserved bacterial transcription factor that relies on cysteine thiol oxidation to sense and respond to both RCS and reactive elec-trophiles. In E. coli, NemR regulates expression of electrophile detoxification genes that play an important role in surviving bleach stress.