JOURNAL OF BACTERIOLOGY, Dec. 2010, p. 6497–6498 0021-9193/10/$12.00 doi:10.1128/JB.01144-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 24
Genome Sequence of the Obligate Methanotroph Methylosinus trichosporium Strain OB3b䌤 Lisa Y. Stein,1* Sukhwan Yoon,2¶ Jeremy D. Semrau,2 Alan A. DiSpirito,3 Andrew Crombie,4 J. Colin Murrell,4 Ste´phane Vuilleumier,5 Marina G. Kalyuzhnaya,6 Huub J. M. Op den Camp,7 Franc¸oise Bringel,5 D. Bruce,8 J.-F. Cheng,9 A. Copeland,9 Lynne Goodwin,8 Shunsheng Han,8 Loren Hauser,10 Mike S. M. Jetten,7 Aure´lie Lajus,11 M. L. Land,10 A. Lapidus,9 S. Lucas,9 Claudine Me´digue,11 S. Pitluck,9 Tanja Woyke,9 Ahmet Zeytun,8 and Martin G. Klotz12 Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada1; Department of Civil and Environmental Engineering, The University of Michigan, 1351 Beal Avenue, Ann Arbor, Michigan 48109-21252; Department of Biochemistry, Biophysics and Molecular Biology, University of Iowa, Ames, Iowa 500113; Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom4; Universite´ de Strasbourg, UMR 7156 CNRS, 67000 Strasbourg, France5; Department of Microbiology, University of Washington, Seattle, Washington 981956; Department of Microbiology, IWWR, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, Netherlands7; Los Alamos National Laboratory, Joint Genome Institute, Biosciences Division Genome Science B6, Los Alamos, New Mexico 875458; U.S. DOE Joint Genome Institute, 2800 Mitchell Drive B310, Walnut Creek, California 94598-16989; Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, Tennessee 3783110; Laboratoire d’Analyses Bioinformatiques pour la Ge´nomique et le Me´tabolisme (LABGeM), Genoscope-IG-CEA, 91057 Evry, France11; and Departments of Biology and Microbiology and Immunology, University of Louisville, Louisville, Kentucky 4029212 Received 24 September 2010/Accepted 5 October 2010
Methylosinus trichosporium OB3b (for “oddball” strain 3b) is an obligate aerobic methane-oxidizing alphaproteobacterium that was originally isolated in 1970 by Roger Whittenbury and colleagues. This strain has since been used extensively to elucidate the structure and function of several key enzymes of methane oxidation, including both particulate and soluble methane monooxygenase (sMMO) and the extracellular copper chelator methanobactin. In particular, the catalytic properties of soluble methane monooxygenase from M. trichosporium OB3b have been well characterized in context with biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of the M. trichosporium OB3b genome is the first reported from a member of the Methylocystaceae family in the order Rhizobiales. karyotic Dynamic Programming Genefinding Algorithm (PRODIGAL) (5). The M. trichosporium OB3b draft genome is 4.9 Mbp in size, contains 66% G⫹C, and encodes a single rRNA operon, a full complement of tRNA genes, and 4,503 predicted protein-encoding gene models. Sequence annotation and comparative genome analysis are under way with assistance from the Microscope annotation platform for annotation and comparative analysis of bacterial genomes at Genoscope (16). Previously studied or predicted genes encoding enzymes and proteins involved in methane oxidation (methane oxidation inventory) were identified; these included both soluble and particulate methane monooxygenases, methanol dehydrogenase, proteins and enzymes involved in pyrroloquinoline quinone synthesis and tetrahydrofolate- and tetrahydromethanopterin-linked pathways, NAD-linked formate dehydrogenases, and hydrogenase. Genes encoding a putative nonribosomal polypeptide synthetase complex implicated in synthesis of peptides involved in metal uptake by methanotrophs were also identified (17). As observed for other alphaproteobacterial methanotrophs (2, 12), but in contrast to Gammaproteobacteria (19), the M. trichosporium OB3b genome encodes the enzymes
Aerobic methanotrophic bacteria appear to be ubiquitous in the terrestrial and aquatic environment (9) and are a major biological sink for methane, the second most important greenhouse gas (10, 14, 15). Methanotrophic bacteria also have considerable potential for use in biotechnology (i.e., protein production) and in bioremediation due to the extensive substrate range of methane monooxygenase enzymes and amenability of these bacteria to large-scale cultivation (6, 8, 11, 14). The genome of the obligate methanotrophic bacterium Methylosinus trichosporium OB3b (18) was sequenced, assembled, and annotated by the U.S. Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/sequencing/). A quality draft of 44 contigs and 9 scaffolds was assembled from Roche 454-FLX, 454-std, 454-Titanium PE, and Illumina Solexa reads using Velvet (21). Automatic annotation was done using the Pro* Corresponding author. Mailing address: Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Centre, Edmonton, Alberta T6G 2E9, Canada. Phone: (780) 492-4782. Fax: (780) 492-9234. E-mail:
[email protected]. ¶ Present address: Max Planck Institute for Terrestrial Microbiology, Marburg, Germany. 䌤 Published ahead of print on 15 October 2010. 6497
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of a complete Embden-Meyerhoff glycolysis pathway and a closed tricarboxylic acid cycle, including ␣-ketoglutarate dehydrogenase. Whereas ribulose phosphokinase and other enzymes required for CO2 fixation via the Calvin-BensonBassham cycle were found in the genome, genes encoding ribulose-bisphosphate carboxylase/oxygenase have not yet been identified (1). As expected from several reports for methanotrophs and related members of the proteobacterial class Alphaproteobacteria (15), a full complement of genes encoding needed inventory for N2 fixation, ammonia transport, and assimilation were identified. In addition, a gene cluster encoding the nsrR response regulator and hybrid cluster (prismane) protein (13) was identified, suggesting operation of a pathway for hydroxylamine detoxification by reduction to ammonium. The M. trichosporium OB3b genome also encodes a cytochrome c⬘-alpha (pfam01322) in the cytochrom_C2 superfamily (c101610) (4) that has been implicated in reduction of NO to nitrous oxide in diverse alphaproteobacteria and betaproteobacteria (3), supporting observed nitrous oxide production by this strain (7, 20). Genes that have not yet been conclusively identified in the inventory include those encoding proteins with the capacity to oxidize hydroxylamine to nitrite and reduce nitrite to NO. Also missing are the high-molecularmass multiheme cytochromes observed in Methylococcus capsulatus Bath (17). These features may well be identified upon closure of the genome. Nucleotide sequence accession number. The M. trichosporium OB3b genome sequence is available in GenBank under accession number ADVE00000000.
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