The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems

Bringel, Françoise

Dispirito, Alan A.

Dunfield, Peter F.

Flynn, James D.

Hirayama, Hisako

Jetten, Mike S. M.

Kalyuzhnaya, Marina G.

Khmelenina, Valentina N.

Klotz, Martin G.

Knief, Claudia

Kyrpides, Nikos

Murrell, J. Colin

Op Den Camp, Huub J. M.

Sakai, Yasuyoshi

Semrau, Jeremy D.

Shapiro, Nicole

Stein, Lisa Y.

Svenning, Mette M.

Trotsenko, Yuri A.

Vuilleumier, Stéphane

Woyke, Tanja

English

PubMed

Flynn, J.D.

Hirayama, H.

Sakai, Y.

Dunfield, P.F.

Klotz, M.G.

Knief, C.

Camp, H.J.M. op den

Jetten, M.S.M.

Khmelenina, V.N.

Trotsenko, Y.A.

NARCIS 

Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine Ecosystems

University of East Anglia digital repository

Draft Genome Sequences of Gammaproteobacterial MethanotrophsIsolated from Marine EcosystemsJames D. Flynn,a Hisako Hirayama,b Yasuyoshi Sakai,c Peter F. Dunﬁeld,d Martin G. Klotz,e Claudia Knief,fHuub J. M. Op den Camp,g Mike S. M. Jetten,g Valentina N. Khmelenina,h Yuri A. Trotsenko,h J. Colin Murrell,i Jeremy D. Semrau,jMette M. Svenning,k Lisa Y. Stein,l Nikos Kyrpides,m Nicole Shapiro,m Tanja Woyke,m Françoise Bringel,n Stéphane Vuilleumier,nAlan A. DiSpirito,o Marina G. KalyuzhnayaaSan Diego State University, Department of Biology, San Diego, California, USAa; Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, Japanb; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Japanc; Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canadad; Division of Math & Natural Sciences, Queens College in theCity University of New York, Flushing, New York, USAe; Institute of Crop Science and Resource Conservation—Molecular Biology of the Rhizosphere, University of Bonn,Bonn, Germanyf; Department of Microbiology, IWWR, Faculty of Science, Radboud University, Nijmegen, The Netherlandsg; GK Skryabin Institute of Biochemistry andPhysiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russian Federationh; School of Environmental Sciences, University of East Anglia, NorwichResearch Park, Norwich, United Kingdomi; Department of Civil and Environmental Engineering, The University of Michigan, Ann Arbor, Michigan, USAj; Department ofArctic and Marine Biology, UiT, The Arctic University of Norway, Tromsø, Norwayk; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada;DOE Joint Genome Institute, Walnut Creek, California, USAm; Department of Microbiology, Genomics and the Environment, Université de Strasbourg, UMR 7156 CNRS,Strasbourg, Francen; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Ames, Iowa, USAoThis is contribution 12 from OMeGA.The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.Received 25 November 2015 Accepted 28 November 2015 Published 21 January 2016Citation Flynn JD, Hirayama H, Sakai Y, Dunfield PF, Klotz MG, Knief C, Op den Camp HJM, Jetten MSM, Khmelenina VN, Trotsenko YA, Murrell JC, Semrau JD, Svenning MM,Stein LY, Kyrpides N, Shapiro N, Woyke T, Bringel F, Vuilleumier S, DiSpirito AA, Kalyuzhnaya MG. 2016. Draft genome sequences of gammaproteobacterial methanotrophsisolated from marine ecosystems. Genome Announc 4(1):e01629-15. doi:10.1128/genomeA.01629-15.Copyright © 2016 Flynn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.Address correspondence to Marina G. Kalyuzhnaya, mkalyuzhnaya@mail.sdsu.edu.Microbial methane oxidation is one of the key drivers of oxy-gen consumption in marine sediments and the overlayingwater column (1). Methanotrophic bacteria are the primary pro-ducers of many cold and hot seep ecosystems (2, 3). Here, wereport three genome sequences of gammaproteobacterial metha-notrophs isolated from three marine ecosystems. MethylobactermarinusA45 (amethanol-adapted strain, formerlyMethylomonasmethanica A4, ACM 4717) was isolated from sewage outfall sedi-ment near Los Angeles, CA (4). Methylobacter sp. strain BBA5.1was isolated from the surface layer of estuary sediment collected atlow tide near Newport, Bay Estuary (CA) (5). Methylomarinumvadi IT-4 ( JCM 13665T  DSM 18976T) was isolated from aTABLE 1 General genome statistics and accession numbersSpeciesSequencingplatform(s)Genome assemblyand annotationGenomecoverage ()Genomesize (Mb)No. of scaffolds(no. of contigs)Core (accessory) metabolicpathwaysaNCBI accessionno.M. marinus A45 Illumina Velvet 1.1.05,AllPaths, Phrap4.24, Prodigal 2.51,237 4.99 9 (49) pMMO, pXmo, Mxa, XoxF1,XoxF2, H4F, H4MPT,FDH, RuMP, EMP, EDD,dPPP, PPP, pSC, TCAARVS00000000Methylobactersp. BBA5.1Illumina,PacBio RSAllPaths, Prodigal 2.5 290 5.07 87 (91) pMMO, pXmo, Mxa, XoxF1,XoxF2, H4F, H4MPT,FDH, RuMP, EMP, EDD,dPPP, PPP, pSC, TCAJQKS00000000M. vadi IT-4 Illumina,PacBio RSProdigal 2.5 272 4.33 1 (1) pMMO, Mxa, XoxF, H4F,H4MPT, FDH, RuMP,EMP, EDD, dPPP, PPP,pSC, TCAJPON00000000a dPPP, dissimilatory pentose-phosphate pathway; EDD, Entner-Doudoroff pathway; EMP, Embden-Meyerhof-Parnas pathway; FDH, formate dehydrogenases; H4F, folate-linkedC1 transfer; H4MPT, methanopterin-linked C1 transfer; Mxa, PQQ-linked methanol dehydrogenases; pMMO, membrane-bound methane monooxygenase; pSC, partial serinecycle; pXmo, methane/ammonia monooxygenase-related proteins of unknown function; PPP, pentose-phosphate pathway; RuMP, assimilatory ribulose monophosphate pathway;Xox, PQQ-linked methanol and formaldehyde dehydrogenases (i.e., no evidence for the glyoxylate regeneration pathway was found); TCA, tricarboxylic acid cycle.crossmarkGenome AnnouncementsJanuary/February 2016 Volume 4 Issue 1 e01629-15 genomea.asm.org 1 on March 15, 2016 by University of East Angliahttp://genomea.asm.org/Downloaded from microbial mat of a shallow submarine hydrothermal system nearTaketomi Island, Okinawa, Japan (6).DNA samples from the three strains were prepared using thestandard phenol-chloroform method (7). DNA sequence datawere obtained at the Joint Genome Institute using a combinationof PacBio (8) and Illumina (9) technologies, and draft genomesequences were assembled. The computational tools used for ge-nome sequencing and assembly are listed in Table 1.All three sequencedmarinemethanotrophs are obligatemethaneand methanol utilizers. All three genomes harbor genes typical fortype I methanotrophs, including genes encoding particulate meth-ane monooxygenase (pmoCAB), the PQQ-dependent methanoldehydrogenases (mxaFI and multiple copies of xoxF), genes for tet-rahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, genes of the ribulose monophos-phate pathway, including its phosphoketolase variant (10), and genesencoding a complete tricarboxylic acid (TCA) cycle and a partial ser-ine cycle (10) (Table 1). The pxmABC gene clusters (11) linked to adistant homologue of the nitrate-nitrite transporter (narK) werefound in theMethylobacter sp. strain BB5.1 andM. marinus A45 ge-nomes. A phosphoenolpyruvate carboxylase gene (ppc) was found inM.vadi IT-4only.Genes encoding solublemethanemonooxygenase,known glyoxylate regeneration pathways, and RubisCO (cbbL andcbbS) were not detected. Genes involved in ammonium and nitrateassimilation are present in all three genomes. The genomes of strainsA45 and BBA5.1 contain all genes necessary to provide for urea hy-drolysis and nitrogen fixation. M. vadi IT-4 has the potential fordissimilatory nitrite reduction to nitric oxide, as suggested by thepresence of nir genes. The NADH:ubiquinone reductase (H)-translocating genes (nuoABCDEFGHIJKLMN) were identified inM. marinus A45 only. All strains possess genes encoding Na-transporting NADH:ubiquinone oxidoreductase (nqrABCDEF),ubiquinol-cytochromebc1 complex, cytochromeb, cytochrome cox-idase, cytochrome P450 and P460, and cytochrome d ubiquinol oxi-dase.Cytochrome bo3 quinol oxidasewas found inM.vadi IT-4only.Both Methylobacter species possess genes encoding the Na-translocating ferredoxin:NAD oxidoreductase complex (rnfAB-CDGE). All genomes contain genes encoding pyruvate-ferredoxin/flavodoxin oxidoreductases, and all three strains possess ectoinebiosynthesis genes.The genome ofM. marinus A45 includes a chromosomally inte-grated complete copy of a bacteriophage genome (predicted size,65 kb) integrated in the chromosome, indicating the possibility oflysogenic infection inmethanotrophic bacteria. These genomes pro-vide a valuable resource to obtain new insights into environmentalcontrols of fitness and diversity in methanotrophs, mechanisms ofgenetic exchange within methanotrophic communities, and the po-tential for the development of new genetic tools for methanotrophs.Nucleotide sequence accession numbers. The genome se-quences have been deposited in GenBank under the accessionnumbers listed in Table 1.ACKNOWLEDGMENTSWe thank all members of the Organization for Methanotroph GenomeAnalysis for collaboration (OMeGA) and Genoscope (France) for accessto itsMicroScope platform for comparative genome analysis (http://www.genoscope.cns.fr/agc/microscope/home/).This report is based upon work supported by the National ScienceFoundation under award MCB-0842686 and by faculty start-up fundsfrom San Diego State University to M. G. Kalyuzhnaya. Work conductedby the U.S. Department of Energy Joint Genome Institute was supportedby the Office of Science of the U.S. Department of Energy under contractno. DE-AC02-05CH11231.FUNDING INFORMATIONThe National Science Foundation (NSF) provided funding to Marina G.Kalyuzhnaya under grant number MCB-0842686. The U.S. Departmentof Energy (DOE) provided funding to TanyaWoyke under grant numberDE-AC02-05CH11231. San Diego State University (SDSU) providedfunding to Marina G. Kalyuzhnaya.REFERENCES1. Boetius A, Wenzhöfer F. 2013. Seafloor oxygen consumption fuelled bymethane from cold seeps. Nat Geosci 6:725–734. http://dx.doi.org/10.1038/ngeo1926.2. Ruff SE, Arnds J, Knittel K, Amann R, Wegener G, Ramette A, BoetiusA. 2013. Microbial communities of deep-sea methane seeps at Hikurangicontinental margin (New Zealand). PLoS One 8:e72627. http://dx.doi.org/10.1371/journal.pone.0072627.3. Ding H, Valentine DL. 2008. Methanotrophic bacteria occupy benthicmicrobial mats in shallow marine hydrocarbon seeps, coal oil Point,Ca l i fornia . J Geophys Res 113: http://dx.doi.org/10.1029/2007JG000537.4. Lidstrom ME. 1988. Isolation and characterization of marine metha-notrophs. Antonie van Leeuwenhoek 54:189–199.5. Smith KS, Costello AM, Lidstrom ME. 1997. Methane and trichloroeth-ylene oxidation by an estuarine methanotroph, Methylobacter sp. strainBB5. 1. Appl Environ Microbiol 63:4617–4620.6. Hirayama H, Fuse H, Abe M, Miyazaki M, Nakamura T, Nunoura T,Furushima Y, Yamamoto H, Takai K. 2013. Methylomarinum vadi gen.nov., sp. nov., amethanotroph isolated from two distinctmarine environ-ments. Int J Syst EvolMicrobiol 63:1073–1082. http://dx.doi.org/10.1099/ijs.0.040568-0.7. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a labora-tory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Har-bor, NY.8. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D,Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B,Christians F, Cicero R, Clark S, Dalal R, deWinter A, Dixon J,Foquet M. 2009. Real-time DNA sequencing from single polymerasemolecules . Science 323:133–138. http://dx.doi.org/10.1126/science.1162986.9. Bennett S. 2004. Solexa Ltd. Pharmacogenomics 5:433–438. http://dx.doi.org/10.1517/14622416.5.4.433.10. Kalyuzhnaya MG. 2016. Methane biocatalysis: selecting the right mi-crobe. In Eckert C, Trinh CT (ed), Biotechnology for biofuel productionand optimization, 1st ed. Elsevier Science and Technology, Amsterdam,The Netherlands.11. Tavormina PL, Orphan VJ, Kalyuzhnaya MG, Jetten MSM, Klotz MG.2011. A novel family of functional operons encoding methane/ammoniamonooxygenase-related proteins in gammaproteobacterial metha-notrophs. Environ Microbiol Rep 3:91–100. http://dx.doi.org/10.1111/j.1758-2229.2010.00192.x.Flynn et al.Genome Announcements2 genomea.asm.org January/February 2016 Volume 4 Issue 1 e01629-15 on March 15, 2016 by University of East Angliahttp://genomea.asm.org/Downloaded from 

Draft genome sequences of gammaproteobacterial methanotrophs isolated from marine ecosystems

Flynn, James D

Dunfield, Peter F

Klotz, Martin G

Camp, Huub JM Op den

Jetten, Mike SM

Khmelenina, Valentina N

Trotsenko, Yuri A

Murrell, J Colin

Semrau, Jeremy D

Svenning, Mette M

Stein, Lisa Y

DiSpirito, Alan A

Kalyuzhnaya, Marina G

eScholarship - University of California

Lawrence Berkeley National LaboratoryLBL PublicationsTitleDraft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine EcosystemsPermalinkhttps://escholarship.org/uc/item/5v60116bJournalMicrobiology Resource Announcements, 4(1)ISSN2576-098XAuthorsFlynn, James DHirayama, HisakoSakai, Yasuyoshiet al.Publication Date2016-02-25DOI10.1128/genomea.01629-15 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaDraft Genome Sequences of Gammaproteobacterial MethanotrophsIsolated from Marine EcosystemsJames D. Flynn,a Hisako Hirayama,b Yasuyoshi Sakai,c Peter F. Dunfield,d Martin G. Klotz,e Claudia Knief,fHuub J. M. Op den Camp,g Mike S. M. Jetten,g Valentina N. Khmelenina,h Yuri A. Trotsenko,h J. Colin Murrell,i Jeremy D. Semrau,jMette M. Svenning,k Lisa Y. Stein,l Nikos Kyrpides,m Nicole Shapiro,m Tanja Woyke,m Françoise Bringel,n Stéphane Vuilleumier,nAlan A. DiSpirito,o Marina G. KalyuzhnayaaSan Diego State University, Department of Biology, San Diego, California, USAa; Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, Japanb; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Japanc; Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canadad; Division of Math & Natural Sciences, Queens College in theCity University of New York, Flushing, New York, USAe; Institute of Crop Science and Resource Conservation—Molecular Biology of the Rhizosphere, University of Bonn,Bonn, Germanyf; Department of Microbiology, IWWR, Faculty of Science, Radboud University, Nijmegen, The Netherlandsg; GK Skryabin Institute of Biochemistry andPhysiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russian Federationh; School of Environmental Sciences, University of East Anglia, NorwichResearch Park, Norwich, United Kingdomi; Department of Civil and Environmental Engineering, The University of Michigan, Ann Arbor, Michigan, USAj; Department ofArctic and Marine Biology, UiT, The Arctic University of Norway, Tromsø, Norwayk; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada;DOE Joint Genome Institute, Walnut Creek, California, USAm; Department of Microbiology, Genomics and the Environment, Université de Strasbourg, UMR 7156 CNRS,Strasbourg, Francen; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Ames, Iowa, USAoThis is contribution 12 from OMeGA.The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.Received 25 November 2015 Accepted 28 November 2015 Published 21 January 2016Citation Flynn JD, Hirayama H, Sakai Y, Dunfield PF, Klotz MG, Knief C, Op den Camp HJM, Jetten MSM, Khmelenina VN, Trotsenko YA, Murrell JC, Semrau JD, Svenning MM,Stein LY, Kyrpides N, Shapiro N, Woyke T, Bringel F, Vuilleumier S, DiSpirito AA, Kalyuzhnaya MG. 2016. Draft genome sequences of gammaproteobacterial methanotrophsisolated from marine ecosystems. Genome Announc 4(1):e01629-15. doi:10.1128/genomeA.01629-15.Copyright © 2016 Flynn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.Address correspondence to Marina G. Kalyuzhnaya, mkalyuzhnaya@mail.sdsu.edu.Microbial methane oxidation is one of the key drivers of oxy-gen consumption in marine sediments and the overlayingwater column (1). Methanotrophic bacteria are the primary pro-ducers of many cold and hot seep ecosystems (2, 3). Here, wereport three genome sequences of gammaproteobacterial metha-notrophs isolated from three marine ecosystems. Methylobactermarinus A45 (a methanol-adapted strain, formerly Methylomonasmethanica A4, ACM 4717) was isolated from sewage outfall sedi-ment near Los Angeles, CA (4). Methylobacter sp. strain BBA5.1was isolated from the surface layer of estuary sediment collected atlow tide near Newport, Bay Estuary (CA) (5). Methylomarinumvadi IT-4 ( JCM 13665T  DSM 18976T) was isolated from aTABLE 1 General genome statistics and accession numbersSpeciesSequencingplatform(s)Genome assemblyand annotationGenomecoverage ()Genomesize (Mb)No. of scaffolds(no. of contigs)Core (accessory) metabolicpathwaysaNCBI accessionno.M. marinus A45 Illumina Velvet 1.1.05,AllPaths, Phrap4.24, Prodigal 2.51,237 4.99 9 (49) pMMO, pXmo, Mxa, XoxF1,XoxF2, H4F, H4MPT,FDH, RuMP, EMP, EDD,dPPP, PPP, pSC, TCAARVS00000000Methylobactersp. BBA5.1Illumina,PacBio RSAllPaths, Prodigal 2.5 290 5.07 87 (91) pMMO, pXmo, Mxa, XoxF1,XoxF2, H4F, H4MPT,FDH, RuMP, EMP, EDD,dPPP, PPP, pSC, TCAJQKS00000000M. vadi IT-4 Illumina,PacBio RSProdigal 2.5 272 4.33 1 (1) pMMO, Mxa, XoxF, H4F,H4MPT, FDH, RuMP,EMP, EDD, dPPP, PPP,pSC, TCAJPON00000000a dPPP, dissimilatory pentose-phosphate pathway; EDD, Entner-Doudoroff pathway; EMP, Embden-Meyerhof-Parnas pathway; FDH, formate dehydrogenases; H4F, folate-linkedC1 transfer; H4MPT, methanopterin-linked C1 transfer; Mxa, PQQ-linked methanol dehydrogenases; pMMO, membrane-bound methane monooxygenase; pSC, partial serinecycle; pXmo, methane/ammonia monooxygenase-related proteins of unknown function; PPP, pentose-phosphate pathway; RuMP, assimilatory ribulose monophosphate pathway;Xox, PQQ-linked methanol and formaldehyde dehydrogenases (i.e., no evidence for the glyoxylate regeneration pathway was found); TCA, tricarboxylic acid cycle.crossmarkGenome AnnouncementsJanuary/February 2016 Volume 4 Issue 1 e01629-15 genomea.asm.org 1microbial mat of a shallow submarine hydrothermal system nearTaketomi Island, Okinawa, Japan (6).DNA samples from the three strains were prepared using thestandard phenol-chloroform method (7). DNA sequence datawere obtained at the Joint Genome Institute using a combinationof PacBio (8) and Illumina (9) technologies, and draft genomesequences were assembled. The computational tools used for ge-nome sequencing and assembly are listed in Table 1.All three sequenced marine methanotrophs are obligate methaneand methanol utilizers. All three genomes harbor genes typical fortype I methanotrophs, including genes encoding particulate meth-ane monooxygenase (pmoCAB), the PQQ-dependent methanoldehydrogenases (mxaFI and multiple copies of xoxF), genes for tet-rahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, genes of the ribulose monophos-phate pathway, including its phosphoketolase variant (10), and genesencoding a complete tricarboxylic acid (TCA) cycle and a partial ser-ine cycle (10) (Table 1). The pxmABC gene clusters (11) linked to adistant homologue of the nitrate-nitrite transporter (narK) werefound in the Methylobacter sp. strain BB5.1 and M. marinus A45 ge-nomes. A phosphoenolpyruvate carboxylase gene (ppc) was found inM. vadi IT-4 only. Genes encoding soluble methane monooxygenase,known glyoxylate regeneration pathways, and RubisCO (cbbL andcbbS) were not detected. Genes involved in ammonium and nitrateassimilation are present in all three genomes. The genomes of strainsA45 and BBA5.1 contain all genes necessary to provide for urea hy-drolysis and nitrogen fixation. M. vadi IT-4 has the potential fordissimilatory nitrite reduction to nitric oxide, as suggested by thepresence of nir genes. The NADH:ubiquinone reductase (H)-translocating genes (nuoABCDEFGHIJKLMN) were identified inM. marinus A45 only. All strains possess genes encoding Na-transporting NADH:ubiquinone oxidoreductase (nqrABCDEF),ubiquinol-cytochrome bc1 complex, cytochrome b, cytochrome c ox-idase, cytochrome P450 and P460, and cytochrome d ubiquinol oxi-dase. Cytochrome bo3 quinol oxidase was found in M. vadi IT-4 only.Both Methylobacter species possess genes encoding the Na-translocating ferredoxin:NAD oxidoreductase complex (rnfAB-CDGE). All genomes contain genes encoding pyruvate-ferredoxin/flavodoxin oxidoreductases, and all three strains possess ectoinebiosynthesis genes.The genome of M. marinus A45 includes a chromosomally inte-grated complete copy of a bacteriophage genome (predicted size,65 kb) integrated in the chromosome, indicating the possibility oflysogenic infection in methanotrophic bacteria. These genomes pro-vide a valuable resource to obtain new insights into environmentalcontrols of fitness and diversity in methanotrophs, mechanisms ofgenetic exchange within methanotrophic communities, and the po-tential for the development of new genetic tools for methanotrophs.Nucleotide sequence accession numbers. The genome se-quences have been deposited in GenBank under the accessionnumbers listed in Table 1.ACKNOWLEDGMENTSWe thank all members of the Organization for Methanotroph GenomeAnalysis for collaboration (OMeGA) and Genoscope (France) for accessto its MicroScope platform for comparative genome analysis (http://www.genoscope.cns.fr/agc/microscope/home/).This report is based upon work supported by the National ScienceFoundation under award MCB-0842686 and by faculty start-up fundsfrom San Diego State University to M. G. Kalyuzhnaya. Work conductedby the U.S. Department of Energy Joint Genome Institute was supportedby the Office of Science of the U.S. Department of Energy under contractno. DE-AC02-05CH11231.FUNDING INFORMATIONThe National Science Foundation (NSF) provided funding to Marina G.Kalyuzhnaya under grant number MCB-0842686. The U.S. Departmentof Energy (DOE) provided funding to Tanya Woyke under grant numberDE-AC02-05CH11231. San Diego State University (SDSU) providedfunding to Marina G. Kalyuzhnaya.REFERENCES1. Boetius A, Wenzhöfer F. 2013. Seafloor oxygen consumption fuelled bymethane from cold seeps. Nat Geosci 6:725–734. http://dx.doi.org/10.1038/ngeo1926.2. Ruff SE, Arnds J, Knittel K, Amann R, Wegener G, Ramette A, BoetiusA. 2013. Microbial communities of deep-sea methane seeps at Hikurangicontinental margin (New Zealand). PLoS One 8:e72627. http://dx.doi.org/10.1371/journal.pone.0072627.3. Ding H, Valentine DL. 2008. Methanotrophic bacteria occupy benthicmicrobial mats in shallow marine hydrocarbon seeps, coal oil Point,Ca l i fornia . J Geophys Res 113: http://dx.doi.org/10.1029/2007JG000537.4. Lidstrom ME. 1988. Isolation and characterization of marine metha-notrophs. Antonie van Leeuwenhoek 54:189 –199.5. Smith KS, Costello AM, Lidstrom ME. 1997. Methane and trichloroeth-ylene oxidation by an estuarine methanotroph, Methylobacter sp. strainBB5. 1. Appl Environ Microbiol 63:4617– 4620.6. Hirayama H, Fuse H, Abe M, Miyazaki M, Nakamura T, Nunoura T,Furushima Y, Yamamoto H, Takai K. 2013. Methylomarinum vadi gen.nov., sp. nov., a methanotroph isolated from two distinct marine environ-ments. Int J Syst Evol Microbiol 63:1073–1082. http://dx.doi.org/10.1099/ijs.0.040568-0.7. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a labora-tory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Har-bor, NY.8. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D,Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B,Christians F, Cicero R, Clark S, Dalal R, deWinter A, Dixon J,Foquet M. 2009. Real-time DNA sequencing from single polymerasemolecules . Science 323:133–138. http://dx.doi.org/10.1126/science.1162986.9. Bennett S. 2004. Solexa Ltd. Pharmacogenomics 5:433– 438. http://dx.doi.org/10.1517/14622416.5.4.433.10. Kalyuzhnaya MG. 2016. Methane biocatalysis: selecting the right mi-crobe. In Eckert C, Trinh CT (ed), Biotechnology for biofuel productionand optimization, 1st ed. Elsevier Science and Technology, Amsterdam,The Netherlands.11. Tavormina PL, Orphan VJ, Kalyuzhnaya MG, Jetten MSM, Klotz MG.2011. A novel family of functional operons encoding methane/ammoniamonooxygenase-related proteins in gammaproteobacterial metha-notrophs. Environ Microbiol Rep 3:91–100. http://dx.doi.org/10.1111/j.1758-2229.2010.00192.x.Flynn et al.Genome Announcements2 genomea.asm.org January/February 2016 Volume 4 Issue 1 e01629-15

Op den Camp, Huub J. M.

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Flynn, J. (James) D. (D)

Hirayama, H. (Hisako)

Sakai, Y. (Yasuyoshi)

Dunfield, P. (Peter) F. (F)

Klotz, M. (Martin) G. (G)

Knief, C. (Claudia)

Op den Camp, H. (Huub) J. (J) M. (M)

Jetten, M. (Mike) S. (S) M. (M)

Khmelenina, V. (Valentina) N. (N)

Trotsenko, Y. (Yuri) A. (A)

Murrell, J. (J) C. (Colin)

Semrau, J. (Jeremy) D. (D)

Svenning, M. (Mette) M. (M)

Stein, L. (Lisa) Y. (Y)

Kyrpides, N. (Nikos)

Shapiro, N. (Nicole)

Woyke, T. (Tanja)

Bringel, F. (Françoise)

Vuilleumier, S. (Stéphane)

DiSpirito, A. (Alan) A. (A)

Kalyuzhnaya, M. (Marina) G. (G)

univOAK

Draft Genome Sequences of Gammaproteobacterial Methanotrophs
Isolated from Marine Ecosystems
James D. Flynn,a Hisako Hirayama,b Yasuyoshi Sakai,c Peter F. Dunﬁeld,d Martin G. Klotz,e Claudia Knief,f
Huub J. M. Op den Camp,g Mike S. M. Jetten,g Valentina N. Khmelenina,h Yuri A. Trotsenko,h J. Colin Murrell,i Jeremy D. Semrau,j
Mette M. Svenning,k Lisa Y. Stein,l Nikos Kyrpides,m Nicole Shapiro,m Tanja Woyke,m Françoise Bringel,n Stéphane Vuilleumier,n
Alan A. DiSpirito,o Marina G. Kalyuzhnayaa
San Diego State University, Department of Biology, San Diego, California, USAa; Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-
Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa, Japanb; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-
Oiwake, Sakyo-ku, Japanc; Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canadad; Division of Math & Natural Sciences, Queens College in the
City University of New York, Flushing, New York, USAe; Institute of Crop Science and Resource Conservation—Molecular Biology of the Rhizosphere, University of Bonn,
Bonn, Germanyf; Department of Microbiology, IWWR, Faculty of Science, Radboud University, Nijmegen, The Netherlandsg; GK Skryabin Institute of Biochemistry and
Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russian Federationh; School of Environmental Sciences, University of East Anglia, Norwich
Research Park, Norwich, United Kingdomi; Department of Civil and Environmental Engineering, The University of Michigan, Ann Arbor, Michigan, USAj; Department of
Arctic and Marine Biology, UiT, The Arctic University of Norway, Tromsø, Norwayk; Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada;
DOE Joint Genome Institute, Walnut Creek, California, USAm; Department of Microbiology, Genomics and the Environment, Université de Strasbourg, UMR 7156 CNRS,
Strasbourg, Francen; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Ames, Iowa, USAo
This is contribution 12 from OMeGA.
The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4
were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.
Received 25 November 2015 Accepted 28 November 2015 Published 21 January 2016
Citation Flynn JD, Hirayama H, Sakai Y, Dunfield PF, Klotz MG, Knief C, Op den Camp HJM, Jetten MSM, Khmelenina VN, Trotsenko YA, Murrell JC, Semrau JD, Svenning MM,
Stein LY, Kyrpides N, Shapiro N, Woyke T, Bringel F, Vuilleumier S, DiSpirito AA, Kalyuzhnaya MG. 2016. Draft genome sequences of gammaproteobacterial methanotrophs
isolated from marine ecosystems. Genome Announc 4(1):e01629-15. doi:10.1128/genomeA.01629-15.
Copyright © 2016 Flynn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Address correspondence to Marina G. Kalyuzhnaya, mkalyuzhnaya@mail.sdsu.edu.
Microbial methane oxidation is one of the key drivers of oxy-gen consumption in marine sediments and the overlaying
water column (1). Methanotrophic bacteria are the primary pro-
ducers of many cold and hot seep ecosystems (2, 3). Here, we
report three genome sequences of gammaproteobacterial metha-
notrophs isolated from three marine ecosystems. Methylobacter
marinusA45 (amethanol-adapted strain, formerlyMethylomonas
methanica A4, ACM 4717) was isolated from sewage outfall sedi-
ment near Los Angeles, CA (4). Methylobacter sp. strain BBA5.1
was isolated from the surface layer of estuary sediment collected at
low tide near Newport, Bay Estuary (CA) (5). Methylomarinum
vadi IT-4 ( JCM 13665T  DSM 18976T) was isolated from a
TABLE 1 General genome statistics and accession numbers
Species
Sequencing
platform(s)
Genome assembly
and annotation
Genome
coverage ()
Genome
size (Mb)
No. of scaffolds
(no. of contigs)
Core (accessory) metabolic
pathwaysa
NCBI accession
no.
M. marinus A45 Illumina Velvet 1.1.05,
AllPaths, Phrap
4.24, Prodigal 2.5
1,237 4.99 9 (49) pMMO, pXmo, Mxa, XoxF1,
XoxF2, H4F, H4MPT,
FDH, RuMP, EMP, EDD,
dPPP, PPP, pSC, TCA
ARVS00000000
Methylobacter
sp. BBA5.1
Illumina,
PacBio RS
AllPaths, Prodigal 2.5 290 5.07 87 (91) pMMO, pXmo, Mxa, XoxF1,
XoxF2, H4F, H4MPT,
FDH, RuMP, EMP, EDD,
dPPP, PPP, pSC, TCA
JQKS00000000
M. vadi IT-4 Illumina,
PacBio RS
Prodigal 2.5 272 4.33 1 (1) pMMO, Mxa, XoxF, H4F,
H4MPT, FDH, RuMP,
EMP, EDD, dPPP, PPP,
pSC, TCA
JPON00000000
a dPPP, dissimilatory pentose-phosphate pathway; EDD, Entner-Doudoroff pathway; EMP, Embden-Meyerhof-Parnas pathway; FDH, formate dehydrogenases; H4F, folate-linked
C1 transfer; H4MPT, methanopterin-linked C1 transfer; Mxa, PQQ-linked methanol dehydrogenases; pMMO, membrane-bound methane monooxygenase; pSC, partial serine
cycle; pXmo, methane/ammonia monooxygenase-related proteins of unknown function; PPP, pentose-phosphate pathway; RuMP, assimilatory ribulose monophosphate pathway;
Xox, PQQ-linked methanol and formaldehyde dehydrogenases (i.e., no evidence for the glyoxylate regeneration pathway was found); TCA, tricarboxylic acid cycle.
crossmark
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microbial mat of a shallow submarine hydrothermal system near
Taketomi Island, Okinawa, Japan (6).
DNA samples from the three strains were prepared using the
standard phenol-chloroform method (7). DNA sequence data
were obtained at the Joint Genome Institute using a combination
of PacBio (8) and Illumina (9) technologies, and draft genome
sequences were assembled. The computational tools used for ge-
nome sequencing and assembly are listed in Table 1.
All three sequencedmarinemethanotrophs are obligatemethane
and methanol utilizers. All three genomes harbor genes typical for
type I methanotrophs, including genes encoding particulate meth-
ane monooxygenase (pmoCAB), the PQQ-dependent methanol
dehydrogenases (mxaFI and multiple copies of xoxF), genes for tet-
rahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-
dependent C1-transfer pathways, genes of the ribulose monophos-
phate pathway, including its phosphoketolase variant (10), and genes
encoding a complete tricarboxylic acid (TCA) cycle and a partial ser-
ine cycle (10) (Table 1). The pxmABC gene clusters (11) linked to a
distant homologue of the nitrate-nitrite transporter (narK) were
found in theMethylobacter sp. strain BB5.1 andM. marinus A45 ge-
nomes. A phosphoenolpyruvate carboxylase gene (ppc) was found in
M.vadi IT-4only.Genes encoding solublemethanemonooxygenase,
known glyoxylate regeneration pathways, and RubisCO (cbbL and
cbbS) were not detected. Genes involved in ammonium and nitrate
assimilation are present in all three genomes. The genomes of strains
A45 and BBA5.1 contain all genes necessary to provide for urea hy-
drolysis and nitrogen fixation. M. vadi IT-4 has the potential for
dissimilatory nitrite reduction to nitric oxide, as suggested by the
presence of nir genes. The NADH:ubiquinone reductase (H)-
translocating genes (nuoABCDEFGHIJKLMN) were identified in
M. marinus A45 only. All strains possess genes encoding Na-
transporting NADH:ubiquinone oxidoreductase (nqrABCDEF),
ubiquinol-cytochromebc1 complex, cytochromeb, cytochrome cox-
idase, cytochrome P450 and P460, and cytochrome d ubiquinol oxi-
dase.Cytochrome bo3 quinol oxidasewas found inM.vadi IT-4only.
Both Methylobacter species possess genes encoding the Na-
translocating ferredoxin:NAD oxidoreductase complex (rnfAB-
CDGE). All genomes contain genes encoding pyruvate-ferredoxin/
flavodoxin oxidoreductases, and all three strains possess ectoine
biosynthesis genes.
The genome ofM. marinus A45 includes a chromosomally inte-
grated complete copy of a bacteriophage genome (predicted size,
65 kb) integrated in the chromosome, indicating the possibility of
lysogenic infection inmethanotrophic bacteria. These genomes pro-
vide a valuable resource to obtain new insights into environmental
controls of fitness and diversity in methanotrophs, mechanisms of
genetic exchange within methanotrophic communities, and the po-
tential for the development of new genetic tools for methanotrophs.
Nucleotide sequence accession numbers. The genome se-
quences have been deposited in GenBank under the accession
numbers listed in Table 1.
ACKNOWLEDGMENTS
We thank all members of the Organization for Methanotroph Genome
Analysis for collaboration (OMeGA) and Genoscope (France) for access
to itsMicroScope platform for comparative genome analysis (http://www
.genoscope.cns.fr/agc/microscope/home/).
This report is based upon work supported by the National Science
Foundation under award MCB-0842686 and by faculty start-up funds
from San Diego State University to M. G. Kalyuzhnaya. Work conducted
by the U.S. Department of Energy Joint Genome Institute was supported
by the Office of Science of the U.S. Department of Energy under contract
no. DE-AC02-05CH11231.
FUNDING INFORMATION
The National Science Foundation (NSF) provided funding to Marina G.
Kalyuzhnaya under grant number MCB-0842686. The U.S. Department
of Energy (DOE) provided funding to TanyaWoyke under grant number
DE-AC02-05CH11231. San Diego State University (SDSU) provided
funding to Marina G. Kalyuzhnaya.
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Flynn et al.
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Draft genome sequences of gammaproteobacterial methanotrophs isolated from marine ecosystems

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