Charting the Complexity of the Marine Microbiome through Single-Cell Genomics

Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.

[1]  Taylor K. Dunivin,et al.  Ecological selection for small microbial genomes along a temperate-to-thermal soil gradient , 2018, Nature Microbiology.

[2]  G. V. van Wezel,et al.  Cracking the regulatory code of biosynthetic gene clusters as a strategy for natural product discovery , 2018, Biochemical pharmacology.

[3]  E. Delong,et al.  The Microbial Engines That Drive Earth's Biogeochemical Cycles , 2008, Science.

[4]  S. Giovannoni,et al.  SAR11 marine bacteria require exogenous reduced sulphur for growth , 2008, Nature.

[5]  Marc Strous,et al.  Archaeal nitrification in the ocean , 2006, Proceedings of the National Academy of Sciences.

[6]  Adina Howe,et al.  Strategies to improve reference databases for soil microbiomes , 2016, The ISME Journal.

[7]  Katherine H. Huang,et al.  Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis , 2012, The ISME Journal.

[8]  O. Gascuel,et al.  An improved general amino acid replacement matrix. , 2008, Molecular biology and evolution.

[9]  N. Kashtan,et al.  Single-Cell Genomics Reveals Hundreds of Coexisting Subpopulations in Wild Prochlorococcus , 2014, Science.

[10]  I. Hewson,et al.  Metagenomic potential of microbial assemblages in the surface waters of the central Pacific Ocean tracks variability in oceanic habitat , 2009 .

[11]  E. Koonin,et al.  Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. , 2000, Science.

[12]  M. Komatsu,et al.  Terpene synthases are widely distributed in bacteria , 2014, Proceedings of the National Academy of Sciences.

[13]  Elaina D. Graham,et al.  Descriptor : The reconstruction of 2 , 631 draft metagenome-assembled genomes from the global oceans , 2018 .

[14]  K. Pollard,et al.  An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial transmission and biogeography , 2016, Genome research.

[15]  Anders Krogh,et al.  Fast and sensitive taxonomic classification for metagenomics with Kaiju , 2016, Nature Communications.

[16]  M. Kimura.  A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences , 1980, Journal of Molecular Evolution.

[17]  Sarah L. R. Stevens,et al.  Contrasting patterns of genome-level diversity across distinct co-occurring bacterial populations , 2016, The ISME Journal.

[18]  M. Könneke,et al.  Isolation of an autotrophic ammonia-oxidizing marine archaeon , 2005, Nature.

[19]  Paul R Jensen,et al.  Developing a new resource for drug discovery: marine actinomycete bacteria , 2006, Nature chemical biology.

[20]  Michael D Lee.  Applications and Considerations of GToTree: A User-Friendly Workflow for Phylogenomics , 2019, Evolutionary bioinformatics online.

[21]  Michael DiCuccio,et al.  Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI , 2018, International journal of systematic and evolutionary microbiology.

[22]  R. Amann,et al.  On-Site Analysis of Bacterial Communities of the Ultraoligotrophic South Pacific Gyre , 2019, Applied and Environmental Microbiology.

[23]  Tanja Woyke,et al.  Genomic sequencing of single microbial cells from environmental samples. , 2008, Current opinion in microbiology.

[24]  Kai Blin,et al.  antiSMASH 4.0—improvements in chemistry prediction and gene cluster boundary identification , 2017, Nucleic Acids Res..

[25]  Devin F R Doud,et al.  The trajectory of microbial single-cell sequencing , 2017, Nature Methods.

[26]  Niels W. Hanson,et al.  Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean , 2013, Proceedings of the National Academy of Sciences.

[27]  J. Beman,et al.  Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Brian C. Thomas,et al.  Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system , 2016, Nature Communications.

[29]  A. Phillippy,et al.  High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries , 2017, Nature Communications.

[30]  G. Amoutzias,et al.  Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes , 2016, Marine drugs.

[31]  Philip D. Blood,et al.  Critical Assessment of Metagenome Interpretation—a benchmark of metagenomics software , 2017, Nature Methods.

[32]  Daniel H. Huson,et al.  CREST – Classification Resources for Environmental Sequence Tags , 2012, PloS one.

[33]  Xiaole Kong,et al.  Chemistry and biology of siderophores. , 2010, Natural product reports.

[34]  R. Ueoka,et al.  Automated structure prediction of trans-acyltransferase polyketide synthase products , 2019, Nature Chemical Biology.

[35]  Z. Yang,et al.  Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. , 2000, Molecular biology and evolution.

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

[37]  J. Fuhrman,et al.  Microbial rhodopsins are major contributors to the solar energy captured in the sea , 2019, Science Advances.

[38]  R. Stepanauskas.  Single cell genomics: an individual look at microbes. , 2012, Current opinion in microbiology.

[39]  E. Delong,et al.  Community Genomics Among Stratified Microbial Assemblages in the Ocean's Interior , 2006, Science.

[40]  Bradley S Moore,et al.  Lessons from the past and charting the future of marine natural products drug discovery and chemical biology. , 2012, Chemistry & biology.

[41]  Peer Bork,et al.  PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments , 2006, Nucleic Acids Res..

[42]  A. Halpern,et al.  The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific , 2007, PLoS biology.

[43]  Koichiro Tamura,et al.  MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. , 2013, Molecular biology and evolution.

[44]  K. Konstantinidis,et al.  Genomic insights that advance the species definition for prokaryotes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Elmar Pruesse,et al.  SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes , 2012, Bioinform..

[46]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[47]  T. Hackl,et al.  Marine microbial metagenomes sampled across space and time , 2018, Scientific Data.

[48]  E. Delong,et al.  Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology , 2016, Microbiology and Molecular Reviews.

[49]  E. Delong,et al.  Potential for Chemolithoautotrophy Among Ubiquitous Bacteria Lineages in the Dark Ocean , 2011, Science.

[50]  Alexandros Stamatakis,et al.  RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies , 2014, Bioinform..

[51]  T. Hackl,et al.  Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments , 2018, Scientific Data.

[52]  D. Higgins,et al.  Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega , 2011, Molecular systems biology.

[53]  M. Koblížek.  Ecology of aerobic anoxygenic phototrophs in aquatic environments. , 2015, FEMS microbiology reviews.

[54]  Torsten Seemann,et al.  Prokka: rapid prokaryotic genome annotation , 2014, Bioinform..

[55]  A. Harvey,et al.  The re-emergence of natural products for drug discovery in the genomics era , 2015, Nature Reviews Drug Discovery.

[56]  P. Schmitt‐Kopplin,et al.  Dissolved organic sulfur in the ocean: Biogeochemistry of a petagram inventory , 2016, Science.

[57]  F. Chavez,et al.  Ammonium Uptake by Phytoplankton Regulates Nitrification in the Sunlit Ocean , 2014, PloS one.

[58]  T. Karpiński.  Marine Macrolides with Antibacterial and/or Antifungal Activity , 2019, Marine drugs.

[59]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[60]  Ziheng Yang.  PAML 4: phylogenetic analysis by maximum likelihood. , 2007, Molecular biology and evolution.

[61]  Donovan H. Parks,et al.  Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life , 2017, Nature Microbiology.

[62]  Steven Salzberg,et al.  BIOINFORMATICS ORIGINAL PAPER , 2004 .

[63]  E. Koonin,et al.  Two fundamentally different classes of microbial genes , 2016, Nature Microbiology.

[64]  Jens Roat Kultima,et al.  An integrated catalog of reference genes in the human gut microbiome , 2014, Nature Biotechnology.

[65]  P. Calder.  Marine omega-3 fatty acids and inflammatory processes: Effects, mechanisms and clinical relevance. , 2015, Biochimica et biophysica acta.

[66]  Otto X. Cordero,et al.  Population Genomics of Early Events in the Ecological Differentiation of Bacteria , 2012, Science.

[67]  O. White,et al.  Environmental Genome Shotgun Sequencing of the Sargasso Sea , 2004, Science.

[68]  Alexander Agafonov,et al.  The MAR databases: development and implementation of databases specific for marine metagenomics , 2017, Nucleic Acids Res..

[69]  P. Bork,et al.  Single-cell genomics of multiple uncultured stramenopiles reveals underestimated functional diversity across oceans , 2018, Nature Communications.

[70]  R. Fulweiler,et al.  Ecological control of nitrite in the upper ocean , 2018, Nature Communications.

[71]  J. Banfield,et al.  Community structure and metabolism through reconstruction of microbial genomes from the environment , 2004, Nature.

[72]  K. Konstantinidis,et al.  The bacterial species definition in the genomic era , 2006, Philosophical Transactions of the Royal Society B: Biological Sciences.

[73]  John D. Hunter,et al.  Matplotlib: A 2D Graphics Environment , 2007, Computing in Science & Engineering.

[74]  S. Hallam,et al.  Major role of nitrite-oxidizing bacteria in dark ocean carbon fixation , 2017, Science.

[75]  Benjamin H. Good,et al.  The Dynamics of Molecular Evolution Over 60,000 Generations , 2017, Nature.

[76]  Joseph P Noel,et al.  The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life. , 2014, Natural product reports.

[77]  M. Touchon,et al.  The chromosomal organization of horizontal gene transfer in bacteria , 2017, Nature Communications.

[78]  S. Giovannoni.  SAR11 Bacteria: The Most Abundant Plankton in the Oceans. , 2017, Annual review of marine science.

[79]  Connor T. Skennerton,et al.  CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes , 2015, Genome research.

[80]  S. Deutsch,et al.  SAR11 bacteria linked to ocean anoxia and nitrogen loss , 2016, Nature.

[81]  J. Handelsman.  Metagenomics: Application of Genomics to Uncultured Microorganisms , 2004, Microbiology and Molecular Biology Reviews.

[82]  David J. States,et al.  Identification of protein coding regions by database similarity search , 1993, Nature Genetics.

[83]  Natalia N. Ivanova,et al.  Insights into the phylogeny and coding potential of microbial dark matter , 2013, Nature.

[84]  Sudhir Kumar,et al.  MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. , 2018, Molecular biology and evolution.

[85]  Brian P. Thompson,et al.  Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles , 2017, Nature Communications.

[86]  Tom O. Delmont,et al.  Nitrogen-fixing populations of Planctomycetes and Proteobacteria are abundant in surface ocean metagenomes , 2018, Nature Microbiology.

[87]  Luis Pedro Coelho,et al.  Structure and function of the global ocean microbiome , 2015, Science.

[88]  S. Giovannoni,et al.  Genetic diversity in Sargasso Sea bacterioplankton , 1990, Nature.

[89]  C. Hertweck,et al.  The biosynthetic logic of polyketide diversity. , 2009, Angewandte Chemie.

[90]  Adam P. Arkin,et al.  FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix , 2009, Molecular biology and evolution.

[91]  D. Canfield,et al.  A Cryptic Sulfur Cycle in Oxygen-Minimum–Zone Waters off the Chilean Coast , 2010, Science.

[92]  D. Mende,et al.  Environmental drivers of a microbial genomic transition zone in the ocean’s interior , 2017, Nature Microbiology.

[93]  S. Giovannoni,et al.  The uncultured microbial majority. , 2003, Annual review of microbiology.

[94]  Johannes Söding,et al.  MMseqs2: sensitive protein sequence searching for the analysis of massive data sets , 2017, bioRxiv.

[95]  J. Eisen,et al.  Assembling the Marine Metagenome, One Cell at a Time , 2009, PloS one.

[96]  M. Wagner,et al.  Complete nitrification by Nitrospira bacteria , 2015, Nature.

[97]  J. Lennon,et al.  Scaling laws predict global microbial diversity , 2016, Proceedings of the National Academy of Sciences.

[98]  S. Giovannoni,et al.  Implications of streamlining theory for microbial ecology , 2014, The ISME Journal.

[99]  N. Kashtan,et al.  Fundamental differences in diversity and genomic population structure between Atlantic and Pacific Prochlorococcus , 2017, The ISME Journal.

[100]  J. Huisman,et al.  Scientists’ warning to humanity: microorganisms and climate change , 2019, Nature Reviews Microbiology.

[101]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[102]  Elaina D. Graham,et al.  Potential for primary productivity in a globally-distributed bacterial phototroph , 2018, The ISME Journal.

[103]  Miriam L. Land,et al.  Trace: Tennessee Research and Creative Exchange Prodigal: Prokaryotic Gene Recognition and Translation Initiation Site Identification Recommended Citation Prodigal: Prokaryotic Gene Recognition and Translation Initiation Site Identification , 2022 .