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1. PPIT: an R package for inferring microbial taxonomy from nifH sequences

   https://doi.org/10.1093/bioinformatics/btab100  

   Kapili, Bennett J ; Dekas, Anne E ( February 2021 , Bioinformatics)

   Birol, Inanc (Ed.)

   Abstract Motivation Linking microbial community members to their ecological functions is a central goal of environmental microbiology. When assigned taxonomy, amplicon sequences of metabolic marker genes can suggest such links, thereby offering an overview of the phylogenetic structure underpinning particular ecosystem functions. However, inferring microbial taxonomy from metabolic marker gene sequences remains a challenge, particularly for the frequently sequenced nitrogen fixation marker gene, nitrogenase reductase (nifH). Horizontal gene transfer in recent nifH evolutionary history can confound taxonomic inferences drawn from the pairwise identity methods used in existing software. Other methods for inferring taxonomy are not standardized and require manual inspection that is difficult to scale. Results We present Phylogenetic Placement for Inferring Taxonomy (PPIT), an R package that infers microbial taxonomy from nifH amplicons using both phylogenetic and sequence identity approaches. After users place query sequences on a reference nifH gene tree provided by PPIT (n = 6317 full-length nifH sequences), PPIT searches the phylogenetic neighborhood of each query sequence and attempts to infer microbial taxonomy. An inference is drawn only if references in the phylogenetic neighborhood are: (1) taxonomically consistent and (2) share sufficient pairwise identity with the query, thereby avoiding erroneous inferences due to known horizontal gene transfer events. We find that PPIT returns a higher proportion of correct taxonomic inferences than BLAST-based approaches at the cost of fewer total inferences. We demonstrate PPIT on deep-sea sediment and find that Deltaproteobacteria are the most abundant potential diazotrophs. Using this dataset we show that emending PPIT inferences based on visual inspection of query sequence placement can achieve taxonomic inferences for nearly all sequences in a query set. We additionally discuss how users can apply PPIT to the analysis of other marker genes. Availability PPIT is freely available to non-commercial users at https://github.com/bkapili/ppit. Installation includes a vignette that demonstrates package use and reproduces the nifH amplicon analysis discussed here. The raw nifH amplicon sequence data have been deposited in the GenBank, EMBL, and DDBJ databases under BioProject number PRJEB37167. Supplementary information Supplementary data are available at Bioinformatics online. 

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2. Seasonal and spatial patterns in diazotroph community composition at Station ALOHA

   https://doi.org/10.3389/fmars.2023.1130158  

   Turk-Kubo, Kendra A. ; Henke, Britt A. ; Gradoville, Mary R. ; Magasin, Jonathan D. ; Church, Matthew J. ; Zehr, Jonathan P. ( May 2023 , Frontiers in Marine Science)

   Dinitrogen (N₂) fixation is carried out by specialized microbes, called diazotrophs, and is a major source of nitrogen supporting primary production in oligotrophic oceans. One of the best-characterized diazotroph habitats is the North Pacific Subtropical Gyre (NPSG), where warm, chronically N-limited surface waters promote year-round N₂fixation. At Station ALOHA (A Long-Term Oligotrophic Habitat Assessment) in the NPSG, N₂fixation is typically ascribed to conspicuous, filamentous cyanobacterial diazotrophs (TrichodesmiumandRichelia), unicellular free-livingCrocosphaera, and the UCYN-A/haptophyte symbiosis, based on using microscopy and quantitative PCR (qPCR). However, the diazotroph community in this ecosystem is diverse and includes non-cyanobacterial diazotrophs (NCDs). We investigated the diversity, depth distributions, and seasonality of diazotroph communities at Stn. ALOHA using high throughput sequencing (HTS) ofnifHgene fragments from samples collected throughout the euphotic zone (0-175 m) at near-monthly intervals from June 2013 to July 2016. The UCYN-A symbioses andTrichodesmiumsp. consistently had the highest relative abundances and seasonal patterns that corroborated qPCR-based analyses. Other prevalent community members included a newCrocosphaera-like species, and several NCDs affiliated with γ- and δ-proteobacteria. Notably, some of the NCDs appear to be stable components of the community at Stn. ALOHA, having also been reported in prior studies. Depth and temporal patterns in microdiversity within two major diazotroph groups (Trichodesmiumand UCYN-A) suggested that sub-populations are adapted to time- and depth-dependent environmental variation. A network analysis of the upper euphotic (0-75 m) HTS data identified two modules that reflect a diazotroph community structure with seasonal turnover between UCYN-A/Gamma A, andTrichodesmium/Crocosphaera. It also reveals the seasonality of several important cyanobacteria and NCDs about which little is known, including a putative δ-proteobacterial phylotype originally discovered at Stn. ALOHA. Collectively, these results underscore the importance of couplingnifHgene HTS with other molecular techniques to obtain a comprehensive view of diazotroph community composition in the marine environment and reveal several understudied diazotroph groups that may contribute to N₂fixation in the NPSG.

    

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3. Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy

   https://doi.org/10.1038/s41396-020-00757-1  

   Metcalfe, Kyle S. ; Murali, Ranjani ; Mullin, Sean W. ; Connon, Stephanie A. ; Orphan, Victoria J. ( October 2020 , The ISME Journal)

   Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

    

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4. Global Grassland Diazotrophic Communities Are Structured by Combined Abiotic, Biotic, and Spatial Distance Factors but Resilient to Fertilization

   https://doi.org/10.3389/fmicb.2022.821030  

   Nepel, Maximilian ; Angel, Roey ; Borer, Elizabeth T. ; Frey, Beat ; MacDougall, Andrew S. ; McCulley, Rebecca L. ; Risch, Anita C. ; Schütz, Martin ; Seabloom, Eric W. ; Woebken, Dagmar ( March 2022 , Frontiers in Microbiology)

   Grassland ecosystems cover around 37% of the ice-free land surface on Earth and have critical socioeconomic importance globally. As in many terrestrial ecosystems, biological dinitrogen (N 2 ) fixation represents an essential natural source of nitrogen (N). The ability to fix atmospheric N 2 is limited to diazotrophs, a diverse guild of bacteria and archaea. To elucidate the abiotic (climatic, edaphic), biotic (vegetation), and spatial factors that govern diazotrophic community composition in global grassland soils, amplicon sequencing of the dinitrogenase reductase gene— nifH —was performed on samples from a replicated standardized nutrient [N, phosphorus (P)] addition experiment in 23 grassland sites spanning four continents. Sites harbored distinct and diverse diazotrophic communities, with most of reads assigned to diazotrophic taxa within the Alphaproteobacteria (e.g., Rhizobiales ), Cyanobacteria (e.g., Nostocales ), and Deltaproteobacteria (e.g., Desulforomonadales ) groups. Likely because of the wide range of climatic and edaphic conditions and spatial distance among sampling sites, only a few of the taxa were present at all sites. The best model describing the variation among soil diazotrophic communities at the OTU level combined climate seasonality (temperature in the wettest quarter and precipitation in the warmest quarter) with edaphic (C:N ratio, soil texture) and vegetation factors (various perennial plant covers). Additionally, spatial variables (geographic distance) correlated with diazotrophic community variation, suggesting an interplay of environmental variables and spatial distance. The diazotrophic communities appeared to be resilient to elevated nutrient levels, as 2–4 years of chronic N and P additions had little effect on the community composition. However, it remains to be seen, whether changes in the community composition occur after exposure to long-term, chronic fertilization regimes. 

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5. Defining the Sphagnum Core Microbiome across the North American Continent Reveals a Central Role for Diazotrophic Methanotrophs in the Nitrogen and Carbon Cycles of Boreal Peatland Ecosystems

   https://doi.org/10.1128/mbio.03714-21  

   Kolton, Max ; Weston, David J. ; Mayali, Xavier ; Weber, Peter K. ; McFarlane, Karis J. ; Pett-Ridge, Jennifer ; Somoza, Mark M. ; Lietard, Jory ; Glass, Jennifer B. ; Lilleskov, Erik A. ; et al ( February 2022 , mBio)

   Martiny, Jennifer B. (Ed.)

   ABSTRACT Peat mosses of the genus Sphagnum are ecosystem engineers that frequently predominate over photosynthetic production in boreal peatlands. Sphagnum spp. host diverse microbial communities capable of nitrogen fixation (diazotrophy) and methane oxidation (methanotrophy), thereby potentially supporting plant growth under severely nutrient-limited conditions. Moreover, diazotrophic methanotrophs represent a possible “missing link” between the carbon and nitrogen cycles, but the functional contributions of the Sphagnum -associated microbiome remain in question. A combination of metagenomics, metatranscriptomics, and dual-isotope incorporation assays was applied to investigate Sphagnum microbiome community composition across the North American continent and provide empirical evidence for diazotrophic methanotrophy in Sphagnum -dominated ecosystems. Remarkably consistent prokaryotic communities were detected in over 250 Sphagnum SSU rRNA libraries from peatlands across the United States (5 states, 17 bog/fen sites, 18 Sphagnum species), with 12 genera of the core microbiome comprising 60% of the relative microbial abundance. Additionally, nitrogenase ( nifH ) and SSU rRNA gene amplicon analysis revealed that nitrogen-fixing populations made up nearly 15% of the prokaryotic communities, predominated by Nostocales cyanobacteria and Rhizobiales methanotrophs. While cyanobacteria comprised the vast majority (>95%) of diazotrophs detected in amplicon and metagenome analyses, obligate methanotrophs of the genus Methyloferula (order Rhizobiales ) accounted for one-quarter of transcribed nifH genes. Furthermore, in dual-isotope tracer experiments, members of the Rhizobiales showed substantial incorporation of 13 CH 4 and 15 N 2 isotopes into their rRNA. Our study characterizes the core Sphagnum microbiome across large spatial scales and indicates that diazotrophic methanotrophs, here defined as obligate methanotrophs of the rare biosphere ( Methyloferula spp. of the Rhizobiales ) that also carry out diazotrophy, play a keystone role in coupling of the carbon and nitrogen cycles in nutrient-poor peatlands. IMPORTANCE Nitrogen availability frequently limits photosynthetic production in Sphagnum moss-dominated high-latitude peatlands, which are crucial carbon-sequestering ecosystems at risk to climate change effects. It has been previously suggested that microbial methane-fueled fixation of atmospheric nitrogen (N 2 ) may occur in these ecosystems, but this process and the organisms involved are largely uncharacterized. A combination of omics (DNA and RNA characterization) and dual-isotope incorporation approaches illuminated the functional diversity of Sphagnum -associated microbiomes and defined 12 bacterial genera in its core microbiome at the continental scale. Moreover, obligate diazotrophic methanotrophs showed high nitrogen fixation gene expression levels and incorporated a substantial amount of atmospheric nitrogen and methane-driven carbon into their biomass. Thus, these results point to a central role for members of the rare biosphere in Sphagnum microbiomes as keystone species that couple nitrogen fixation to methane oxidation in nutrient-poor peatlands. 

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