Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph.

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined deoxyribonucleic acid-stable isotope probing (DNA-SIP) with metagenomics and metaproteomics to characterize an uncultivated marine methylotroph that actively incorporated carbon from (13) C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which the culture-independent techniques of DNA-SIP and protein-SIP have been used to characterize the metabolism of a naturally occurring Methylophaga-like bacterium in the marine environment (i.e. Methylophaga thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment.

[1]  Natalia Ivanova,et al.  The Expanded Diversity of Methylophilaceae from Lake Washington through Cultivation and Genomic Sequencing of Novel Ecotypes , 2014, PloS one.

[2]  J. Murrell,et al.  Oxidation of dimethylsulfide to tetrathionate by Methylophaga thiooxidans sp. nov.: a new link in the sulfur cycle. , 2010, Environmental microbiology.

[3]  M. Lidstrom,et al.  Comparative transcriptomics in three Methylophilaceae species uncover different strategies for environmental adaptation , 2013, PeerJ.

[4]  M. Lidstrom,et al.  Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1. , 1997, Microbiology.

[5]  P. Steudler,et al.  The Methanol Dehydrogenase Gene, mxaF, as a Functional and Phylogenetic Marker for Proteobacterial Methanotrophs in Natural Environments , 2013, PloS one.

[6]  Victor Markowitz,et al.  High-resolution metagenomics targets specific functional types in complex microbial communities , 2008, Nature Biotechnology.

[7]  Steffen Kolb,et al.  Prerequisites for amplicon pyrosequencing of microbial methanol utilizers in the environment , 2013, Front. Microbiol..

[8]  Martin Taubert,et al.  Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology , 2013, The ISME Journal.

[9]  M. Lidstrom,et al.  Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation. , 1998, FEMS microbiology letters.

[10]  C. Blake,et al.  The structure and function of methanol dehydrogenase and related quinoproteins containing pyrrolo-quinoline quinone. , 1994, The Biochemical journal.

[11]  Advanced MudPIT as a next step toward high proteome coverage , 2011, Proteomics.

[12]  J. Murrell,et al.  Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave , 2014, The ISME Journal.

[13]  Knut Reinert,et al.  TOPP - the OpenMS proteomics pipeline , 2007, Bioinform..

[14]  S. Bryant,et al.  Open mass spectrometry search algorithm. , 2004, Journal of proteome research.

[15]  Martin Hartmann,et al.  Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities , 2009, Applied and Environmental Microbiology.

[16]  S. Kleinsteuber,et al.  Protein-SIP enables time-resolved analysis of the carbon flux in a sulfate-reducing, benzene-degrading microbial consortium , 2012, The ISME Journal.

[17]  R. Villemur,et al.  Methylophaga nitratireducenticrescens sp. nov. and Methylophaga frappieri sp. nov., isolated from the biofilm of the methanol-fed denitrification system treating the seawater at the Montreal Biodome. , 2013, International journal of systematic and evolutionary microbiology.

[18]  Daniel Patrick Smith,et al.  One Carbon Metabolism in SAR11 Pelagic Marine Bacteria , 2011, PloS one.

[19]  Knut Reinert,et al.  OpenMS – An open-source software framework for mass spectrometry , 2008, BMC Bioinformatics.

[20]  Josh D Neufeld,et al.  Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. , 2008, Environmental microbiology.

[21]  Philip Ineson,et al.  Stable-isotope probing as a tool in microbial ecology , 2000, Nature.

[22]  V. Davidson.  Pyrroloquinoline quinone (PQQ) from methanol dehydrogenase and tryptophan tryptophylquinone (TTQ) from methylamine dehydrogenase. , 2001, Advances in protein chemistry.

[23]  J. Murrell,et al.  XoxF encoding an alternative methanol dehydrogenase is widespread in coastal marine environments. , 2015, Environmental microbiology.

[24]  Sergey I. Nikolenko,et al.  SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing , 2012, J. Comput. Biol..

[25]  M. Lidstrom,et al.  A new cofactor in a prokaryotic enzyme: tryptophan tryptophylquinone as the redox prosthetic group in methylamine dehydrogenase , 1991, Science.

[26]  Josh D Neufeld,et al.  DNA stable-isotope probing , 2007, Nature Protocols.

[27]  Ludmila Chistoserdova,et al.  Modularity of methylotrophy, revisited. , 2011, Environmental microbiology.

[28]  Lynne A. Goodwin,et al.  Genome of Methylobacillus flagellatus, Molecular Basis for Obligate Methylotrophy, and Polyphyletic Origin of Methylotrophy , 2007, Journal of bacteriology.

[29]  M. Lidstrom,et al.  Analysis of two formaldehyde oxidation pathways in Methylobacillus flagellatus KT, a ribulose monophosphate cycle methylotroph. , 2000, Microbiology.

[30]  Sergey I. Nikolenko,et al.  BayesHammer: Bayesian clustering for error correction in single-cell sequencing , 2012, BMC Genomics.

[31]  Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica , 2012, Biochemistry (Moscow).

[32]  Dawn Field,et al.  The seasonal structure of microbial communities in the Western English Channel. , 2009, Environmental microbiology.

[33]  J. Murrell,et al.  The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs , 1997, Applied and environmental microbiology.

[34]  Ludmila Chistoserdova,et al.  The expanding world of methylotrophic metabolism. , 2009, Annual review of microbiology.

[35]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[36]  S. Dowd,et al.  Survey of fungi and yeast in polymicrobial infections in chronic wounds. , 2011, Journal of wound care.

[37]  Manesh Shah,et al.  Environmental proteomics of microbial plankton in a highly productive coastal upwelling system , 2011, The ISME Journal.

[38]  Huub J. M. Op den Camp,et al.  PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference , 2014, Applied Microbiology and Biotechnology.

[39]  F. Grimont,et al.  Methylophaga marina gen. nov., sp. nov. and Methylophaga thalassica sp. nov., marine methylotrophs , 1985 .

[40]  T. Smyth,et al.  Annual study of oxygenated volatile organic compounds in UK shelf waters , 2015 .

[41]  Rick L. Stevens,et al.  The RAST Server: Rapid Annotations using Subsystems Technology , 2008, BMC Genomics.

[42]  Matthew Z. DeMaere,et al.  Metaproteogenomic analysis of a dominant green sulfur bacterium from Ace Lake, Antarctica , 2010, The ISME Journal.

[43]  V. Davidson,et al.  MauG, a diheme enzyme that catalyzes tryptophan tryptophylquinone biosynthesis by remote catalysis. , 2014, Archives of biochemistry and biophysics.

[44]  S. Ferriera,et al.  Draft Genome Sequence of the Chemolithoheterotrophic, Halophilic Methylotroph Methylophaga thiooxydans DMS010 , 2011, Journal of bacteriology.

[45]  P. Nightingale,et al.  Microbial methanol uptake in northeast Atlantic waters , 2011, The ISME Journal.

[46]  J. Murrell,et al.  γ-Glutamylmethylamide Is an Essential Intermediate in the Metabolism of Methylamine by Methylocella silvestris , 2010, Applied and Environmental Microbiology.

[47]  J. Tiedje,et al.  Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy , 2007, Applied and Environmental Microbiology.

[48]  C. Anthony,et al.  The methanol oxidation genes mxaFJGIR (S) ACKLD in Methylobacterium extorquens. , 1997, FEMS microbiology letters.

[49]  J. Murrell,et al.  Metabolic aspects of aerobic obligate methanotrophy. , 2008, Advances in applied microbiology.

[50]  M. Dumont,et al.  Community-level analysis: key genes of aerobic methane oxidation. , 2005, Methods in enzymology.

[51]  T. Barends,et al.  Rare earth metals are essential for methanotrophic life in volcanic mudpots. , 2014, Environmental microbiology.

[52]  J. R. Quayle,et al.  MICROBIAL GROWTH ON C1 COMPOUNDS. SYNTHESIS OF CELL CONSTITUENTS BY METHANE- AND METHANOL-GROWN PSEUDOMONAS METHANICA. , 1965, The Biochemical journal.

[53]  H. Schäfer.  Isolation of Methylophaga spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide , 2007, Applied and Environmental Microbiology.

[54]  J. Neufeld,et al.  Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism , 2007, The ISME Journal.

[55]  M. Lidstrom,et al.  Novel Formaldehyde-Activating Enzyme inMethylobacterium extorquens AM1 Required for Growth on Methanol , 2000, Journal of bacteriology.

[56]  M. Kalyuzhnaya,et al.  Genetics of the glutamate‐mediated methylamine utilization pathway in the facultative methylotrophic beta‐proteobacterium Methyloversatilis universalis FAM5 , 2010, Molecular microbiology.

[57]  Robert C. Edgar,et al.  UPARSE: highly accurate OTU sequences from microbial amplicon reads , 2013, Nature Methods.

[58]  N. Shapiro,et al.  Multiphyletic origins of methylotrophy in Alphaproteobacteria, exemplified by comparative genomics of Lake Washington isolates. , 2015, Environmental microbiology.

[59]  Andreas Wilke,et al.  phylogenetic and functional analysis of metagenomes , 2022 .

[60]  C. Anthony,et al.  The Biochemistry of Methylotrophs , 1982 .

[61]  J. Gilbert,et al.  Metagenomes and metatranscriptomes from the L4 long-term coastal monitoring station in the Western English Channel , 2010, Standards in genomic sciences.

[62]  R. Villemur,et al.  Complete Genome Sequences of Methylophaga sp. Strain JAM1 and Methylophaga sp. Strain JAM7 , 2012, Journal of bacteriology.

[63]  J. Neufeld,et al.  Substrate-Specific Clades of Active Marine Methylotrophs Associated with a Phytoplankton Bloom in a Temperate Coastal Environment , 2008, Applied and Environmental Microbiology.

[64]  U. Völker,et al.  Protein-based stable isotope probing (protein-SIP) in functional metaproteomics. , 2012, Mass spectrometry reviews.