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Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species

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Abstract

The genome of the green sulfur bacterium Chlorobaculum (Cba.) tepidum, a strictly anaerobic photolithoautotroph, is predicted to encode more than ten genes whose products are potentially involved in protection from reactive oxygen species and an oxidative stress response. The encoded proteins include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were insertionally inactivated. Wild-type Cba. tepidum cells were very sensitive to oxygen in the light but were remarkably resistant to oxygen in the dark. When wild-type and mutant cells were subjected to air for various times under dark or light condition, significant decreases in viability were detected in most of the mutants relative to wild type. Treatments with hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BOOH) and methyl viologen resulted in more severe effects in most of the mutants than in the wild type. The results demonstrated that these putative antioxidant proteins combine to form an effective defense against oxygen and reactive oxygen species. Reverse-transcriptase polymerase chain reaction studies showed that the genes with functions in oxidative stress protection were constitutively transcribed under anoxic growth conditions.

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References

  • Alphey MS, Bond CS, Tetaud E, Fairlamb AH, Hunter WN (2000) The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins. J Mol Biol 300:903–916

    Article  CAS  PubMed  Google Scholar 

  • Benov L, Kredich NM, Fridovich I (1996) The mechanism of auxotrophy for sulfur-containing amino acids imposed upon Escherichia coli by superoxide. J Biol Chem 271:21037–21040

    Article  CAS  PubMed  Google Scholar 

  • Blankenship RE, Cheng P, Causgrove TP, Brune DC, Wang S-H, Choh JU, Wang J (1993) Redox regulation of energy transfer efficiency in antennas of green photosynthetic bacteria. Photochem Photobiol 57:103–107

    Article  CAS  PubMed  Google Scholar 

  • Briviba K, Klotz LO, Sies H (1997) Toxic and signaling effects of photochemically or chemically generated singlet oxygen in biological systems. Biol Chem 378:1259–1265

    CAS  PubMed  Google Scholar 

  • Carlioz A, Touati D (1986) Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J 5:623–630

    CAS  PubMed  Google Scholar 

  • Cha MK, Kim WC, Lim CJ, Kim K, Kim IH (2004) Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth. J Biol Chem 279:8769–8778

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Liu MY, LeGall J, Fareleira P, Santos H, Xavier AV (1993) Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the “strict anaerobe” Desulfovibrio gigas. Biochem Biophys Res Commun 193:100–105

    Article  CAS  PubMed  Google Scholar 

  • Choi J, Choi S, Choi J, Cha MK, Kim IH, Shin W (2003) Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins. J Biol Chem 278:49478–49486

    Article  CAS  PubMed  Google Scholar 

  • Claiborne A, Yeh JI, Mallett TC, Luba J, Crane EJ III, Charrier V, Parsonage D (1999) Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 38:15407–15416

    Article  CAS  PubMed  Google Scholar 

  • Das A, Silaghi-Dumitrescu R, Ljungdahl LG, Kurtz DM Jr (2005) Cytochrome bd oxidase, oxidative stress, and dioxygen tolerance of the strictly anaerobic bacterium Moorella thermoacetica. J Bacteriol 187:2020–2029

    Article  CAS  PubMed  Google Scholar 

  • Dos Santos WG, Pacheco I, Liu MY, Teixeira M, Xavier AV, LeGall J (2000) Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas. J Bacteriol 182:796–804

    Article  CAS  PubMed  Google Scholar 

  • Eisen JA, Nelson KE, Paulsen IT, Heidelberg JF, Wu M, Dodson RJ, Deboy R, Gwinn ML, Nelson WC, Haft DH, Hickey EK, Peterson JD, Durkin AS, Kolonay JL, Yang F, Holt I, Umayam LA, Mason T, Brenner M, Shea TP, Parksey D, Nierman WC, Feldblyum TV, Hansen CL, Craven MB, Radune D, Vamathevan J, Khouri H, White O, Gruber TM, Ketchum KA, Venter JC, Tettelin H, Bryant DA, Fraser CM (2002) The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proc Natl Acad Sci USA 99:9509–9514

    Article  CAS  PubMed  Google Scholar 

  • Flint D, Tuminello J, Emptage M (1993) The inactivation of Fe-S cluster containing hydro-lyses by superoxide. J Biol Chem 268:22369–22376

    CAS  PubMed  Google Scholar 

  • Frazão C, Silva G, Gomes CM, Matias P, Coelho R, Sieker L, Macedo S, Liu MY, Oliveira S, Teixeira M, Xavier AV, Rodrigues-Pousada C, Carrondo MA, LeGall J (2000) Structure of a dioxygen reduction enzyme from Desulfovibrio gigas. Nat Struct Biol 7:1041–1045

    Article  PubMed  Google Scholar 

  • Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64:97–112

    Article  CAS  PubMed  Google Scholar 

  • Benov L, Fridovich I (1999) Why superoxide imposes an aromatic amino acid auxotrophy on Escherichia coli. The transketolase connection. J Biol Chem 274:4202–4206

    Article  CAS  PubMed  Google Scholar 

  • Frigaard N-U, Bryant DA (2001) Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation. Appl Environ Microbiol 67:2538–2544

    Article  CAS  PubMed  Google Scholar 

  • Frigaard N-U, Bryant DA (2006) Chlorosomes: antenna organelles in green photosynthetic bacteria. In: Shively JM (ed) Microbiology monographs, vol 2 complex intracellular structures in Prokaryotes. Springer, Berlin, pp 79–114

    Chapter  Google Scholar 

  • Frigaard N-U, Matsuura K (1999) Oxygen uncouples light absorption by the chlorosome antenna and photosynthetic electron transfer in the green sulfur bacterium Chlorobium tepidum. Biochim Biophys Acta 1412:108–117

    Article  CAS  PubMed  Google Scholar 

  • Frigaard N-U, Maresca JA, Yunker CE, Jones AD, Bryant DA (2004a) Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum. J Bacteriol 186:5210–5220

    Article  CAS  PubMed  Google Scholar 

  • Frigaard N-U, Sakuragi Y, Bryant DA (2004b) Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation. Meth Mol Biol 274:325–340

    CAS  Google Scholar 

  • Gennis RB, Stewart V (1996) Respiration. In: Neidhardt FC (ed) Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, pp 217–261

    Google Scholar 

  • Hauska G, Schoedl T, Remigy H, Tsiotis G (2001) The reaction center of green sulfur bacteria. Biochim Biophys Acta 1507:260–277

    Article  CAS  PubMed  Google Scholar 

  • Hillmann F, Fischer R-J, Saint-Prix F, Girbal L, Bahl H (2008) PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum. Mol Microbiol 68:848–860

    Article  CAS  PubMed  Google Scholar 

  • Hirotsu S, Abe Y, Okada K, Nagahara N, Hori H, Nishino T, Hakoshima T (1999) Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product. Proc Natl Acad Sci USA 96:12333–12338

    Article  CAS  PubMed  Google Scholar 

  • Iino T, Mori K, Uchino Y, Nakagawa T, Harayama S, Suzuki K (2009) Ignavibacterium album gen. nov., sp. nov., a moderately thermophilic anaerobic bacterium isolated from microbial mats at a terrestrial hot spring in Japan and proposal of Ignavibacteria classis nov. for a novel lineage at the periphery of green sulfur bacteria. Int J Syst Evol Microbiol. doi:10.1099/ijs.0.012484-0

  • Ikonen TP, Li H, Psencik J, Laurinmäki PA, Butcher SJ, Frigaard N-U, Serimaa RE, Bryant DA, Tuma R (2007) X-ray scattering and electron cryomicroscopy study on the effect of carotenoid biosynthesis to the structure of Chlorobium tepidum chlorosomes. Biophys J 93:620–628

    Article  CAS  PubMed  Google Scholar 

  • Imhoff JF (2003) Phylogenetic taxonomy of the family Chlorobiaceae on the basis of 16S rRNA and fmo (Fenna–Matthews–Olson protein) gene sequences. Int J Syst Evol Microbiol 53:941–951

    Article  CAS  PubMed  Google Scholar 

  • Imlay JA (2002) How oxygen damages microbes: oxygen tolerance and obligate anaerobiosis. Adv Microb Physiol 46:111–153

    Article  CAS  PubMed  Google Scholar 

  • Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol 57:395–418

    Article  CAS  PubMed  Google Scholar 

  • Imlay JA (2006) Iron-sulphur clusters and the problem with oxygen. Mol Microbiol 59:1073–1082

    Article  PubMed  Google Scholar 

  • Imlay JA (2008) How obligatory is anaerobiosis? Mol Microbiol 68:801–804

    Article  CAS  PubMed  Google Scholar 

  • Seaver LC, Imlay JA (2001) Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J Bacteriol 183:7173–7181

    Article  CAS  PubMed  Google Scholar 

  • Imlay JA, Linn S (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240:640–642

    Article  CAS  PubMed  Google Scholar 

  • Jenney FE Jr, Verhagen MF, Cui X, Adams MW (1999) Anaerobic microbes: oxygen detoxification without superoxide dismutase. Science 286:306–309

    Article  CAS  PubMed  Google Scholar 

  • Jeong W, Cha MK, Kim IH (2000) Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family. J Biol Chem 275:2924–2930

    Article  CAS  PubMed  Google Scholar 

  • Jeong W, Cha MK, Kim IH (2006) Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress. FEBS J 273:721–731

    Article  Google Scholar 

  • Jiang Q, Yan YH, Hu GK, Zhang YZ (2005) Molecular cloning and characterization of a peroxiredoxin from Phanerochaete chrysosporium. Cell Mol Biol Lett 10:659–668

    CAS  PubMed  Google Scholar 

  • Juty NS, Moshiri F, Merrick M, Anthony C, Hill S (1997) The Klebsiella pneumoniae cytochrome bd terminal oxidase complex and its role in microaerobic nitrogen fixation. Microbiology 143:2673–2683

    Article  CAS  PubMed  Google Scholar 

  • Kämpf C, Pfennig N (1980) Capacity of Chromatiaceae for chemotrophic growth. Specific respiration rates of Thiocystis violacea and Chromatium vinosum. Arch Microbiol 127:125–135

    Article  Google Scholar 

  • Kawasaki S, Ishikura J, Chiba D, Nishino T, Niimura Y (2004) Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria. Arch Microbiol 181:324–330

    Article  CAS  PubMed  Google Scholar 

  • Kaysser TM, Ghaim JB, Georgiou C, Gennis RB (1995) Methionine-393 is an axial ligand of the heme b 558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli. Biochemistry 34:13491–13501

    Article  CAS  PubMed  Google Scholar 

  • Kim H, Li H, Maresca JA, Bryant DA, Savikhin S (2007) Triplet exciton formation as a novel photoprotection mechanism in chlorosomes of Chlorobium tepidum. Biophys J 93:192–201

    Article  CAS  PubMed  Google Scholar 

  • Kurtz DM Jr (2006) Avoiding high valent iron intermediates: superoxide reductase and rubrerythrin. J Inorg Biochem 100:679–693

    Article  CAS  PubMed  Google Scholar 

  • Limauro D, Pedone E, Pirone L, Bartolucci S (2006) Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress. FEBS J 273:721–731

    Article  CAS  PubMed  Google Scholar 

  • Marklund S (1976) Spectrophotometric study of spontaneous disproportionation of superoxide anion radical and sensitive direct assay for superoxide dismutase. J Biol Chem 251:7504–7507

    CAS  PubMed  Google Scholar 

  • McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055

    CAS  PubMed  Google Scholar 

  • Mizohata E, Sakai H, Fusatomi E, Terada T, Murayama K, Shirouzu M, Yokoyama S (2005) Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1. J Mol Biol 354:317–329

    Article  CAS  PubMed  Google Scholar 

  • Moshiri F, Chawla A, Maier RJ (1991a) Cloning, characterization, and expression in Escherichia coli of the genes encoding the cytochrome d oxidase complex from Azotobacter vinelandii. J Bacteriol 173:6230–6241

    CAS  PubMed  Google Scholar 

  • Moshiri F, Smith EG, Taormino JP, Maier RJ (1991b) Transcriptional regulation of cytochrome d in nitrogen-fixing Azotobacter vinelandii. Evidence that up-regulation during N2 fixation is independent of nifA but dependent on ntrA. J Biol Chem 266:23169–23174

    CAS  PubMed  Google Scholar 

  • Moskovitz J, Rahman MA, Strassman J, Yancey SO, Kushner SR, Brot N, Weissbach H (1995) Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J Bacteriol 177:502–507

    CAS  PubMed  Google Scholar 

  • Niimura Y, Nishiyama Y, Saito D, Tsuji H, Hidaka M, Miyaji T, Watanabe T, Massey V (2000) A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus. J Bacteriol 182:5046–5051

    Article  CAS  PubMed  Google Scholar 

  • Oberley LW, Spitz DR (1984) Assay of superoxide dismutase activity in tumor tissue. Meth Enzymol 105:457–464

    Article  CAS  PubMed  Google Scholar 

  • Wang G, Olczak AA, Walton JP, Maier RJ (2005) Contribution of the Helicobacter pylori thiol peroxidase bacterioferritin comigratory protein to oxidative stress resistance and host colonization. Infect Immun 73:378–384

    Article  CAS  PubMed  Google Scholar 

  • Overmann J (2001) Green sulfur bacteria. In: Boone DR, Castenholz RW (eds) Bergey’s manual of systematic bacteriology, vol 1, 2nd edn. Springer, New York, pp 601–604

    Google Scholar 

  • Overmann J (2006) The family Chlorobiaceae. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes. Springer, New York. doi:10.1007/0-387-30747-8_13

    Google Scholar 

  • Papinutto E, Windle HJ, Cendron L, Battistutta R, Kelleher D, Zanotti G (2005) Crystal structure of alkyl hydroperoxide-reductase (AhpC) from Helicobacter pylori. Biochim Biophys Acta 1753:240–246

    CAS  PubMed  Google Scholar 

  • Poole LB (2005) Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases. Arch Biochem Biophys 433:240–254

    Article  CAS  PubMed  Google Scholar 

  • Presig O, Zufferey R, Thony-Meyer L, Hennecke H (1996) A high-affinity cbb 3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. J Bacteriol 178:1532–1538

    Google Scholar 

  • Ross RP, Claiborne A (1992) Molecular cloning and analysis of the gene encoding the NADH oxidase from Streptococcus faecalis 10C1. Comparison with NADH peroxidase and the flavoprotein disulfide reductases. J Mol Biol 227:658–671

    Article  CAS  PubMed  Google Scholar 

  • Storz G, Imlay JA (1999) Oxidative stress. Curr Opin Microbiol 2:188–194

    Article  CAS  PubMed  Google Scholar 

  • Sturr MG, Krulwich TA, Hicks DB (1996) Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a Δcyo Δcyd strain complemented by genes from Bacillus firmus OF4. J Bacteriol 176:1742–1749

    Google Scholar 

  • Vassiliev IR, Ronan MT, Hauska G, Golbeck JH (2000) The bound electron acceptors in green sulfur bacteria: resolution of the g-tensor of the FX iron-sulfur cluster in Chlorobium tepidum. Biophys J 78:3160–3169

    Article  CAS  PubMed  Google Scholar 

  • Wahlund TM, Madigan MT (1995) Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum. J Bacteriol 177:2583–2588

    CAS  PubMed  Google Scholar 

  • Wahlund TM, Woese CR, Castenholz RW, Madigan MT (1991) A thermophilic green sulfur bacterium from New Zealand hot springs, Chlorobium tepidum sp. Nov. Arch Microbiol 156:81–90

    Article  CAS  Google Scholar 

  • Wang J, Brune DC, Blankenship RE (1990) Effects of oxidants and reductants on the efficiency of excitation transfer in green photosynthetic bacteria. Biochim Biophys Acta 1015:457–463

    Article  CAS  PubMed  Google Scholar 

  • Ward DE, Donnelly CJ, Mullendore ME, van der Oost J, de Vos WM, Crane EJ III (2001) The NADH oxidase from Pyrococcus furiosus. Implications for the protection of anaerobic hyperthermophiles against oxidative stress. Eur J Biochem 268:5816–5823

    Article  CAS  PubMed  Google Scholar 

  • Winterbourn CC, Metodiewa D (1999) Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radic Biol Med 27:322–328

    Article  CAS  PubMed  Google Scholar 

  • Wood ZA, Schroder E, Harris JR, Poole LB (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28:32–40

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto H, Miyake C, Dietz KJ, Tomizawa K, Murata N, Yokota A (1999) Thioredoxin peroxidase in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett 447:269–273

    Article  CAS  PubMed  Google Scholar 

  • Yoon KS, Hille R, Hemann C, Tabita FR (1999) Rubredoxin from the green sulfur bacterium Chlorobium tepidum functions as an electron acceptor for pyruvate ferredoxin oxidoreductase. J Biol Chem 274:29772–29778

    Article  CAS  PubMed  Google Scholar 

  • Yoon KS, Bobst C, Hemann CF, Hille R, Tabita FR (2001) Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum. J Biol Chem 276:44027–44036

    Article  CAS  PubMed  Google Scholar 

  • Yu BP (1994) Cellular defense against damage from reactive oxygen species. Physiol Rev 74:139–162

    CAS  PubMed  Google Scholar 

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Acknowledgments

This research was supported by grant DE-FG-2-94ER20137 to D.A.B. from the US Department of Energy. Sequence data for the Cba. tepidum genome and for the other green sulfur bacterial genomes were obtained from the Joint Genome Institute Web site at http://www.jgi.doe.gov/. Sequencing of these genomes was accomplished with support from the US Department of Energy and grant MCB-0523100 from the National Science Foundation to D.A.B.

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  1. Hui Li

    Present address: Department of Laboratory Medicine, University of Washington, Box 359690, Seattle, WA, 98105, USA

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  1. Department of Biochemistry and Molecular Biology and Center for Biomolecular Structure and Function, The Pennsylvania State University , S-235 Frear Building, University Park, PA, 16802, USA

    Hui Li, Sara Jubelirer, Amaya M. Garcia Costas & Donald A. Bryant

  2. Department of Biology, University of Copenhagen, 2200, Copenhagen N, Denmark

    Niels-Ulrik Frigaard

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Correspondence to Donald A. Bryant.

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Communicated by Jörg Overmann.

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Li, H., Jubelirer, S., Garcia Costas, A.M. et al. Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species. Arch Microbiol 191, 853–867 (2009). https://doi.org/10.1007/s00203-009-0514-7

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