Permafrost environments play a crucial role in global carbon and methane cycling. We report here the draft genome sequence of Methylocella silvestris TVC, a new facultative methanotroph strain, isolated from the Siksik Creek catchment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada)

Crombie, Andrew

Geng, Kan

Ma, Ke

Murrell, J. Colin

Pratscher, Jennifer

Street, Lorna

Ul Haquea, Muhammad Farhan

Wang, JIng

Wookey, Philip A.

Wang, Jing

Farhan Ul Haque, Muhammad

Street, Lorna E.

University of East Anglia digital repository

Draft Genome Sequence of Methylocella silvestris TVC, aFacultative Methanotroph Isolated from PermafrostJing Wang,a,b Kan Geng,a,b Muhammad Farhan Ul Haque,a Andrew Crombie,c Lorna E. Street,d Philip A. Wookey,e Ke Ma,bJ. Colin Murrell,a Jennifer PratscherfaSchool of Environmental Sciences, University of East Anglia, Norwich, United KingdombCollege of Resources and Environmental Sciences, China Agricultural University, Beijing, ChinacSchool of Biological Sciences, University of East Anglia, Norwich, United KingdomdSchool of GeoSciences, University of Edinburgh, Edinburgh, United KingdomeBiological and Environmental Sciences, University of Stirling, Stirling, United KingdomfLyell Centre, Heriot-Watt University, Edinburgh, United KingdomABSTRACT Permafrost environments play a crucial role in global carbon and meth-ane cycling. We report here the draft genome sequence of Methylocella silvestrisTVC, a new facultative methanotroph strain, isolated from the Siksik Creek catch-ment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada).Methanotrophic bacteria utilize methane as sole carbon and energy sources, thusplaying a major role in the global methane cycle (1). They are widespread in theenvironment, including lakes, rivers, sediments, rice paddies, sewage sludge, forests,and landﬁll soils (2, 3). All described methanotrophic species belonging to the genusMethylocella (family Beijerinckiaceae) possess a soluble methane monooxygenase(sMMO) that catalyzes the oxidation of methane to methanol, a key methane-oxidizingenzyme that is present in only a subset of methanotrophs (4–6). Methanotrophs of thegenus Methylocella do not possess a particulate methane monooxygenase (pMMO),which is present in most other methanotrophs (7). Methylocella species can also utilizemulticarbon compounds, including acetate, pyruvate, succinate, ethane, and propane(7, 8). Three species of Methylocella have so far been described, M. palustris, M. silvestris,and M. tundrae (4–6). Type strain M. silvestris BL2 was isolated from an acidic forestcambisol, and its genome sequence was reported previously (9). We now report theisolation and draft genome of a new strain, M. silvestris TVC.Samples for isolation were taken from a middle hill slope to stream channel transectof the Siksik Creek catchment (68°44=54.5 N, 133°29=41.7 W), a tributary of Trail ValleyCreek (TVC), Canada (10). After initial enrichment of the soil samples with CH4 (18%vol/vol in the headspace), subsamples were transferred to liquid medium and platedrepeatedly until the culture was pure.Genome sequencing of M. silvestris TVC was performed by MicrobesNG (Birming-ham, UK) using Illumina HiSeq technology (1,151,332 trimmed reads, 109-fold meancoverage) and assembled, using SPAdes version 3.11.1 (11), into 82 contigs with agenome size of 4,292,072 bp and a GC content of 62.94%. Annotation was performedusing Prokka version 1.12 (12).Comparative genome analysis revealed that although the 16S rRNA gene se-quences of strain TVC and strain BL2 shared 99% nucleotide identity, the similarityof mmoXYBZDC, encoding the sMMO, and mxaF, encoding methanol dehydroge-nase, was 90 to 95% between the two strains. Average nucleotide identity betweenstrains BL2 and TVC was calculated using JSpecies (13), showing 89.80% and 90.59%similarities based on the BLAST algorithm and MUMmer ultrarapid aligning tool,respectively.Received 11 January 2018 Accepted 30January 2018 Published 22 February 2018CitationWang J, Geng K, Farhan Ul Haque M,Crombie A, Street LE, Wookey PA, Ma K, MurrellJC, Pratscher J. 2018. Draft genome sequenceofMethylocella silvestris TVC, a facultativemethanotroph isolated from permafrost.Genome Announc 6:e00040-18. https://doi.org/10.1128/genomeA.00040-18.Copyright © 2018 Wang et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to J. Colin Murrell,j.c.murrell@uea.ac.uk.PROKARYOTEScrossmVolume 6 Issue 8 e00040-18 genomea.asm.org 1 on March 2, 2018 by University of East Angliahttp://genomea.asm.org/Downloaded from Strain TVC used C1 substrates, including methane and methanol, as sole carbonsources. In addition to genes encoding the sMMO and methanol dehydrogenase,genes encoding the tetrahydrofolate (H4F)-dependent and tetrahydromethanop-terin (H4MPT)-dependent pathways of formaldehyde oxidation and those of theserine cycle are present in the genome. Like M. silvestris BL2, strain TVC was afacultative methanotroph and was able to utilize multicarbon compounds, includ-ing acetate, ethanol, succinate, and propane. Genes encoding the glyoxylate bypassenzymes isocitrate lyase and malate synthase are present. Also identiﬁed were theprmA, prmB, prmC, and prmD genes encoding a propane monooxygenase, showing91%, 84%, 86%, and 86% identities to those of M. silvestris BL2, respectively,enabling growth on propane. Further analyses of the genome and comparison withother strains will lead to a better understanding of the phylogeny and evolution offacultative methanotrophs.Accession number(s). This whole-genome shotgun project has been deposited atDDBJ/ENA/GenBank under the accession number PDZR00000000. The version de-scribed in this paper is the ﬁrst version, PDZR01000000.ACKNOWLEDGMENTSThis work was supported by Natural Environment Research Council (NERC) Inde-pendent Research Fellowship NE/L010771/1, Leverhulme Trust Research Project grantRPG2016-050, Leverhulme Early Career Fellowship ECF-2016-626, and NERC grantNE/K000284/1.Gwen Lancashire provided important help with ﬁeld sampling. We are also gratefulfor the logistical assistance provided by Philip Marsh and his research group, as well asby the staff at the Aurora Research Institute, Inuvik.REFERENCES1. Hanson RS, Hanson TE. 1996. Methanotrophic bacteria. Microbiol Rev60:439–471.2. Trotsenko YA, Murrell JC. 2008. Metabolic aspects of aerobic obligatemethanotrophy. Adv Appl Microbiol 63:183–229. https://doi.org/10.1016/S0065-2164(07)00005-6.3. Semrau JD, DiSpirito AA, Yoon S. 2010. Methanotrophs and copper.FEMS Microbiol Rev 34:496–531. https://doi.org/10.1111/j.1574-6976.2010.00212.x.4. Dedysh SN, Liesack W, Khmelenina VN, Suzina NE, Trotsenko YA, SemrauJD, Bares AM, Panikov NS, Tiedje JM. 2000. Methylocella palustris gen.nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peatbogs, representing a novel subtype of serine-pathway methanotrophs.Int J Syst Evol Microbiol 50:955–969. https://doi.org/10.1099/00207713-50-3-955.5. Dunﬁeld PF, Khmelenina VN, Suzina NE, Trotsenko YA, Dedysh SN. 2003.Methylocella silvestris sp. nov., a novel methanotroph isolated from anacidic forest cambisol. Int J Syst Evol Microbiol 53:1231–1239. https://doi.org/10.1099/ijs.0.02481-0.6. Dedysh SN, Berestovskaya YY, Vasylieva LV, Belova SE, Khmelenina VN,Suzina NE, Trotsenko YA, Liesack W, Zavarzin GA. 2004. Methylocellatundrae sp. nov., a novel methanotrophic bacterium from acidic tundrapeatlands. Int J Syst Evol Microbiol 54:151–156. https://doi.org/10.1099/ijs.0.02805-0.7. Semrau JD, DiSpirito AA, Vuilleumier S. 2011. Facultative methanotrophy:false leads, true results, and suggestions for future research. FEMS MicrobiolLett 323:1–12. https://doi.org/10.1111/j.1574-6968.2011.02315.x.8. Crombie AT, Murrell JC. 2014. Trace-gas metabolic versatility of thefacultative methanotroph Methylocella silvestris. Nature 510:148–151.https://doi.org/10.1038/nature13192.9. Chen Y, Crombie A, Rahman MT, Dedysh SN, Liesack W, Stott MB, AlamM, Theisen AR, Murrell JC, Dunﬁeld PF. 2010. Complete genome se-quence of the aerobic facultative methanotroph Methylocella silvestrisBL2. J Bacteriol 192:3840–3841. https://doi.org/10.1128/JB.00506-10.10. Street LE, Dean JF, Billett MF, Baxter R, Dinsmore KJ, Lessels JS, Subke J,Tetzlaff D, Wookey PA. 2016. Redox dynamics in the active layer of anarctic headwater catchment; examining the potential for transfer ofdissolved methane from soils to stream water. J Geophys Res Biogeosci121:2776–2792. https://doi.org/10.1002/2016JG003387.11. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS,Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV,Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a newgenome assembly algorithm and its applications to single-cell sequenc-ing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.12. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.13. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard forthe prokaryotic species deﬁnition. Proc Natl Acad Sci U S A 106:19126–19131. https://doi.org/10.1073/pnas.0906412106.Wang et al.Volume 6 Issue 8 e00040-18 genomea.asm.org 2 on March 2, 2018 by University of East Angliahttp://genomea.asm.org/Downloaded from 

Draft genome sequence of Methylocella silvestris TVC, a facultative methanotroph isolated from permafrost

Permafrost environments play a crucial role in global carbon and methane cycling. We report here the draft genome sequence of Methylocella silvestris TVC, a new facultative methanotroph strain, isolated from the Siksik Creek catchment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada).</p

Heriot Watt Pure

ABSTRACT
          
            Permafrost environments play a crucial role in global carbon and methane cycling. We report here the draft genome sequence of
            Methylocella silvestris
            TVC, a new facultative methanotroph strain, isolated from the Siksik Creek catchment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada).
          </jats:p

Jing Wang

Kan Geng

Muhammad Farhan Ul Haque

Andrew Crombie

Lorna E. Street

Philip A. Wookey

Ke Ma

J. Colin Murrell

Jennifer Pratscher

Crossref

Genome Announcements

Draft Genome Sequence of Methylocella silvestris TVC, a Facultative Methanotroph Isolated from Permafrost

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Draft Genome Sequence of Methylocella silvestris TVC, aFacultative Methanotroph Isolated from PermafrostCitation for published version:Wang, JI, Geng, K, Ul Haquea, MF, Crombie, A, Street, L, Wookey, PA, Ma, K, Murrell, JC & Pratscher, J2018, 'Draft Genome Sequence of Methylocella silvestris TVC, a Facultative Methanotroph Isolated fromPermafrost' Genome announcements. DOI: 10.1128/genomeA.00040-18.Digital Object Identifier (DOI):10.1128/genomeA.00040-18.Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Genome announcementsGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 07. Jul. 2018Draft Genome Sequence of Methylocella silvestris TVC, aFacultative Methanotroph Isolated from PermafrostJing Wang,a,b Kan Geng,a,b Muhammad Farhan Ul Haque,a Andrew Crombie,c Lorna E. Street,d Philip A. Wookey,e Ke Ma,bJ. Colin Murrell,a Jennifer PratscherfaSchool of Environmental Sciences, University of East Anglia, Norwich, United KingdombCollege of Resources and Environmental Sciences, China Agricultural University, Beijing, ChinacSchool of Biological Sciences, University of East Anglia, Norwich, United KingdomdSchool of GeoSciences, University of Edinburgh, Edinburgh, United KingdomeBiological and Environmental Sciences, University of Stirling, Stirling, United KingdomfLyell Centre, Heriot-Watt University, Edinburgh, United KingdomABSTRACT Permafrost environments play a crucial role in global carbon and meth-ane cycling. We report here the draft genome sequence of Methylocella silvestrisTVC, a new facultative methanotroph strain, isolated from the Siksik Creek catch-ment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada).Methanotrophic bacteria utilize methane as sole carbon and energy sources, thusplaying a major role in the global methane cycle (1). They are widespread in theenvironment, including lakes, rivers, sediments, rice paddies, sewage sludge, forests,and landﬁll soils (2, 3). All described methanotrophic species belonging to the genusMethylocella (family Beijerinckiaceae) possess a soluble methane monooxygenase(sMMO) that catalyzes the oxidation of methane to methanol, a key methane-oxidizingenzyme that is present in only a subset of methanotrophs (4–6). Methanotrophs of thegenus Methylocella do not possess a particulate methane monooxygenase (pMMO),which is present in most other methanotrophs (7). Methylocella species can also utilizemulticarbon compounds, including acetate, pyruvate, succinate, ethane, and propane(7, 8). Three species of Methylocella have so far been described, M. palustris, M. silvestris,and M. tundrae (4–6). Type strain M. silvestris BL2 was isolated from an acidic forestcambisol, and its genome sequence was reported previously (9). We now report theisolation and draft genome of a new strain, M. silvestris TVC.Samples for isolation were taken from a middle hill slope to stream channel transectof the Siksik Creek catchment (68°44=54.5 N, 133°29=41.7 W), a tributary of Trail ValleyCreek (TVC), Canada (10). After initial enrichment of the soil samples with CH4 (18%vol/vol in the headspace), subsamples were transferred to liquid medium and platedrepeatedly until the culture was pure.Genome sequencing of M. silvestris TVC was performed by MicrobesNG (Birming-ham, UK) using Illumina HiSeq technology (1,151,332 trimmed reads, 109-fold meancoverage) and assembled, using SPAdes version 3.11.1 (11), into 82 contigs with agenome size of 4,292,072 bp and a GC content of 62.94%. Annotation was performedusing Prokka version 1.12 (12).Comparative genome analysis revealed that although the 16S rRNA gene se-quences of strain TVC and strain BL2 shared 99% nucleotide identity, the similarityof mmoXYBZDC, encoding the sMMO, and mxaF, encoding methanol dehydroge-nase, was 90 to 95% between the two strains. Average nucleotide identity betweenstrains BL2 and TVC was calculated using JSpecies (13), showing 89.80% and 90.59%similarities based on the BLAST algorithm and MUMmer ultrarapid aligning tool,respectively.Received 11 January 2018 Accepted 30January 2018 Published 22 February 2018CitationWang J, Geng K, Farhan Ul Haque M,Crombie A, Street LE, Wookey PA, Ma K, MurrellJC, Pratscher J. 2018. Draft genome sequenceofMethylocella silvestris TVC, a facultativemethanotroph isolated from permafrost.Genome Announc 6:e00040-18. https://doi.org/10.1128/genomeA.00040-18.Copyright © 2018 Wang et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to J. Colin Murrell,j.c.murrell@uea.ac.uk.PROKARYOTEScrossmVolume 6 Issue 8 e00040-18 genomea.asm.org 1 on March 27, 2018 by UNIVERSITY OF EDINBURGHhttp://genomea.asm.org/Downloaded from Strain TVC used C1 substrates, including methane and methanol, as sole carbonsources. In addition to genes encoding the sMMO and methanol dehydrogenase,genes encoding the tetrahydrofolate (H4F)-dependent and tetrahydromethanop-terin (H4MPT)-dependent pathways of formaldehyde oxidation and those of theserine cycle are present in the genome. Like M. silvestris BL2, strain TVC was afacultative methanotroph and was able to utilize multicarbon compounds, includ-ing acetate, ethanol, succinate, and propane. Genes encoding the glyoxylate bypassenzymes isocitrate lyase and malate synthase are present. Also identiﬁed were theprmA, prmB, prmC, and prmD genes encoding a propane monooxygenase, showing91%, 84%, 86%, and 86% identities to those of M. silvestris BL2, respectively,enabling growth on propane. Further analyses of the genome and comparison withother strains will lead to a better understanding of the phylogeny and evolution offacultative methanotrophs.Accession number(s). This whole-genome shotgun project has been deposited atDDBJ/ENA/GenBank under the accession number PDZR00000000. The version de-scribed in this paper is the ﬁrst version, PDZR01000000.ACKNOWLEDGMENTSThis work was supported by Natural Environment Research Council (NERC) Inde-pendent Research Fellowship NE/L010771/1, Leverhulme Trust Research Project grantRPG2016-050, Leverhulme Early Career Fellowship ECF-2016-626, and NERC grantNE/K000284/1.Gwen Lancashire provided important help with ﬁeld sampling. We are also gratefulfor the logistical assistance provided by Philip Marsh and his research group, as well asby the staff at the Aurora Research Institute, Inuvik.REFERENCES1. Hanson RS, Hanson TE. 1996. Methanotrophic bacteria. Microbiol Rev60:439–471.2. Trotsenko YA, Murrell JC. 2008. Metabolic aspects of aerobic obligatemethanotrophy. Adv Appl Microbiol 63:183–229. https://doi.org/10.1016/S0065-2164(07)00005-6.3. Semrau JD, DiSpirito AA, Yoon S. 2010. Methanotrophs and copper.FEMS Microbiol Rev 34:496–531. https://doi.org/10.1111/j.1574-6976.2010.00212.x.4. Dedysh SN, Liesack W, Khmelenina VN, Suzina NE, Trotsenko YA, SemrauJD, Bares AM, Panikov NS, Tiedje JM. 2000. Methylocella palustris gen.nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peatbogs, representing a novel subtype of serine-pathway methanotrophs.Int J Syst Evol Microbiol 50:955–969. https://doi.org/10.1099/00207713-50-3-955.5. Dunﬁeld PF, Khmelenina VN, Suzina NE, Trotsenko YA, Dedysh SN. 2003.Methylocella silvestris sp. nov., a novel methanotroph isolated from anacidic forest cambisol. Int J Syst Evol Microbiol 53:1231–1239. https://doi.org/10.1099/ijs.0.02481-0.6. Dedysh SN, Berestovskaya YY, Vasylieva LV, Belova SE, Khmelenina VN,Suzina NE, Trotsenko YA, Liesack W, Zavarzin GA. 2004. Methylocellatundrae sp. nov., a novel methanotrophic bacterium from acidic tundrapeatlands. Int J Syst Evol Microbiol 54:151–156. https://doi.org/10.1099/ijs.0.02805-0.7. Semrau JD, DiSpirito AA, Vuilleumier S. 2011. Facultative methanotrophy:false leads, true results, and suggestions for future research. FEMS MicrobiolLett 323:1–12. https://doi.org/10.1111/j.1574-6968.2011.02315.x.8. Crombie AT, Murrell JC. 2014. Trace-gas metabolic versatility of thefacultative methanotroph Methylocella silvestris. Nature 510:148–151.https://doi.org/10.1038/nature13192.9. Chen Y, Crombie A, Rahman MT, Dedysh SN, Liesack W, Stott MB, AlamM, Theisen AR, Murrell JC, Dunﬁeld PF. 2010. Complete genome se-quence of the aerobic facultative methanotroph Methylocella silvestrisBL2. J Bacteriol 192:3840–3841. https://doi.org/10.1128/JB.00506-10.10. Street LE, Dean JF, Billett MF, Baxter R, Dinsmore KJ, Lessels JS, Subke J,Tetzlaff D, Wookey PA. 2016. Redox dynamics in the active layer of anarctic headwater catchment; examining the potential for transfer ofdissolved methane from soils to stream water. J Geophys Res Biogeosci121:2776–2792. https://doi.org/10.1002/2016JG003387.11. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS,Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV,Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a newgenome assembly algorithm and its applications to single-cell sequenc-ing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.12. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.13. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard forthe prokaryotic species deﬁnition. Proc Natl Acad Sci U S A 106:19126–19131. https://doi.org/10.1073/pnas.0906412106.Wang et al.Volume 6 Issue 8 e00040-18 genomea.asm.org 2 on March 27, 2018 by UNIVERSITY OF EDINBURGHhttp://genomea.asm.org/Downloaded from 

Haque, Muhammad Farhan Ul

Street, Lorna E

Wookey, Philip

Murrell, J Colin

Stirling Online Research Repository (RIOXX)

Draft Genome Sequence of Methylocella silvestris TVC, aFacultative Methanotroph Isolated from PermafrostJing Wang,a,b Kan Geng,a,b Muhammad Farhan Ul Haque,a Andrew Crombie,c Lorna E. Street,d Philip A. Wookey,e Ke Ma,bJ. Colin Murrell,a Jennifer PratscherfaSchool of Environmental Sciences, University of East Anglia, Norwich, United KingdombCollege of Resources and Environmental Sciences, China Agricultural University, Beijing, ChinacSchool of Biological Sciences, University of East Anglia, Norwich, United KingdomdSchool of GeoSciences, University of Edinburgh, Edinburgh, United KingdomeBiological and Environmental Sciences, University of Stirling, Stirling, United KingdomfLyell Centre, Heriot-Watt University, Edinburgh, United KingdomABSTRACT Permafrost environments play a crucial role in global carbon and meth-ane cycling. We report here the draft genome sequence of Methylocella silvestrisTVC, a new facultative methanotroph strain, isolated from the Siksik Creek catch-ment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada).Methanotrophic bacteria utilize methane as sole carbon and energy sources, thusplaying a major role in the global methane cycle (1). They are widespread in theenvironment, including lakes, rivers, sediments, rice paddies, sewage sludge, forests,and landfill soils (2, 3). All described methanotrophic species belonging to the genusMethylocella (family Beijerinckiaceae) possess a soluble methane monooxygenase(sMMO) that catalyzes the oxidation of methane to methanol, a key methane-oxidizingenzyme that is present in only a subset of methanotrophs (4–6). Methanotrophs of thegenus Methylocella do not possess a particulate methane monooxygenase (pMMO),which is present in most other methanotrophs (7). Methylocella species can also utilizemulticarbon compounds, including acetate, pyruvate, succinate, ethane, and propane(7, 8). Three species of Methylocella have so far been described, M. palustris, M. silvestris,and M. tundrae (4–6). Type strain M. silvestris BL2 was isolated from an acidic forestcambisol, and its genome sequence was reported previously (9). We now report theisolation and draft genome of a new strain, M. silvestris TVC.Samples for isolation were taken from a middle hill slope to stream channel transectof the Siksik Creek catchment (68°44=54.5 N, 133°29=41.7 W), a tributary of Trail ValleyCreek (TVC), Canada (10). After initial enrichment of the soil samples with CH4 (18%vol/vol in the headspace), subsamples were transferred to liquid medium and platedrepeatedly until the culture was pure.Genome sequencing of M. silvestris TVC was performed by MicrobesNG (Birming-ham, UK) using Illumina HiSeq technology (1,151,332 trimmed reads, 109-fold meancoverage) and assembled, using SPAdes version 3.11.1 (11), into 82 contigs with agenome size of 4,292,072 bp and a GC content of 62.94%. Annotation was performedusing Prokka version 1.12 (12).Comparative genome analysis revealed that although the 16S rRNA gene se-quences of strain TVC and strain BL2 shared 99% nucleotide identity, the similarityof mmoXYBZDC, encoding the sMMO, and mxaF, encoding methanol dehydroge-nase, was 90 to 95% between the two strains. Average nucleotide identity betweenstrains BL2 and TVC was calculated using JSpecies (13), showing 89.80% and 90.59%similarities based on the BLAST algorithm and MUMmer ultrarapid aligning tool,respectively.Received 11 January 2018 Accepted 30January 2018 Published 22 February 2018Citation Wang J, Geng K, Farhan Ul Haque M,Crombie A, Street LE, Wookey PA, Ma K, MurrellJC, Pratscher J. 2018. Draft genome sequenceof Methylocella silvestris TVC, a facultativemethanotroph isolated from permafrost.Genome Announc 6:e00040-18. https://doi.org/10.1128/genomeA.00040-18.Copyright © 2018 Wang et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to J. Colin Murrell,j.c.murrell@uea.ac.uk.PROKARYOTEScrossmVolume 6 Issue 8 e00040-18 genomea.asm.org 1 on March 9, 2018 by guesthttp://genomea.asm.org/Downloaded from Strain TVC used C1 substrates, including methane and methanol, as sole carbonsources. In addition to genes encoding the sMMO and methanol dehydrogenase,genes encoding the tetrahydrofolate (H4F)-dependent and tetrahydromethanop-terin (H4MPT)-dependent pathways of formaldehyde oxidation and those of theserine cycle are present in the genome. Like M. silvestris BL2, strain TVC was afacultative methanotroph and was able to utilize multicarbon compounds, includ-ing acetate, ethanol, succinate, and propane. Genes encoding the glyoxylate bypassenzymes isocitrate lyase and malate synthase are present. Also identified were theprmA, prmB, prmC, and prmD genes encoding a propane monooxygenase, showing91%, 84%, 86%, and 86% identities to those of M. silvestris BL2, respectively,enabling growth on propane. Further analyses of the genome and comparison withother strains will lead to a better understanding of the phylogeny and evolution offacultative methanotrophs.Accession number(s). This whole-genome shotgun project has been deposited atDDBJ/ENA/GenBank under the accession number PDZR00000000. The version de-scribed in this paper is the first version, PDZR01000000.ACKNOWLEDGMENTSThis work was supported by Natural Environment Research Council (NERC) Inde-pendent Research Fellowship NE/L010771/1, Leverhulme Trust Research Project grantRPG2016-050, Leverhulme Early Career Fellowship ECF-2016-626, and NERC grantNE/K000284/1.Gwen Lancashire provided important help with field sampling. We are also gratefulfor the logistical assistance provided by Philip Marsh and his research group, as well asby the staff at the Aurora Research Institute, Inuvik.REFERENCES1. Hanson RS, Hanson TE. 1996. Methanotrophic bacteria. Microbiol Rev60:439 – 471.2. Trotsenko YA, Murrell JC. 2008. Metabolic aspects of aerobic obligatemethanotrophy. Adv Appl Microbiol 63:183–229. https://doi.org/10.1016/S0065-2164(07)00005-6.3. Semrau JD, DiSpirito AA, Yoon S. 2010. Methanotrophs and copper.FEMS Microbiol Rev 34:496 –531. https://doi.org/10.1111/j.1574-6976.2010.00212.x.4. Dedysh SN, Liesack W, Khmelenina VN, Suzina NE, Trotsenko YA, SemrauJD, Bares AM, Panikov NS, Tiedje JM. 2000. Methylocella palustris gen.nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peatbogs, representing a novel subtype of serine-pathway methanotrophs.Int J Syst Evol Microbiol 50:955–969. https://doi.org/10.1099/00207713-50-3-955.5. Dunfield PF, Khmelenina VN, Suzina NE, Trotsenko YA, Dedysh SN. 2003.Methylocella silvestris sp. nov., a novel methanotroph isolated from anacidic forest cambisol. Int J Syst Evol Microbiol 53:1231–1239. https://doi.org/10.1099/ijs.0.02481-0.6. Dedysh SN, Berestovskaya YY, Vasylieva LV, Belova SE, Khmelenina VN,Suzina NE, Trotsenko YA, Liesack W, Zavarzin GA. 2004. Methylocellatundrae sp. nov., a novel methanotrophic bacterium from acidic tundrapeatlands. Int J Syst Evol Microbiol 54:151–156. https://doi.org/10.1099/ijs.0.02805-0.7. Semrau JD, DiSpirito AA, Vuilleumier S. 2011. Facultative methanotrophy:false leads, true results, and suggestions for future research. FEMS MicrobiolLett 323:1–12. https://doi.org/10.1111/j.1574-6968.2011.02315.x.8. Crombie AT, Murrell JC. 2014. Trace-gas metabolic versatility of thefacultative methanotroph Methylocella silvestris. Nature 510:148 –151.https://doi.org/10.1038/nature13192.9. Chen Y, Crombie A, Rahman MT, Dedysh SN, Liesack W, Stott MB, AlamM, Theisen AR, Murrell JC, Dunfield PF. 2010. Complete genome se-quence of the aerobic facultative methanotroph Methylocella silvestrisBL2. J Bacteriol 192:3840 –3841. https://doi.org/10.1128/JB.00506-10.10. Street LE, Dean JF, Billett MF, Baxter R, Dinsmore KJ, Lessels JS, Subke J,Tetzlaff D, Wookey PA. 2016. Redox dynamics in the active layer of anarctic headwater catchment; examining the potential for transfer ofdissolved methane from soils to stream water. J Geophys Res Biogeosci121:2776 –2792. https://doi.org/10.1002/2016JG003387.11. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS,Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV,Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a newgenome assembly algorithm and its applications to single-cell sequenc-ing. J Comput Biol 19:455– 477. https://doi.org/10.1089/cmb.2012.0021.12. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068 –2069. https://doi.org/10.1093/bioinformatics/btu153.13. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard forthe prokaryotic species definition. Proc Natl Acad Sci U S A 106:19126 –19131. https://doi.org/10.1073/pnas.0906412106.Wang et al.Volume 6 Issue 8 e00040-18 genomea.asm.org 2 on March 9, 2018 by guesthttp://genomea.asm.org/Downloaded from 

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Draft Genome Sequence of Methylocella silvestris TVC, aFacultative Methanotroph Isolated from PermafrostJing Wang,a,b Kan Geng,a,b Muhammad Farhan Ul Haque,a Andrew Crombie,c Lorna E. Street,d Philip A. Wookey,e Ke Ma,bJ. Colin Murrell,a Jennifer PratscherfaSchool of Environmental Sciences, University of East Anglia, Norwich, United KingdombCollege of Resources and Environmental Sciences, China Agricultural University, Beijing, ChinacSchool of Biological Sciences, University of East Anglia, Norwich, United KingdomdSchool of GeoSciences, University of Edinburgh, Edinburgh, United KingdomeBiological and Environmental Sciences, University of Stirling, Stirling, United KingdomfLyell Centre, Heriot-Watt University, Edinburgh, United KingdomABSTRACT Permafrost environments play a crucial role in global carbon and meth-ane cycling. We report here the draft genome sequence of Methylocella silvestrisTVC, a new facultative methanotroph strain, isolated from the Siksik Creek catch-ment in the continuous permafrost zone of Inuvik (Northwest Territories, Canada).Methanotrophic bacteria utilize methane as sole carbon and energy sources, thusplaying a major role in the global methane cycle (1). They are widespread in theenvironment, including lakes, rivers, sediments, rice paddies, sewage sludge, forests,and landﬁll soils (2, 3). All described methanotrophic species belonging to the genusMethylocella (family Beijerinckiaceae) possess a soluble methane monooxygenase(sMMO) that catalyzes the oxidation of methane to methanol, a key methane-oxidizingenzyme that is present in only a subset of methanotrophs (4–6). Methanotrophs of thegenus Methylocella do not possess a particulate methane monooxygenase (pMMO),which is present in most other methanotrophs (7). Methylocella species can also utilizemulticarbon compounds, including acetate, pyruvate, succinate, ethane, and propane(7, 8). Three species of Methylocella have so far been described, M. palustris, M. silvestris,and M. tundrae (4–6). Type strain M. silvestris BL2 was isolated from an acidic forestcambisol, and its genome sequence was reported previously (9). We now report theisolation and draft genome of a new strain, M. silvestris TVC.Samples for isolation were taken from a middle hill slope to stream channel transectof the Siksik Creek catchment (68°44=54.5 N, 133°29=41.7 W), a tributary of Trail ValleyCreek (TVC), Canada (10). After initial enrichment of the soil samples with CH4 (18%vol/vol in the headspace), subsamples were transferred to liquid medium and platedrepeatedly until the culture was pure.Genome sequencing of M. silvestris TVC was performed by MicrobesNG (Birming-ham, UK) using Illumina HiSeq technology (1,151,332 trimmed reads, 109-fold meancoverage) and assembled, using SPAdes version 3.11.1 (11), into 82 contigs with agenome size of 4,292,072 bp and a GC content of 62.94%. Annotation was performedusing Prokka version 1.12 (12).Comparative genome analysis revealed that although the 16S rRNA gene se-quences of strain TVC and strain BL2 shared 99% nucleotide identity, the similarityof mmoXYBZDC, encoding the sMMO, and mxaF, encoding methanol dehydroge-nase, was 90 to 95% between the two strains. Average nucleotide identity betweenstrains BL2 and TVC was calculated using JSpecies (13), showing 89.80% and 90.59%similarities based on the BLAST algorithm and MUMmer ultrarapid aligning tool,respectively.Received 11 January 2018 Accepted 30January 2018 Published 22 February 2018CitationWang J, Geng K, Farhan Ul Haque M,Crombie A, Street LE, Wookey PA, Ma K, MurrellJC, Pratscher J. 2018. Draft genome sequenceofMethylocella silvestris TVC, a facultativemethanotroph isolated from permafrost.Genome Announc 6:e00040-18. https://doi.org/10.1128/genomeA.00040-18.Copyright © 2018 Wang et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to J. Colin Murrell,j.c.murrell@uea.ac.uk.PROKARYOTEScrossmVolume 6 Issue 8 e00040-18 genomea.asm.org 1 on March 9, 2018 by guesthttp://genomea.asm.org/Downloaded from Strain TVC used C1 substrates, including methane and methanol, as sole carbonsources. In addition to genes encoding the sMMO and methanol dehydrogenase,genes encoding the tetrahydrofolate (H4F)-dependent and tetrahydromethanop-terin (H4MPT)-dependent pathways of formaldehyde oxidation and those of theserine cycle are present in the genome. Like M. silvestris BL2, strain TVC was afacultative methanotroph and was able to utilize multicarbon compounds, includ-ing acetate, ethanol, succinate, and propane. Genes encoding the glyoxylate bypassenzymes isocitrate lyase and malate synthase are present. Also identiﬁed were theprmA, prmB, prmC, and prmD genes encoding a propane monooxygenase, showing91%, 84%, 86%, and 86% identities to those of M. silvestris BL2, respectively,enabling growth on propane. Further analyses of the genome and comparison withother strains will lead to a better understanding of the phylogeny and evolution offacultative methanotrophs.Accession number(s). This whole-genome shotgun project has been deposited atDDBJ/ENA/GenBank under the accession number PDZR00000000. The version de-scribed in this paper is the ﬁrst version, PDZR01000000.ACKNOWLEDGMENTSThis work was supported by Natural Environment Research Council (NERC) Inde-pendent Research Fellowship NE/L010771/1, Leverhulme Trust Research Project grantRPG2016-050, Leverhulme Early Career Fellowship ECF-2016-626, and NERC grantNE/K000284/1.Gwen Lancashire provided important help with ﬁeld sampling. We are also gratefulfor the logistical assistance provided by Philip Marsh and his research group, as well asby the staff at the Aurora Research Institute, Inuvik.REFERENCES1. Hanson RS, Hanson TE. 1996. Methanotrophic bacteria. Microbiol Rev60:439–471.2. Trotsenko YA, Murrell JC. 2008. Metabolic aspects of aerobic obligatemethanotrophy. Adv Appl Microbiol 63:183–229. https://doi.org/10.1016/S0065-2164(07)00005-6.3. Semrau JD, DiSpirito AA, Yoon S. 2010. Methanotrophs and copper.FEMS Microbiol Rev 34:496–531. https://doi.org/10.1111/j.1574-6976.2010.00212.x.4. Dedysh SN, Liesack W, Khmelenina VN, Suzina NE, Trotsenko YA, SemrauJD, Bares AM, Panikov NS, Tiedje JM. 2000. Methylocella palustris gen.nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peatbogs, representing a novel subtype of serine-pathway methanotrophs.Int J Syst Evol Microbiol 50:955–969. https://doi.org/10.1099/00207713-50-3-955.5. Dunﬁeld PF, Khmelenina VN, Suzina NE, Trotsenko YA, Dedysh SN. 2003.Methylocella silvestris sp. nov., a novel methanotroph isolated from anacidic forest cambisol. Int J Syst Evol Microbiol 53:1231–1239. https://doi.org/10.1099/ijs.0.02481-0.6. Dedysh SN, Berestovskaya YY, Vasylieva LV, Belova SE, Khmelenina VN,Suzina NE, Trotsenko YA, Liesack W, Zavarzin GA. 2004. Methylocellatundrae sp. nov., a novel methanotrophic bacterium from acidic tundrapeatlands. Int J Syst Evol Microbiol 54:151–156. https://doi.org/10.1099/ijs.0.02805-0.7. Semrau JD, DiSpirito AA, Vuilleumier S. 2011. Facultative methanotrophy:false leads, true results, and suggestions for future research. FEMS MicrobiolLett 323:1–12. https://doi.org/10.1111/j.1574-6968.2011.02315.x.8. Crombie AT, Murrell JC. 2014. Trace-gas metabolic versatility of thefacultative methanotroph Methylocella silvestris. Nature 510:148–151.https://doi.org/10.1038/nature13192.9. Chen Y, Crombie A, Rahman MT, Dedysh SN, Liesack W, Stott MB, AlamM, Theisen AR, Murrell JC, Dunﬁeld PF. 2010. Complete genome se-quence of the aerobic facultative methanotroph Methylocella silvestrisBL2. J Bacteriol 192:3840–3841. https://doi.org/10.1128/JB.00506-10.10. Street LE, Dean JF, Billett MF, Baxter R, Dinsmore KJ, Lessels JS, Subke J,Tetzlaff D, Wookey PA. 2016. Redox dynamics in the active layer of anarctic headwater catchment; examining the potential for transfer ofdissolved methane from soils to stream water. J Geophys Res Biogeosci121:2776–2792. https://doi.org/10.1002/2016JG003387.11. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS,Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV,Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a newgenome assembly algorithm and its applications to single-cell sequenc-ing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.12. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.13. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard forthe prokaryotic species deﬁnition. Proc Natl Acad Sci U S A 106:19126–19131. https://doi.org/10.1073/pnas.0906412106.Wang et al.Volume 6 Issue 8 e00040-18 genomea.asm.org 2 on March 9, 2018 by guesthttp://genomea.asm.org/Downloaded from 

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Draft Genome Sequence of Methylocella silvestris TVC, a Facultative Methanotroph Isolated from Permafrost

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