Multiomics in the central Arctic Ocean for benchmarking biodiversity change.

Multiomics approaches need to be applied in the central Arctic Ocean to benchmark biodiversity change and to identify novel species and their genes. As part of MOSAiC, EcoOmics will therefore be essential for conservation and sustainable bioprospecting in one of the least explored ecosystems on Earth.

Twenty-five years of Genomes OnLine Database (GOLD): data updates and new features in v.9.

The Genomes OnLine Database (GOLD) (https://gold.jgi.doe.gov/) at the Department of Energy Joint Genome Institute (DOE-JGI) continues to maintain its role as one of the flagship genomic metadata repositories of the world. The ever-increasing number of projects and metadata are freely available to the user community world-wide. GOLD's metadata is consumed by scientists and remains an important source for large-scale comparative genomics analysis initiatives. Encouraged by this active user engagement and growth, GOLD has continued to add new components and capabilities. The new features such as a public Application Programming Interface (API) and Ecosystem landing page as well as the growth of different entities in this current GOLD v.9 edition are described in detail in this manuscript.

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).

IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata.

Viruses are widely recognized as critical members of all microbiomes. Metagenomics enables large-scale exploration of the global virosphere, progressively revealing the extensive genomic diversity of viruses on Earth and highlighting the myriad of ways by which viruses impact biological processes. IMG/VR provides access to the largest collection of viral sequences obtained from (meta)genomes, along with functional annotation and rich metadata. A web interface enables users to efficiently browse and search viruses based on genome features and/or sequence similarity. Here, we present the fourth version of IMG/VR, composed of >15 million virus genomes and genome fragments, a ≈6-fold increase in size compared to the previous version. These clustered into 8.7 million viral operational taxonomic units, including 231 408 with at least one high-quality representative. Viral sequences in IMG/VR are now systematically identified from genomes, metagenomes, and metatranscriptomes using a new detection approach (geNomad), and IMG standard annotation are complemented with genome quality estimation using CheckV, taxonomic classification reflecting the latest taxonomic standards, and microbial host taxonomy prediction. IMG/VR v4 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR.

Genomes OnLine Database (GOLD) v.8: overview and updates.

The Genomes OnLine Database (GOLD) (https://gold.jgi.doe.gov/) is a manually curated, daily updated collection of genome projects and their metadata accumulated from around the world. The current version of the database includes over 1.17 million entries organized broadly into Studies (45 770), Organisms (387 382) or Biosamples (101 207), Sequencing Projects (355 364) and Analysis Projects (283 481). These four levels contain over 600 metadata fields, which includes 76 controlled vocabulary (CV) tables containing 3873 terms. GOLD provides an interactive web user interface for browsing and searching by a wide range of project and metadata fields. Users can enter details about their own projects in GOLD, which acts as a gatekeeper to ensure that metadata is accurately documented before submitting sequence information to the Integrated Microbial Genomes (IMG) system for analysis. In order to maintain a reference dataset for use by members of the scientific community, GOLD also imports projects from public repositories such as GenBank and SRA. The current status of the database, along with recent updates and improvements are described in this manuscript.

Genomes OnLine database (GOLD) v.7: updates and new features.

The Genomes Online Database (GOLD) (https://gold.jgi.doe.gov) is an open online resource, which maintains an up-to-date catalog of genome and metagenome projects in the context of a comprehensive list of associated metadata. Information in GOLD is organized into four levels: Study, Biosample/Organism, Sequencing Project and Analysis Project. Currently GOLD hosts information on 33 415 Studies, 49 826 Biosamples, 313 324 Organisms, 215 881 Sequencing Projects and 174 454 Analysis Projects with a total of 541 metadata fields, of which 80 are based on controlled vocabulary (CV) terms. GOLD provides a user-friendly web interface to browse sequencing projects and launch advanced search tools across four classification levels. Users submit metadata on a wide range of Sequencing and Analysis Projects in GOLD before depositing sequence data to the Integrated Microbial Genomes (IMG) system for analysis. GOLD conforms with and supports the rules set by the Genomic Standards Consortium (GSC) Minimum Information standards. The current version of GOLD (v.7) has seen the number of projects and associated metadata increase exponentially over the years. This paper provides an update on the current status of GOLD and highlights the new features added over the last two years.

Genomes OnLine Database (GOLD) v.6: data updates and feature enhancements.

The Genomes Online Database (GOLD) (https://gold.jgi.doe.gov) is a manually curated data management system that catalogs sequencing projects with associated metadata from around the world. In the current version of GOLD (v.6), all projects are organized based on a four level classification system in the form of a Study, Organism (for isolates) or Biosample (for environmental samples), Sequencing Project and Analysis Project. Currently, GOLD provides information for 26 117 Studies, 239 100 Organisms, 15 887 Biosamples, 97 212 Sequencing Projects and 78 579 Analysis Projects. These are integrated with over 312 metadata fields from which 58 are controlled vocabularies with 2067 terms. The web interface facilitates submission of a diverse range of Sequencing Projects (such as isolate genome, single-cell genome, metagenome, metatranscriptome) and complex Analysis Projects (such as genome from metagenome, or combined assembly from multiple Sequencing Projects). GOLD provides a seamless interface with the Integrated Microbial Genomes (IMG) system and supports and promotes the Genomic Standards Consortium (GSC) Minimum Information standards. This paper describes the data updates and additional features added during the last two years.

Loss of virulence in NAD(P)H cytochrome b5 oxidoreductase deficient Leishmania major.

Leishmania promastigotes have the ability to synthesize essential polyunsaturated fatty acids de novo and can grow in lipid free media. Recently, we have shown that NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) enzyme in Leishmania acts as the redox partner for Δ12 fatty acid desaturase, which catalyses the conversion of oleate to linoleate. So far, the exact role of Leishmania derived linoleate synthesis is still incomplete in the literature. The viability assay by flow cytometry as well as microscopic studies suggests that linoleate is an absolute requirement for Leishmania promastigote survival in delipidated media. Western blot analysis suggested that infection with log phase linoleate deficient mutant (KO) results in increased level of NF-κBp65, IκB and IKKß phosphorylation in RAW264.7 cells. Similarly, the log phase KO infected RAW264.7 cells show dramatic increment of COX-2 expression and TNF-α secretion, compared to control or Ncb5or complement (CM) cell lines. The activation of inflammatory signaling pathways by KO mutant is significantly reduced when the RAW264.7 cells are pre-treated with BSA bound linoleate. Together, these findings confirmed that the leishmanial linoleate inhibits both COX-2 and TNF-α expression in macrophage via the inactivation of NF-κB signaling pathway. The stationary phase of KO promastigotes shows avirulence after infection in macrophages as well as inoculation into BALB/c mice; whereas CM cell lines show virulence. Collectively, these data provide strong evidence that de novo linoleate synthesis in Leishmania is an essential for parasite survival at extracellular promastigote stage as well as intracellular amastigote stage.

Microbial species delineation using whole genome sequences.

Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.

Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O2-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O2-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O2 tensions.

The ferrous-dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: the role of proximal histidine on its stability.

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O2 dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790-16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O2 are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25°C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O2 at a rate of ~10s(-1). To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate=6.1s(-1)). Decomposition of the oxygenated intermediate is independent of O2 concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.

The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections.

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is "to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind." Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.

Metatranscriptomes of California grassland soil microbial communities in response to rewetting.

When very dry soil is rewet, rapid stimulation of microbial activity has important implications for ecosystem biogeochemistry, yet associated changes in microbial transcription are poorly known. Here, we present metatranscriptomes of California annual grassland soil microbial communities, collected over 1 week from soils rewet after a summer drought-providing a time series of short-term transcriptional response during rewetting.

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.

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.

A Practical Approach to Using the Genomic Standards Consortium MIxS Reporting Standard for Comparative Genomics and Metagenomics.

Comparative analysis of (meta)genomes necessitates aggregation, integration, and synthesis of well-annotated data using standards. The Genomic Standards Consortium (GSC) collaborates with the research community to develop and maintain the Minimum Information about any (x) Sequence (MIxS) reporting standard for genomic data. To facilitate the use of the GSC's MIxS reporting standard, we provide a description of the structure and terminology, how to navigate ontologies for required terms in MIxS, and demonstrate practical usage through a soil metagenome example.

NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death.

NAD(P)H cytochrome b(5) oxidoreductase (Ncb5or), comprising cytochrome b(5) and cytochrome b(5) reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b(5) oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O(2) uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H(2)O(2), indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania.

Standardized naming of microbiome samples in Genomes OnLine Database.

The power of next-generation sequencing has resulted in an explosive growth in the number of projects aiming to understand the metagenomic diversity of complex microbial environments. The interdisciplinary nature of this microbiome research community, along with the absence of reporting standards for microbiome data and samples, poses a significant challenge for follow-up studies. Commonly used names of metagenomes and metatranscriptomes in public databases currently lack the essential information necessary to accurately describe and classify the underlying samples, which makes a comparative analysis difficult to conduct and often results in misclassified sequences in data repositories. The Genomes OnLine Database (GOLD) (https:// gold.jgi.doe.gov/) at the Department of Energy Joint Genome Institute has been at the forefront of addressing this challenge by developing a standardized nomenclature system for naming microbiome samples. GOLD, currently in its twenty-fifth anniversary, continues to enrich the research community with hundreds of thousands of metagenomes and metatranscriptomes with well-curated and easy-to-understand names. Through this manuscript, we describe the overall naming process that can be easily adopted by researchers worldwide. Additionally, we propose the use of this naming system as a best practice for the scientific community to facilitate better interoperability and reusability of microbiome data.

Plant microbiomes harbor potential to promote nutrient turnover in impoverished substrates of a Brazilian biodiversity hotspot.

The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

Draft Metagenome Sequences of the Sphagnum (Peat Moss) Microbiome from Ambient and Warmed Environments across Europe.

We present 49 metagenome assemblies of the microbiome associated with Sphagnum (peat moss) collected from ambient, artificially warmed, and geothermally warmed conditions across Europe. These data will enable further research regarding the impact of climate change on plant-microbe symbiosis, ecology, and ecosystem functioning of northern peatland ecosystems.