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DOE OSTI2020

Amylases in the Human Vagina

ABSTRACT Dominance of Lactobacillus species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and Lactobacillus in the vagina is complex. IMPORTANCE In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as “common goods” that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

DRAM example narrative

DRAM example narrative DRAM on KBase let's anyone run annotations using DRAM in the cloud. DRAM is an annotation tool that can annotate bacterial, archaeal and viral genomes and distills those annotatios into represetations of the functional genomic potential of those organisms. If you want to read more about DRAM you can check out the GitHub, wiki and journal article. DRAM annotate assemblies In KBase Assembly objects contain nucleotide sequences from genomes or metagenomes. DRAM can predict genes and annotate their function from KBase Assembly objects which may be microbial isolate genomes, metagenome assembled genomes or metagenomes. This is done with the Annotate and Distill Assemblies with DRAM app. This app can also anntoate AssemblySet objects which contain collection of Assembly objects. It also generates a Genome object and a GenomeSet object which can be used for further analysis with other KBase apps. The full annotations and other DRAM files are also available for download in the app.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics

Abstract Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Microbial Taxonomy Run Amok

DNA sequencing has led to an explosion in discovery of microbial phylogenetic novelty, especially that represented by uncultivated taxa, to which the traditional system of prokaryotic taxonomy has not adapted. A lack of expansion of the International Code of Nomenclature of Prokaryotes (ICNP, 'the Code') to effectively capture this information has created a 'wild west' situation where names are published or appear in popular reference databases without further verification or validation. The rapid propagation of variant and questionable naming methods has led to widespread confusion and undermines prior accomplishments. Furthermore, we exemplify inconsistencies that have arisen from this practice and endanger the interoperability of scientific findings. The immediate solution to this problem is to develop and adopt universal best practices that are accepted by expert researchers, major publishers, the International Committee on Systematics of Prokaryotes (ICSP), and international microbiological societies.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Alternative strategies of nutrient acquisition and energy conservation map to the biogeography of marine ammonia-oxidizing archaea

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Ecological divergence of syntopic marine bacterial species is shaped by gene content and expression

Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species’ divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species’ pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Integrated Phage-Host Prediction tool (iPHoP) v1.0.0

iPHoP is a bioinformatic tools that uses a set of approaches to predict the potential host of novel bacteriophages (viruses infecting bacteria) that are only known by their genome sequence, and not cultivated in the laboratory. Existing technologies typically rely on a single method, and the main advantage of iPHoP is its ability to integrate the results from multiple methods into a single prediction. This is of interest for microbial ecology researchers, as they often analyze novel bacteriophage genomes that they were able to assemble from metagenomes, but they don't know which bacteria these phages infect.

Roux, Simon↗

DOE OSTI2026

Untargeted, tandem mass spectrometry (LC/MS-MS) metaproteomes from soil samples in control and warming plots in Blodgett Forest, CA (2014-2021)

The pathways of carbon transport and loss through and from soils—soil organic matter (SOM) depolymerization to dissolved organic carbon and mineralization to carbon dioxide (CO2)—are fundamentally driven by microbial activity, which is strongly regulated by environmental conditions. As part of Lawrence Berkeley National Laboratory (LBNL) Terrestrial Ecosystem Science (TES) Belowground Biogeochemistry Science Focus Area (SFA), we have established a novel whole-soil long-term warming experiment at the University of California (UC) Blodgett Forest Research Station (Sierra Nevada) in 2014, where we study the role of biogeochemical, microbial and geochemical process interactions in SOM decomposition and stabilization. This package contains soil metaproteomics data in the context of site specific metagenomes from soil depth profiles in three paired control and warming plots from a temperate mixed forest in Northern California. Each paired plot had been subjected to experimental warming since June 2014 to simulate a predicted climate change scenario for northern California. These metaproteomes were collected in 2018 after 4.5 years of warming from five depth intervals (0-10 cm, 10-30 cm, 30-45 cm, 45-60 cm, 60-80 cm). For protein identification, the collected spectra were searched following a target-decoy search strategy against a database of metagenome predicted proteins (covering 96 samples from 2014 to 2021) representing the complete sequence diversity at the site. Data was searched with mass spectrometry database search tool (MS-GF+) using Pacific Northwest National Laboratory (PNNL)'s Data Management System (DMS) Processing pipeline. The metagenomes are published as part of another data package. Raw metaproteomic data and the data products from MS-GF+ are deposited in the Mass Spectrometry Interactive Virtual Environment (MassIVE) database under accession no. MSV000097826. Here we present a dataset that includes spectral counts for the detected proteins across samples (EMSL50964_BrodieAllMAGs_Globals_SC.txt), the sequences of the detected proteins, and sample metadata file that contains site information for the soil metaproteome samples.

Belowground Biogeochemistry Science Focus Area↗

DOE OSTI2025

LACSD BNR Pilot Plant Metagenomes

In this study, we aimed to evaluate Biological Nutrient Removal (BNR) and investigate microbial community changes when the DO is reduced in the aerated portions of wastewater treatment trains. We present a dataset of metagenomes obtained from activated sludge collected from the Los Angeles Sanitary District BNR pilot plant at the beginning of operation, when the DO was high, and at the end of operation, when the DO was low.

metagenome↗

DOE OSTI2020

ATLAS: a Snakemake workflow for assembly, annotation, and genomic binning of metagenome sequence data

Background: Metagenomics and metatranscriptomics studies provide valuable insight into the composition and function of microbial populations from diverse environments, however the data processing pipelines that rely on mapping reads to gene catalogs or genome databases for cultured strains yield results that underrepresent the genes and functional potential of uncultured microbes. Recent improvements in sequence assembly methods have eased the reliance on genome databases, thereby allowing the recovery of genomes from uncultured microbes. However, configuring these tools, linking them with advanced binning and annotation tools, and maintaining provenance of the processing continues to be challenging for researchers. Results: Here we present ATLAS, a software package for customizable data processing from raw sequence reads to functional and taxonomic annotations using state-of-the-art tools to assemble, annotate, quantify, and bin metagenome and metatranscriptome data. Genome-centric resolution and abundance estimates are provided for each sample in a dataset. ATLAS is written in Python and the workflow implemented in Snakemake; it operates in a Linux environment, and is compatible with Python 3.5+ and Anaconda 3+ versions. The source code for ATLAS is freely available, distributed under a BSD-3 license. Conclusions: ATLAS provides a user-friendly, modular and customizable Snakemake workflow for metagenome and metatranscriptome data processing; it is easily installable with conda and maintained as open-source on GitHub at https://github.com/metagenome-atlas/atlas.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Targeted assemblies of cas1 suggest CRISPR-Cas’s response to soil warming

Abstract There is an increasing interest in the clustered regularly interspaced short palindromic repeats CRISPR-associated protein (CRISPR-Cas) system to reveal potential virus–host dynamics. The universal and most conserved Cas protein, cas1 is an ideal marker to elucidate CRISPR-Cas ecology. We constructed eight Hidden Markov Models (HMMs) and assembled cas1 directly from metagenomes by a targeted-gene assembler, Xander, to improve detection capacity and resolve the diverse CRISPR-Cas systems. The eight HMMs were first validated by recovering all 17 cas1 subtypes from the simulated metagenome generated from 91 prokaryotic genomes across 11 phyla. We challenged the targeted method with 48 metagenomes from a tallgrass prairie in Central Oklahoma recovering 3394 cas1. Among those, 88 were near full length, 5 times more than in de-novo assemblies from the Oklahoma metagenomes. To validate the host assignment by cas1, the targeted-assembled cas1 was mapped to the de-novo assembled contigs. All the phylum assignments of those mapped contigs were assigned independent of CRISPR-Cas genes on the same contigs and consistent with the host taxonomies predicted by the mapped cas1. We then investigated whether 8 years of soil warming altered cas1 prevalence within the communities. A shift in microbial abundances was observed during the year with the biggest temperature differential (mean 4.16 °C above ambient). cas1 prevalence increased and even in the phyla with decreased microbial abundances over the next 3 years, suggesting increasing virus–host interactions in response to soil warming. This targeted method provides an alternative means to effectively mine cas1 from metagenomes and uncover the host communities.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

A pipeline for targeted metagenomics of environmental bacteria

Background:Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. Methods:Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. Results: Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. Conclusions: With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

From soil to sequence: filling the critical gap in genome-resolved metagenomics is essential to the future of soil microbial ecology

Abstract Soil microbiomes are heterogeneous, complex microbial communities. Metagenomic analysis is generating vast amounts of data, creating immense challenges in sequence assembly and analysis. Although advances in technology have resulted in the ability to easily collect large amounts of sequence data, soil samples containing thousands of unique taxa are often poorly characterized. These challenges reduce the usefulness of genome-resolved metagenomic (GRM) analysis seen in other fields of microbiology, such as the creation of high quality metagenomic assembled genomes and the adoption of genome scale modeling approaches. The absence of these resources restricts the scale of future research, limiting hypothesis generation and the predictive modeling of microbial communities. Creating publicly available databases of soil MAGs, similar to databases produced for other microbiomes, has the potential to transform scientific insights about soil microbiomes without requiring the computational resources and domain expertise for assembly and binning.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Biases in genome reconstruction from metagenomic data

Background Advances in sequencing, assembly, and assortment of contigs into species-specific bins has enabled the reconstruction of genomes from metagenomic data (MAGs). Though a powerful technique, it is difficult to determine whether assembly and binning techniques are accurate when applied to environmental metagenomes due to a lack of complete reference genome sequences against which to check the resulting MAGs. Methods We compared MAGs derived from an enrichment culture containing ~20 organisms to complete genome sequences of 10 organisms isolated from the enrichment culture. Factors commonly considered in binning software—nucleotide composition and sequence repetitiveness—were calculated for both the correctly binned and not-binned regions. This direct comparison revealed biases in sequence characteristics and gene content in the not-binned regions. Additionally, the composition of three public data sets representing MAGs reconstructed from the Tara Oceans metagenomic data was compared to a set of representative genomes available through NCBI RefSeq to verify that the biases identified were observable in more complex data sets and using three contemporary binning software packages. Results Repeat sequences were frequently not binned in the genome reconstruction processes, as were sequence regions with variant nucleotide composition. Genes encoded on the not-binned regions were strongly biased towards ribosomal RNAs, transfer RNAs, mobile element functions and genes of unknown function. Our results support genome reconstruction as a robust process and suggest that reconstructions determined to be >90% complete are likely to effectively represent organismal function; however, population-level genotypic heterogeneity in natural populations, such as uneven distribution of plasmids, can lead to incorrect inferences.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

A method for achieving complete microbial genomes and improving bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity. However, less than 100 circularized bacterial and archaeal genomes have been assembled and published from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a semi-automated method called Jorg to help circularize small bacterial, archaeal, and viral genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. In addition to 34 circular CPR genomes, we present one circular Margulisbacteria genome, one circular Chloroflexi genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg and is available on the DOE Systems Biology KnowledgeBase as a beta app.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Supervised extraction of near-complete genomes from metagenomic samples: A new service in PATRIC

Large amounts of metagenomically-derived data are submitted to PATRIC for analysis. In the future, we expect even more jobs submitted to PATRIC will use metagenomic data. One in-demand use case is the extraction of near-complete draft genomes from assembled contigs of metagenomic origin. The PATRIC metagenome binning service utilizes the PATRIC database to furnish a large, diverse set of reference genomes. We provide a new service for supervised extraction and annotation of high-quality, near-complete genomes from metagenomically-derived contigs. Reference genomes are assigned to putative draft genome bins based on the presence of single-copy universal marker roles in the sample, and contigs are sorted into these bins by their similarity to reference genomes in PATRIC. Each set of binned contigs represents a draft genome that will be annotated by RASTtk in PATRIC. A structured-language binning report is provided containing quality measurements and taxonomic information about the contig bins. The PATRIC metagenome binning service emphasizes extraction of high-quality genomes for downstream analysis using other PATRIC tools and services. Due to its supervised nature, the binning service is not appropriate for mining novel or extremely low-coverage genomes from metagenomic samples.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides.

09 BIOMASS FUELS↗