Whole community shotgun metagenomes of two biological soil crust types from the Mojave Desert.

We present six whole community shotgun metagenomic sequencing data sets of two types of biological soil crusts sampled at the ecotone of the Mojave Desert and Colorado Desert in California. These data will help us understand the diversity and function of biocrust microbial communities, which are essential for desert ecosystems.

Metatranscriptomes of two biological soil crust types from the Mojave desert in response to wetting.

We present eight metatranscriptomic datasets of light algal and cyanolichen biological soil crusts from the Mojave Desert in response to wetting. These data will help us understand gene expression patterns in desert biocrust microbial communities after they have been reactivated by the addition of water.

Terabase-Scale Coassembly of a Tropical Soil Microbiome.

Petabases of environmental metagenomic data are publicly available, presenting an opportunity to characterize complex environments and discover novel lineages of life. Metagenome coassembly, in which many metagenomic samples from an environment are simultaneously analyzed to infer the underlying genomes' sequences, is an essential tool for achieving this goal. We applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 terabases (Tbp) of metagenome data from a tropical soil in the Luquillo Experimental Forest (LEF), Puerto Rico. The resulting coassembly yielded 39 high-quality (>90% complete, <5% contaminated, with predicted 23S, 16S, and 5S rRNA genes and ≥18 tRNAs) metagenome-assembled genomes (MAGs), including two from the candidate phylum Eremiobacterota. Another 268 medium-quality (≥50% complete, <10% contaminated) MAGs were extracted, including the candidate phyla Dependentiae, Dormibacterota, and Methylomirabilota. In total, 307 medium- or higher-quality MAGs were assigned to 23 phyla, compared to 294 MAGs assigned to nine phyla in the same samples individually assembled. The low-quality (<50% complete, <10% contaminated) MAGs from the coassembly revealed a 49% complete rare biosphere microbe from the candidate phylum FCPU426 among other low-abundance microbes, an 81% complete fungal genome from the phylum Ascomycota, and 30 partial eukaryotic MAGs with ≥10% completeness, possibly representing protist lineages. A total of 22,254 viruses, many of them low abundance, were identified. Estimation of metagenome coverage and diversity indicates that we may have characterized ≥87.5% of the sequence diversity in this humid tropical soil and indicates the value of future terabase-scale sequencing and coassembly of complex environments. IMPORTANCE Petabases of reads are being produced by environmental metagenome sequencing. An essential step in analyzing these data is metagenome assembly, the computational reconstruction of genome sequences from microbial communities. "Coassembly" of metagenomic sequence data, in which multiple samples are assembled together, enables more complete detection of microbial genomes in an environment than "multiassembly," in which samples are assembled individually. To demonstrate the potential for coassembling terabases of metagenome data to drive biological discovery, we applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 Tbp of reads from a humid tropical soil environment. The resulting coassembly, its functional annotation, and analysis are presented here. The coassembly yielded more, and phylogenetically more diverse, microbial, eukaryotic, and viral genomes than the multiassembly of the same data. Our resource may facilitate the discovery of novel microbial biology in tropical soils and demonstrates the value of terabase-scale metagenome sequencing.

A standardized quantitative analysis strategy for stable isotope probing metagenomics.

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg, an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, "who is eating what?" and "who is active?" within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism's taxonomic identity and genome composition while providing quantitative estimates of the microorganism's isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

Dataset of 143 metagenome-assembled genomes from the Arctic and Atlantic Oceans, including 21 for eukaryotic organisms.

This article presents metagenome-assembled genomes (MAGs) for both eukaryotic and prokaryotic organisms originating from the Arctic and Atlantic oceans, along with gene prediction and functional annotation for MAGs from both domains. Eleven samples from the chlorophyll-a maximum layer of the surface ocean were collected during two cruises in 2012; six from the Arctic in June-July on ARK-XXVII/1 (PS80), and five from the Atlantic in November on ANT-XXIX/1 (PS81). Sequencing and assembly was carried out by the Joint Genome Institute (JGI), who provide annotation of the assembled sequences, and 122 MAGs for prokaryotic organisms. A subsequent binning process identified 21 MAGs for eukaryotic organisms, mostly identified as Mamiellophyceae or Bacillariophyceae. The data for each MAG includes sequences in FASTA format, and tables of functional annotation of genes. For eukaryotic MAGs, transcript and protein sequences for predicted genes are available. A spreadsheet is provided summarising quality measures and taxonomic classifications for each MAG. These data provide draft genomes for uncultured marine microbes, including some of the first MAGs for polar eukaryotes, and can provide reference genetic data for these environments, or used in genomics-based comparison between environments.

Discovery of a novel filamentous prophage in the genome of the Mimosa pudica microsymbiont Cupriavidus taiwanensis STM 6018.

Integrated virus genomes (prophages) are commonly found in sequenced bacterial genomes but have rarely been described in detail for rhizobial genomes. Cupriavidus taiwanensis STM 6018 is a rhizobial Betaproteobacteria strain that was isolated in 2006 from a root nodule of a Mimosa pudica host in French Guiana, South America. Here we describe features of the genome of STM 6018, focusing on the characterization of two different types of prophages that have been identified in its genome. The draft genome of STM 6018 is 6,553,639 bp, and consists of 80 scaffolds, containing 5,864 protein-coding genes and 61 RNA genes. STM 6018 contains all the nodulation and nitrogen fixation gene clusters common to symbiotic Cupriavidus species; sharing >99.97% bp identity homology to the nod/nif/noeM gene clusters from C. taiwanensis LMG19424T and "Cupriavidus neocalidonicus" STM 6070. The STM 6018 genome contains the genomes of two prophages: one complete Mu-like capsular phage and one filamentous phage, which integrates into a putative dif site. This is the first characterization of a filamentous phage found within the genome of a rhizobial strain. Further examination of sequenced rhizobial genomes identified filamentous prophage sequences in several Beta-rhizobial strains but not in any Alphaproteobacterial rhizobia.

The IMG/M data management and analysis system v.7: content updates and new features.

The Integrated Microbial Genomes & Microbiomes system (IMG/M: https://img.jgi.doe.gov/m/) at the Department of Energy (DOE) Joint Genome Institute (JGI) continues to provide support for users to perform comparative analysis of isolate and single cell genomes, metagenomes, and metatranscriptomes. In addition to datasets produced by the JGI, IMG v.7 also includes datasets imported from public sources such as NCBI Genbank, SRA, and the DOE National Microbiome Data Collaborative (NMDC), or submitted by external users. In the past couple years, we have continued our effort to help the user community by improving the annotation pipeline, upgrading the contents with new reference database versions, and adding new analysis functionalities such as advanced scaffold search, Average Nucleotide Identity (ANI) for high-quality metagenome bins, new cassette search, improved gene neighborhood display, and improvements to metatranscriptome data display and analysis. We also extended the collaboration and integration efforts with other DOE-funded projects such as NMDC and DOE Biology Knowledgebase (KBase).

Sodalis ligni Strain 159R Isolated from an Anaerobic Lignin-Degrading Consortium.

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Mechanistic insights into consumption of the food additive xanthan gum by the human gut microbiota.

Processed foods often include food additives such as xanthan gum, a complex polysaccharide with unique rheological properties, that has established widespread use as a stabilizer and thickening agent. Xanthan gum's chemical structure is distinct from those of host and dietary polysaccharides that are more commonly expected to transit the gastrointestinal tract, and little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other dietary fibre polysaccharides. Here we show that the ability to digest xanthan gum is common in human gut microbiomes from industrialized countries and appears contingent on a single uncultured bacterium in the family Ruminococcaceae. Our data reveal that this primary degrader cleaves the xanthan gum backbone before processing the released oligosaccharides using additional enzymes. Some individuals harbour Bacteroides intestinalis that is incapable of consuming polymeric xanthan gum but grows on oligosaccharide products generated by the Ruminococcaceae. Feeding xanthan gum to germfree mice colonized with a human microbiota containing the uncultured Ruminococcaceae supports the idea that the additive xanthan gum can drive expansion of the primary degrader Ruminococcaceae, along with exogenously introduced B. intestinalis. Our work demonstrates the existence of a potential xanthan gum food chain involving at least two members of different phyla of gut bacteria and provides an initial framework for understanding how widespread consumption of a recently introduced food additive influences human microbiomes.

Metagenome-assembled genomes of phytoplankton microbiomes from the Arctic and Atlantic Oceans.

BACKGROUND: Phytoplankton communities significantly contribute to global biogeochemical cycles of elements and underpin marine food webs. Although their uncultured genomic diversity has been estimated by planetary-scale metagenome sequencing and subsequent reconstruction of metagenome-assembled genomes (MAGs), this approach has yet to be applied for complex phytoplankton microbiomes from polar and non-polar oceans consisting of microbial eukaryotes and their associated prokaryotes. RESULTS: Here, we have assembled MAGs from chlorophyll a maximum layers in the surface of the Arctic and Atlantic Oceans enriched for species associations (microbiomes) with a focus on pico- and nanophytoplankton and their associated heterotrophic prokaryotes. From 679 Gbp and estimated 50 million genes in total, we recovered 143 MAGs of medium to high quality. Although there was a strict demarcation between Arctic and Atlantic MAGs, adjacent sampling stations in each ocean had 51-88% MAGs in common with most species associations between Prasinophytes and Proteobacteria. Phylogenetic placement revealed eukaryotic MAGs to be more diverse in the Arctic whereas prokaryotic MAGs were more diverse in the Atlantic Ocean. Approximately 70% of protein families were shared between Arctic and Atlantic MAGs for both prokaryotes and eukaryotes. However, eukaryotic MAGs had more protein families unique to the Arctic whereas prokaryotic MAGs had more families unique to the Atlantic. CONCLUSION: Our study provides a genomic context to complex phytoplankton microbiomes to reveal that their community structure was likely driven by significant differences in environmental conditions between the polar Arctic and warm surface waters of the tropical and subtropical Atlantic Ocean. Video Abstract.

Diverse events have transferred genes for edible seaweed digestion from marine to human gut bacteria.

Humans harbor numerous species of colonic bacteria that digest fiber polysaccharides in commonly consumed terrestrial plants. More recently in history, regional populations have consumed edible macroalgae seaweeds containing unique polysaccharides. It remains unclear how extensively gut bacteria have adapted to digest these nutrients. Here, we show that the ability of gut bacteria to digest seaweed polysaccharides is more pervasive than previously appreciated. Enrichment-cultured Bacteroides harbor previously discovered genes for seaweed degradation, which have mobilized into several members of this genus. Additionally, other examples of marine bacteria-derived genes, and their mobile DNA elements, are involved in gut microbial degradation of seaweed polysaccharides, including genes in gut-resident Firmicutes. Collectively, these results uncover multiple separate events that have mobilized the genes encoding seaweed-degrading-enzymes into gut bacteria. This work further underscores the metabolic plasticity of the human gut microbiome and global exchange of genes in the context of dietary selective pressures.

Expanding the genomic encyclopedia of Actinobacteria with 824 isolate reference genomes.

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%-50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences.

The biogeographic differentiation of algal microbiomes in the upper ocean from pole to pole.

Eukaryotic phytoplankton are responsible for at least 20% of annual global carbon fixation. Their diversity and activity are shaped by interactions with prokaryotes as part of complex microbiomes. Although differences in their local species diversity have been estimated, we still have a limited understanding of environmental conditions responsible for compositional differences between local species communities on a large scale from pole to pole. Here, we show, based on pole-to-pole phytoplankton metatranscriptomes and microbial rDNA sequencing, that environmental differences between polar and non-polar upper oceans most strongly impact the large-scale spatial pattern of biodiversity and gene activity in algal microbiomes. The geographic differentiation of co-occurring microbes in algal microbiomes can be well explained by the latitudinal temperature gradient and associated break points in their beta diversity, with an average breakpoint at 14 °C ± 4.3, separating cold and warm upper oceans. As global warming impacts upper ocean temperatures, we project that break points of beta diversity move markedly pole-wards. Hence, abrupt regime shifts in algal microbiomes could be caused by anthropogenic climate change.

Metagenome Sequencing to Explore Phylogenomics of Terrestrial Cyanobacteria.

Cyanobacteria are ubiquitous microorganisms with crucial ecosystem functions, yet most knowledge of their biology relates to aquatic taxa. We have constructed metagenomes for 50 taxonomically well-characterized terrestrial cyanobacterial cultures. These data will support phylogenomic studies of evolutionary relationships and gene content among these unique algae and their aquatic relatives.

DOE JGI Metagenome Workflow.

The DOE Joint Genome Institute (JGI) Metagenome Workflow performs metagenome data processing, including assembly; structural, functional, and taxonomic annotation; and binning of metagenomic data sets that are subsequently included into the Integrated Microbial Genomes and Microbiomes (IMG/M) (I.-M. A. Chen, K. Chu, K. Palaniappan, A. Ratner, et al., Nucleic Acids Res, 49:D751-D763, 2021, https://doi.org/10.1093/nar/gkaa939) comparative analysis system and provided for download via the JGI data portal (https://genome.jgi.doe.gov/portal/). This workflow scales to run on thousands of metagenome samples per year, which can vary by the complexity of microbial communities and sequencing depth. Here, we describe the different tools, databases, and parameters used at different steps of the workflow to help with the interpretation of metagenome data available in IMG and to enable researchers to apply this workflow to their own data. We use 20 publicly available sediment metagenomes to illustrate the computing requirements for the different steps and highlight the typical results of data processing. The workflow modules for read filtering and metagenome assembly are available as a workflow description language (WDL) file (https://code.jgi.doe.gov/BFoster/jgi_meta_wdl). The workflow modules for annotation and binning are provided as a service to the user community at https://img.jgi.doe.gov/submit and require filling out the project and associated metadata descriptions in the Genomes OnLine Database (GOLD) (S. Mukherjee, D. Stamatis, J. Bertsch, G. Ovchinnikova, et al., Nucleic Acids Res, 49:D723-D733, 2021, https://doi.org/10.1093/nar/gkaa983).IMPORTANCE The DOE JGI Metagenome Workflow is designed for processing metagenomic data sets starting from Illumina fastq files. It performs data preprocessing, error correction, assembly, structural and functional annotation, and binning. The results of processing are provided in several standard formats, such as fasta and gff, and can be used for subsequent integration into the Integrated Microbial Genomes and Microbiomes (IMG/M) system where they can be compared to a comprehensive set of publicly available metagenomes. As of 30 July 2020, 7,155 JGI metagenomes have been processed by the DOE JGI Metagenome Workflow. Here, we present a metagenome workflow developed at the JGI that generates rich data in standard formats and has been optimized for downstream analyses ranging from assessment of the functional and taxonomic composition of microbial communities to genome-resolved metagenomics and the identification and characterization of novel taxa. This workflow is currently being used to analyze thousands of metagenomic data sets in a consistent and standardized manner.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing "Candidatus Thermoflexus japonica," "Candidatus Thermoflexus tengchongensis," and "Candidatus Thermoflexus sinensis." Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Challenges in Bioinformatics Workflows for Processing Microbiome Omics Data at Scale.

The nascent field of microbiome science is transitioning from a descriptive approach of cataloging taxa and functions present in an environment to applying multi-omics methods to investigate microbiome dynamics and function. A large number of new tools and algorithms have been designed and used for very specific purposes on samples collected by individual investigators or groups. While these developments have been quite instructive, the ability to compare microbiome data generated by many groups of researchers is impeded by the lack of standardized application of bioinformatics methods. Additionally, there are few examples of broad bioinformatics workflows that can process metagenome, metatranscriptome, metaproteome and metabolomic data at scale, and no central hub that allows processing, or provides varied omics data that are findable, accessible, interoperable and reusable (FAIR). Here, we review some of the challenges that exist in analyzing omics data within the microbiome research sphere, and provide context on how the National Microbiome Data Collaborative has adopted a standardized and open access approach to address such challenges.

The IMG/M data management and analysis system v.6.0: new tools and advanced capabilities.

The Integrated Microbial Genomes & Microbiomes system (IMG/M: https://img.jgi.doe.gov/m/) contains annotated isolate genome and metagenome datasets sequenced at the DOE's Joint Genome Institute (JGI), submitted by external users, or imported from public sources such as NCBI. IMG v 6.0 includes advanced search functions and a new tool for statistical analysis of mixed sets of genomes and metagenome bins. The new IMG web user interface also has a new Help page with additional documentation and webinar tutorials to help users better understand how to use various IMG functions and tools for their research. New datasets have been processed with the prokaryotic annotation pipeline v.5, which includes extended protein family assignments.