The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts1,2,3. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Boschi-Muller, S., Gand, A. & Branlant, G. The methionine sulfoxide reductases: catalysis and substrate specificities. Arch. Biochem. Biophys. 474, 266–273 (2008)
Ezraty, B., Aussel, L. & Barras, F. Methionine sulfoxide reductases in prokaryotes. Biochim. Biophys. Acta 1703, 221–229 (2005)
Lee, B. C. & Gladyshev, V. N. The biological significance of methionine sulfoxide stereochemistry. Free Radic. Biol. Med. 50, 221–227 (2011)
Urano, H., Umezawa, Y., Yamamoto, K., Ishihama, A. & Ogasawara, H. Cooperative regulation of the common target genes between hydrogen peroxide-response YedVW and copper-response CusSR in Escherichia coli. Microbiology 161, 729–738 (2015)
Loschi, L. et al. Structural and biochemical identification of a novel bacterial oxidoreductase. J. Biol. Chem. 279, 50391–50400 (2004)
Workun, G. J., Moquin, K., Rothery, R. A. & Weiner, J. H. Evolutionary persistence of the molybdopyranopterin-containing sulfite oxidase protein fold. Microbiol. Mol. Biol. Rev. 72, 228–248 (2008)
Melnyk, R. A. et al. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase. MBio 6, e00233–15 (2015)
Stewart, E. J., Aslund, F. & Beckwith, J. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J. 17, 5543–5550 (1998)
Brokx, S. J., Rothery, R. A., Zhang, G., Ng, D. P. & Weiner, J. H. Characterization of an Escherichia coli sulfite oxidase homologue reveals the role of a conserved active site cysteine in assembly and function. Biochemistry 44, 10339–10348 (2005)
Tarrago, L. & Gladyshev, V. N. Recharging oxidative protein repair: catalysis by methionine sulfoxide reductases towards their amino acid, protein, and model substrates. Biokhimiia 77, 1097–1107 (2012)
Lowe, R. H. & Evans, H. J. Preparation and some properties of a soluble nitrate reductase from Rhizobium japonicum. Biochim. Biophys. Acta 85, 377–389 (1964)
Le, D. T. et al. Analysis of methionine/selenomethionine oxidation and methionine sulfoxide reductase function using methionine-rich proteins and antibodies against their oxidized forms. Biochemistry 47, 6685–6694 (2008)
Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010)
Goemans, C., Denoncin, K. & Collet, J. F. Folding mechanisms of periplasmic proteins. Biochim. Biophys. Acta 1843, 1517–1528 (2014)
Sklar, J. G., Wu, T., Kahne, D. & Silhavy, T. J. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev. 21, 2473–2484 (2007)
Denoncin, K., Schwalm, J., Vertommen, D., Silhavy, T. J. & Collet, J. F. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics. Proteomics 12, 1391–1401 (2012)
Ruiz, N., Falcone, B., Kahne, D. & Silhavy, T. J. Chemical conditionality: a genetic strategy to probe organelle assembly. Cell 121, 307–317 (2005)
Brot, N. et al. The thioredoxin domain of Neisseria gonorrhoeae PilB can use electrons from DsbD to reduce downstream methionine sulfoxide reductases. J. Biol. Chem. 281, 32668–32675 (2006)
Cho, S. H. & Collet, J. F. Many roles of the bacterial envelope reducing pathways. Antioxid. Redox Signal. 18, 1690–1698 (2013)
Hitchcock, A. et al. Roles of the twin-arginine translocase and associated chaperones in the biogenesis of the electron transport chains of the human pathogen Campylobacter jejuni. Microbiology 156, 2994–3010 (2010)
Spector, D., Etienne, F., Brot, N. & Weissbach, H. New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli. Biochem. Biophys. Res. Commun. 302, 284–289 (2003)
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2, 2006.0008 (2006)
Bremer, E., Silhavy, T. J., Weisemann, J. M. & Weinstock, G. M. Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step. J. Bacteriol. 158, 1084–1093 (1984)
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)
Mandin, P. & Gottesman, S. A genetic approach for finding small RNAs regulators of genes of interest identifies RybC as regulating the DpiA/DpiB two-component system. Mol. Microbiol. 72, 551–565 (2009)
Gottlieb, H. E., Kotlyar, V. & Nudelman, A. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 62, 7512–7515 (1997)
Holland, H. L., Andreana, P. R. & Brown, F. M. Biocatalytic and chemical routes to all the stereoisomers of methionine and ethionine sulfoxides. Tetrahedron Asymm. 10, 2833–2843 (1999)
Lavine, T. F. The formation, resolution, and optical properties of the diastereoisomeric sulfoxides derived from l-methionine. J. Biol. Chem. 169, 477–491 (1947)
Koc, A., Gasch, A. P., Rutherford, J. C., Kim, H. Y. & Gladyshev, V. N. Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen species-dependent and -independent components of aging. Proc. Natl Acad. Sci. USA 101, 7999–8004 (2004)
Lherbet, C., Gravel, C. & Keillor, J. W. Synthesis of S-alkyl l-homocysteine analogues of glutathione and their kinetic studies with γ-glutamyl transpeptidase. Bioorg. Med. Chem. Lett. 14, 3451–3455 (2004)
Vertommen, D. et al. The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner. Mol. Microbiol. 67, 336–349 (2008)
Arts, I. S. et al. Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa. MBio 4, e00912–13 (2013)
Vizcaíno, J. A. et al. ProteomeXchange provides globally co-ordinated proteomics data submission and dissemination. Nature Biotechnol. 30, 223–226 (2004)
Roberts, D. M. et al. Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis. Biochemistry 24, 5090–5098 (1985)
Grimaud, R. et al. Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase. J. Biol. Chem. 276, 48915–48920 (2001)
Tsvetkov, P. O. et al. Calorimetry and mass spectrometry study of oxidized calmodulin interaction with target and differential repair by methionine sulfoxide reductases. Biochimie 87, 473–480 (2005)
Cascales, E., Bernadac, A., Gavioli, M., Lazzaroni, J. C. & Lloubes, R. Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity. J. Bacteriol. 184, 754–759 (2002)
Miller, J. A Short Course in Bacterial Genetics Unit 3, 72–74 (Cold Spring Harbor Laboratory Press, 1992)
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013)
Philippe, H. MUST, a computer package of Management Utilities for Sequences and Trees. Nucleic Acids Res. 21, 5264–5272 (1993)
Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010)
Overmars, L., Kerkhoven, R., Siezen, R. J. & Francke, C. MGcV: the microbial genomic context viewer for comparative genome analysis. BMC Genomics 14, 209 (2013)
Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014)
Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods 8, 785–786 (2011)
Sonnhammer, E. L., von Heijne, G. & Krogh, A. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 6, 175–182 (1998)
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2 – approximately maximum-likelihood trees for large alignments. PLoS One 5, e9490 (2010)
Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010)
Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012)
We thank A. Boujtat, G. Herinckx and J.-P. Szikora for technical and computational help and the members of the Barras and Collet laboratories for discussions. We are indebted to M. Sabaty and D. Pignol for sharing unpublished information, to T. Silhavy, T. Palmer, E. Bouveret and D. Hughes for providing strains and plasmids, to T. Lowther and M. Réglier for advice and discussions and to N. Typas, T. Mignot, J. Messens, J. Bardwell and F.-A. Wollman for reading the manuscript and providing comments. A.G. and J.S. are research fellows of the Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture, P.L. is ‘Chargée de Recherche’ and J.-F.C. is ‘Maître de Recherche’ of the Fonds de la Recherche Scientifique-FNRS (FRS-FNRS). E.O. is supported by a grant from the Indo-French Center for the Promotion of Advanced Research CEFIPRA ‘(5105-2)’. R.A. is supported by the Fonds Maurange, Fondation Roi Baudouin. This work was supported, in part, by grants from the FRS-FNRS and from the European Research Council (FP7/2007–2013) ERC independent researcher starting grant 282335–Sulfenic to J.-F.C. and funding by the Centre National de la Recherche Scientifique (CNRS), Fondation pour la Recherche Médicale (FRM) and Aix-Marseille Université to the F.B. team.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Induction of MsrPQ by HOCl is dependent on the presence of a functional YedVW two-component system.
Top, immunoblot analysis shows that the induction of MsrP synthesis by HOCl (0.2 mM) is yedW-dependent. The image is representative of experiments made in biological triplicate. Bottom, an msrP::lacZ fusion was used as a read-out for msrP expression. Deletion of yedV upregulates msrP expression, while deletion of yedW prevents its induction by HOCl. Error bars, mean ± s.e.m.; n = 4. The uncropped blot is shown in Supplementary Fig. 4.
Extended Data Figure 2 Respiratory chain-powered, non-stereospecific reduction of Met-O in periplasmic proteins by the MsrPQ system maintains envelope integrity.
Upon exposure to reactive species of chlorine (RCS) and/or reactive species of oxygen (ROS), methionine residues (Met) in periplasmic proteins such as SurA and Pal get oxidized and randomly form either the R- or the S- diastereoisomer of Met-O. This results in the loss of function of some proteins important for maintaining the integrity of the envelope, such as SurA, giving rise to envelope defects. MsrP catalyses the reduction of both diastereoisomers of Met-O with the help of its molybdenum-molybdopterin (Mo-MPT) cofactor. Electrons for reduction are provided by the quinone (Q) pool of the respiratory chain through MsrQ, the inner membrane haem b-containing partner of MsrP. PG, peptidoglycan.
a, MsrP reduces N-acetyl-Met-O (NacMet-O), a substrate mimicking protein-bound Met-O, with Km = 3.8 ± 1.2 mM, turnover number (kcat) = 30.5 ± 3.1 s–1 and Vmax = 56.3 ± 5.8 μmol min−1 per milligram protein (error bars, mean ± s.d.; n = 3). b, MsrP is a non-stereospecific Msr, being able to reduce both S-Met-O (with Km = 8.0 ± 2.7 mM, kcat = 36.0 ± 3.6 s–1 and Vmax = 67.2 ± 6.4 μmol min−1 per milligram protein) and R-Met-O (with Km = 25.7 ± 4.7 mM, kcat = 168.3 ± 15.0 s–1 and Vmax = 313.4 ± 27.6 μmol min−1 per milligram protein). Error bars, mean ± s.d.; n = 3. c, Strain JB08 (Met– MsrA– MsrB– BisC–, producing MsrC) is able to grow only on R-Met-O, whereas strain CH193 (Met– MsrA– MsrB– MsrC–, producing BisC) in only able to grow on S-Met-O. Deletion of msrP in strain BE100 (Met– Msr– SupMet-O+) prevents its growth on R- and S-Met-O (strain BE104 = Met– Msr– SupMet-O+ ΔmsrP, compare with growth of BE100 in Fig. 2e). Images are representative of experiments made in biological triplicate. d, The periplasmic chaperone SurA was treated with H2O2, giving rise to SurA ox, a sample of which was subsequently incubated with MsrP and the inorganic reducing system in vitro. The oxidation state of specific Met residues (Met 136, 231 and 298) in the various samples was determined by LC–MS/MS analysis. Error bars, mean ± s.e.m.; n = 4.
a, The Oak Ridge Thermal Ellipsoid Plot (ORTEP ellipsoid) representation with 50% probability level of the crystal structure for the isolated salt of l-methionine-S-sulfoxide (right) picrate (left). The grey, blue, red, white and yellow spheres respectively represent carbon, nitrogen, oxygen, hydrogen and sulfur atoms. b, Chemdraw representation of l-methionine-R,S-sulfoxide with proton and carbon positioning (relative to NMR assignment). c, Zoom on the 1H NMR spectra of ~150 mM solutions of l-methionine sulfoxide in D2O pD 6.5, either as a mixture of R- and S- diastereoisomers (top), isolated S- (middle) or isolated R- (bottom) (containing 30 mM dioxane as an internal reference). d, Zoom on the 13C NMR spectra of ~150 mM solutions of l-methionine sulfoxide in D2O pD 6.5, either as a mixture of R- and S- diastereoisomers (top), isolated S- (middle) or isolated R- (bottom) (containing 30 mM dioxane as an internal reference).
Shown are unrooted Bayesian phylogenetic trees for YedY (b1971, 310 sequences, 260 positions). Numbers at nodes indicate posterior probabilities computed by MrBayes49 and bootstrap values computed by PhyML48. Only posterior probabilities and bootstrap values above 0.5 and 50%, respectively, are shown. Scale bars, average number of substitutions per site. In the phylogenetic tree, YedY from E. coli is highlighted in grey.
Shown are unrooted Bayesian phylogenetic trees for YedZ (b1972, 369 sequences, 135 positions). Numbers at nodes indicate posterior probabilities computed by MrBayes49 and bootstrap values computed by PhyML48. Only posterior probabilities and bootstrap values above 0.5 and 50%, respectively, are shown. Scale bars, average number of substitutions per site. In the phylogenetic tree, YedZ from E. coli is highlighted in grey.
This file contains Supplementary Tables 1-3, Supplementary Figures 1-6, legends for Supplementary Data 1-3 (see separate files), and Supplementary References. (PDF 885 kb)
This file contains an exhaustive list of the homologues of YedY and YedZ proteins found in complete genomes (see Supplementary Information file for legend). (PDF 142 kb)
This file contains multiple sequence alignment of YedY homologs (see Supplementary Information file for legend). (PDF 9081 kb)
This file contains multiple sequence alignment of YedZ homologs (see Supplementary Information file for legend). (PDF 16141 kb)
About this article
Cite this article
Gennaris, A., Ezraty, B., Henry, C. et al. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons. Nature 528, 409–412 (2015). https://doi.org/10.1038/nature15764
Influence of rehydration on transcriptome during resuscitation of desiccated Pseudomonas putida KT2440
Annals of Microbiology (2020)
Nature Communications (2020)
High-resolution in situ transcriptomics of Pseudomonas aeruginosa unveils genotype independent patho-phenotypes in cystic fibrosis lungs
Nature Communications (2018)
Nature Reviews Microbiology (2017)
Proteome wide identification of iron binding proteins of Xanthomonas translucens pv. undulosa: focus on secretory virulent proteins