Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria

Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

[1]  D. Karl,et al.  Vertical distributions of nitrogen-fixing phylotypes at Stn ALOHA in the oligotrophic North Pacific Ocean , 2005 .

[2]  Tracy K. Teal,et al.  Systematic artifacts in metagenomes from complex microbial communities , 2009, The ISME Journal.

[3]  J. LaRoche,et al.  Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean , 2005, Applied and Environmental Microbiology.

[4]  J. LaRoche,et al.  Abundances and Distributions of the Dominant nifH Phylotypes in the Northern Atlantic Ocean , 2008, Applied and Environmental Microbiology.

[5]  S. Giovannoni,et al.  Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR , 1996, Applied and environmental microbiology.

[6]  S. Acinas,et al.  Fine-scale phylogenetic architecture of a complex bacterial community , 2004, Nature.

[7]  L. Riemann,et al.  Identity and dynamics of putative N2 -fixing picoplankton in the Baltic Sea proper suggest complex patterns of regulation. , 2009, Environmental microbiology reports.

[8]  A. Chao Nonparametric estimation of the number of classes in a population , 1984 .

[9]  T. Sørensen,et al.  A method of establishing group of equal amplitude in plant sociobiology based on similarity of species content and its application to analyses of the vegetation on Danish commons , 1948 .

[10]  G. Steward,et al.  Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray , 2004, Applied and Environmental Microbiology.

[11]  H. Paerl,et al.  Molecular Ecological Aspects of Nitrogen Fixation in the Marine Environment , 2008 .

[12]  I. Berman‐Frank,et al.  Molecular ecology of nifH genes and transcripts in the eastern Mediterranean Sea. , 2007, Environmental microbiology.

[13]  A. Paytan,et al.  Seasonality of N 2 fixation and nifH gene diversity in the Gulf of Aqaba ( Red Sea ) , 2008 .

[14]  E. Delong,et al.  Low genomic diversity in tropical oceanic N2-fixing cyanobacteria , 2007, Proceedings of the National Academy of Sciences.

[15]  M. C. Horner-Devine,et al.  News About Nitrogen , 2008, Science.

[16]  I. Hewson,et al.  Diazotrophic bacterioplankton in a coral reef lagoon: phylogeny, diel nitrogenase expression and response to phosphate enrichment , 2007, The ISME Journal.

[17]  S. Sørensen,et al.  Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults , 2010, PloS one.

[18]  R. Beinart,et al.  Diversity and abundance of diazotrophic microorganisms in the South China Sea during intermonsoon , 2008, The ISME Journal.

[19]  Peer Bork,et al.  Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation , 2007, Bioinform..

[20]  L. Riemann,et al.  Nitrogenase genes in non-cyanobacterial plankton: prevalence, diversity and regulation in marine waters , 2010 .

[21]  L. Riemann,et al.  Putative N-2-fixing heterotrophic bacteria associated with dinoflagellate-Cyanobacteria consortia in the low-nitrogen Indian Ocean , 2010 .

[22]  R. Knight,et al.  UniFrac: a New Phylogenetic Method for Comparing Microbial Communities , 2005, Applied and Environmental Microbiology.

[23]  Sang Joon Kim,et al.  A Mathematical Theory of Communication , 2006 .

[24]  K. Schneider,et al.  Identification of Two New Genes Involved in Diazotrophic Growth via the Alternative Fe-Only Nitrogenase in the Phototrophic Purple Bacterium Rhodobacter capsulatus , 2005, Journal of bacteriology.

[25]  C. M. Short,et al.  Temporal Patterns of Nitrogenase Gene (nifH) Expression in the Oligotrophic North Pacific Ocean , 2005, Applied and Environmental Microbiology.

[26]  Adam Godzik,et al.  Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences , 2006, Bioinform..

[27]  E. Delong,et al.  Unsuspected diversity among marine aerobic anoxygenic phototrophs , 2002, Nature.

[28]  J. Gilbert,et al.  Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic , 2011, The ISME Journal.

[29]  Edward J. Carpenter,et al.  Trichodesmium, a Globally Significant Marine Cyanobacterium , 1997 .

[30]  B. Jenkins,et al.  Spatial-temporal variability in diazotroph assemblages in Chesapeake Bay using an oligonucleotide nifH microarray. , 2007, Environmental microbiology.

[31]  H. Paerl,et al.  Distribution of nitrogen-fixing microorganisms along the Neuse River Estuary, North Carolina , 2001, Microbial Ecology.

[32]  L. Riemann,et al.  Global patterns of diversity and community structure in marine bacterioplankton , 2006, Molecular ecology.

[33]  I. Hewson,et al.  Characteristics of diazotrophs in surface to abyssopelagic waters of the Sargasso Sea , 2007 .

[34]  W. C. Lin,et al.  Geobacter sulfurreducens Can Grow with Oxygen as a Terminal Electron Acceptor , 2004, Applied and Environmental Microbiology.

[35]  G. Asner,et al.  Nitrogen Cycles: Past, Present, and Future , 2004 .

[36]  J. Zehr,et al.  Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii , 1989, Applied and environmental microbiology.

[37]  Joaquín Martínez Martínez,et al.  Spatial Distribution and Transcriptional Activity of an Uncultured Clade of Planktonic Diazotrophic γ-Proteobacteria in the Arabian Sea , 2005, Applied and Environmental Microbiology.

[38]  M. Church,et al.  Nitrogenase genes in PCR and RT-PCR reagents: implications for studies of diversity of functional genes. , 2003, BioTechniques.

[39]  S. Zinder,et al.  Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227 , 1996, Journal of bacteriology.

[40]  J. Pinhassi,et al.  Biogeographic analysis of ribosomal RNA clusters from marine bacterioplankton , 2005 .

[41]  A. Paytan,et al.  Seasonality of N2 fixation and nifH gene diversity in the Gulf of Aqaba (Red Sea) , 2009 .

[42]  U. Göbel,et al.  Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. , 1997, FEMS microbiology reviews.

[43]  Paul G. Falkowski,et al.  Evolution of the nitrogen cycle and its influence on the biological sequestration of CO2 in the ocean , 1997, Nature.

[44]  E. Carpenter,et al.  Extensive bloom of a N2-fixing diatom/cyanobacterial association in the tropical Atlantic Ocean , 1999 .

[45]  L. Riemann,et al.  Nodularia sp. nifH gene transcripts in the Baltic Sea proper , 2007 .

[46]  Angelicque E. White,et al.  Unicellular Cyanobacterial Distributions Broaden the Oceanic N2 Fixation Domain , 2010, Science.

[47]  Jonathan P Zehr,et al.  Nitrogenase gene diversity and microbial community structure: a cross-system comparison. , 2003, Environmental microbiology.

[48]  T. Hurek,et al.  Evaluation of PCR primers for universal nifH gene targeting and for assessment of transcribed nifH pools in roots of Oryza longistaminata with and without low nitrogen input. , 2008, FEMS microbiology ecology.

[49]  J. Zehr,et al.  Nitrogen fixation: Nitrogenase genes and gene expression , 2001 .

[50]  D. Kirchman Microbial ecology of the oceans , 2008 .

[51]  C. Lovejoy,et al.  Water masses and biogeography of picoeukaryote assemblages in a cold hydrographically complex system , 2008 .

[52]  L. Proctor,et al.  Molecular evidence for zooplankton-associated nitrogen-fixing anaerobes based on amplification of the nifH gene , 1999 .

[53]  Susan M. Huse,et al.  Accuracy and quality of massively parallel DNA pyrosequencing , 2007, Genome Biology.

[54]  Yan Sun,et al.  Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) , 2008, BMC Microbiology.

[55]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[56]  Shotaro Ando,et al.  Contamination of diverse nifH and nifH-like DNA into commercial PCR primers. , 2005, FEMS microbiology letters.

[57]  V. Kunin,et al.  Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. , 2009, Environmental microbiology.

[58]  M. Saito,et al.  Regional distributions of nitrogen‐fixing bacteria in the Pacific Ocean , 2008 .

[59]  Andrew Hansen,et al.  Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean , 2001, Nature.

[60]  K. Schleifer,et al.  ARB: a software environment for sequence data. , 2004, Nucleic acids research.

[61]  C. Kurland,et al.  Evolution of microbial genomes: sequence acquisition and loss. , 2002, Molecular biology and evolution.

[62]  Jonathan P. Zehr,et al.  Globally Distributed Uncultivated Oceanic N2-Fixing Cyanobacteria Lack Oxygenic Photosystem II , 2008, Science.

[63]  J. Oelze,et al.  Control of nitrogen fixation by oxygen in purple nonsulfur bacteria , 1996, Archives of Microbiology.

[64]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..