Deletion of a gene cluster for [Ni-Fe] hydrogenase maturation in the anaerobic hyperthermophilic bacterium Caldicellulosiruptor bescii identifies its role in hydrogen metabolism
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The anaerobic, hyperthermophlic, cellulolytic bacterium Caldicellulosiruptor bescii grows optimally at ∼80 °C and effectively degrades plant biomass without conventional pretreatment. It utilizes a variety of carbohydrate carbon sources, including both C5 and C6 sugars, released from plant biomass and produces lactate, acetate, CO2, and H2 as primary fermentation products. The C. bescii genome encodes two hydrogenases, a bifurcating [Fe-Fe] hydrogenase and a [Ni-Fe] hydrogenase. The [Ni-Fe] hydrogenase is the most widely distributed in nature and is predicted to catalyze hydrogen production and to pump protons across the cellular membrane creating proton motive force. Hydrogenases are the key enzymes in hydrogen metabolism and their crystal structure reveals complexity in the organization of their prosthetic groups suggesting extensive maturation of the primary protein. Here, we report the deletion of a cluster of genes, hypABFCDE, required for maturation of the [Ni-Fe] hydrogenase. These proteins are specific for the hydrogenases they modify and are required for hydrogenase activity. The deletion strain grew more slowly than the wild type or the parent strain and produced slightly less hydrogen overall, but more hydrogen per mole of cellobiose. Acetate yield per mole of cellobiose was increased ∼67 % and ethanol yield per mole of cellobiose was decreased ∼39 %. These data suggest that the primary role of the [Ni-Fe] hydrogenase is to generate a proton gradient in the membrane driving ATP synthesis and is not the primary enzyme for hydrogen catalysis. In its absence, ATP is generated from increased acetate production resulting in more hydrogen produced per mole of cellobiose.
KeywordsAnaerobe Hyperthermophile Caldicellulosiruptor bescii Hydrogen Bifurcating [Fe-Fe] hydrogenase [Ni-Fe] hydrogenase Hydrogenase maturation proteins
We thank Jennifer Copeland and Elise Snyder for the outstanding technical assistance, Brian Davison for providing the switchgrass used in this study, Sidney Kushner for the expert technical advice, William Whitman for the advice and use of his GC, Joe Groom and Jenna Young for the critical review of the manuscript. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
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Conflict of interest
The authors declare that they have no competing interests.
- Carere CR, Rydzak T, Verbeke TJ, Cicek N, Levin DB, Sparling R (2012) Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria. BMC Microbiol 12:295. doi: 10.1186/1471-2180-12-295 PubMedCentralCrossRefPubMedGoogle Scholar
- Chou CJ, Jenney Jr FE, Adams MW, Kelly RM (2008) Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels. Metab Eng 10(6):394–404. doi: 10.1016/j.ymben.2008.06.007
- Chung D, Cha M, Farkas J, Westpheling J (2013a) Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus. PLoS One 8(5):e62881. doi: 10.1371/journal.pone.0062881 PubMedCentralCrossRefPubMedGoogle Scholar
- Chung D, Farkas J, Huddleston JR, Olivar E, Westpheling J (2012) Methylation by a unique alpha-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725. PLoS One 7(8):e43844. doi: 10.1371/journal.pone.0043844 PubMedCentralCrossRefPubMedGoogle Scholar
- Das D, Dutta T, Nath K, Kotya SM, Das AK, Veziroglu TN (2006) Role of hydrogenase in biological hydrogen production. Curr Sci 90(12):1627–1637Google Scholar
- Farkas J, Chung D, Cha M, Copeland J, Grayeski P, Westpheling J (2013) Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol 40(1):41–49. doi: 10.1007/s10295-012-1202-1 PubMedCentralCrossRefPubMedGoogle Scholar
- Green MR, Sambrook J, Sambrook J (2012) Molecular cloning : a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Kadar Z, de Vrije T, van Noorden GE, Budde MA, Szengyel Z, Reczey K, Claassen PA (2004) Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus. Appl Biochem Biotechnol 113-116:497–508CrossRefPubMedGoogle Scholar
- van de Werken HJ, Verhaart MR, VanFossen AL, Willquist K, Lewis DL, Nichols JD, Goorissen HP, Mongodin EF, Nelson KE, Van Niel EW, Stams AJ, Ward DE, de Vos WM, van der Oost J, Kelly RM, Kengen SW (2008) Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. Appl Environ Microbiol 74(21):6720–6729. doi: 10.1128/AEM.00968-08 PubMedCentralCrossRefPubMedGoogle Scholar
- White D (2007) The physiology and biochemistry of prokaryotes, 3rd edn. Oxford University Press, New YorkGoogle Scholar
- Yang SJ, Kataeva I, Hamilton-Brehm SD, Engle NL, Tschaplinske TJ, Doeppke C, Davis M, Wespheling J, Adams MW (2009) Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe “Anaerocellum thermophilum” DSM 6725. Appl Environ Microbiol 75(14):4762–4769. doi: 10.1128/AEM.00236-09 PubMedCentralCrossRefPubMedGoogle Scholar