RefSeq and the prokaryotic genome annotation pipeline in the age of metagenomes.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains over 315 000 bacterial and archaeal genomes and 236 million proteins with up-to-date and consistent annotation. In the past 3 years, we have expanded the diversity of the RefSeq collection by including the best quality metagenome-assembled genomes (MAGs) submitted to INSDC (DDBJ, ENA and GenBank), while maintaining its quality by adding validation checks. Assemblies are now more stringently evaluated for contamination and for completeness of annotation prior to acceptance into RefSeq. MAGs now account for over 17000 assemblies in RefSeq, split over 165 orders and 362 families. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP), which is used to annotate nearly all RefSeq assemblies include better detection of protein-coding genes. Nearly 83% of RefSeq proteins are now named by a curated Protein Family Model, a 4.7% increase in the past three years ago. In addition to literature citations, Enzyme Commission numbers, and gene symbols, Gene Ontology terms are now assigned to 48% of RefSeq proteins, allowing for easier multi-genome comparison. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/. PGAP is available as a stand-alone tool able to produce GenBank-ready files at https://github.com/ncbi/pgap.

Environment and taxonomy shape the genomic signature of prokaryotic extremophiles.

This study provides comprehensive quantitative evidence suggesting that adaptations to extreme temperatures and pH imprint a discernible environmental component in the genomic signature of microbial extremophiles. Both supervised and unsupervised machine learning algorithms were used to analyze genomic signatures, each computed as the k-mer frequency vector of a 500 kbp DNA fragment arbitrarily selected to represent a genome. Computational experiments classified/clustered genomic signatures extracted from a curated dataset of [Formula: see text] extremophile (temperature, pH) bacteria and archaea genomes, at multiple scales of analysis, [Formula: see text]. The supervised learning resulted in high accuracies for taxonomic classifications at [Formula: see text], and medium to medium-high accuracies for environment category classifications of the same datasets at [Formula: see text]. For [Formula: see text], our findings were largely consistent with amino acid compositional biases and codon usage patterns in coding regions, previously attributed to extreme environment adaptations. The unsupervised learning of unlabelled sequences identified several exemplars of hyperthermophilic organisms with large similarities in their genomic signatures, in spite of belonging to different domains in the Tree of Life.

Prediction of DNA-Binding Transcription Factors in Bacteria and Archaea Genomes.

DNA-binding transcription factors (TFs) play a central role in the gene expression of all organisms, from viruses to humans, including bacteria and archaea. The role of these proteins is the fate of gene expression in the context of environmental challenges. Because thousands of genomes have been sequenced to date, predictions of the encoded proteins are validated through the use of bioinformatics tools to obtain the necessary experimental, posterior knowledge. In this chapter, we describe three approaches to identify TFs in protein sequences. The first approach integrates the results of sequence comparisons and PFAM assignments, using as reference a manually curated collection of TFs. The second approach considers the prediction of DNA-binding structures, such as the classical helix-turn-helix (HTH); and the third approach considers a deep learning model. We suggest that all approaches must be considered together to increase the possibility of identifying new TFs in bacterial and archaeal genomes.

Recovery of Lutacidiplasmatales archaeal order genomes suggests convergent evolution in Thermoplasmatota.

The Terrestrial Miscellaneous Euryarchaeota Group has been identified in various environments, and the single genome investigated thus far suggests that these archaea are anaerobic sulfite reducers. We assemble 35 new genomes from this group that, based on genome analysis, appear to possess aerobic and facultative anaerobic lifestyles and may oxidise rather than reduce sulfite. We propose naming this order (representing 16 genera) "Lutacidiplasmatales" due to their occurrence in various acidic environments and placement within the phylum Thermoplasmatota. Phylum-level analysis reveals that Thermoplasmatota evolution had been punctuated by several periods of high levels of novel gene family acquisition. Several essential metabolisms, such as aerobic respiration and acid tolerance, were likely acquired independently by divergent lineages through convergent evolution rather than inherited from a common ancestor. Ultimately, this study describes the terrestrially prevalent Lutacidiciplasmatales and highlights convergent evolution as an important driving force in the evolution of archaeal lineages.

The Ancient Origins of Death Domains Support the 'Original Sin' Hypothesis for the Evolution of Programmed Cell Death.

The discovery of caspase homologs in bacteria highlighted the relationship between programmed cell death (PCD) evolution and eukaryogenesis. However, the origin of PCD genes in prokaryotes themselves (bacteria and archaea) is poorly understood and a source of controversy. Whether archaea also contain C14 peptidase enzymes and other death domains is largely unknown because of a historical dearth of genomic data. Archaeal genomic databases have grown significantly in the last decade, which allowed us to perform a detailed comparative study of the evolutionary histories of PCD-related death domains in major archaeal phyla, including the deepest branching phyla of Candidatus Aenigmarchaeota, Candidatus Woesearchaeota, and Euryarchaeota. We identified death domains associated with executioners of PCD, like the caspase homologs of the C14 peptidase family, in 321 archaea sequences. Of these, 15.58% were metacaspase type I orthologues and 84.42% were orthocaspases. Maximum likelihood phylogenetic analyses revealed a scattered distribution of orthocaspases and metacaspases in deep-branching bacteria and archaea. The tree topology was incongruent with the prokaryote 16S phylogeny suggesting a common ancestry of PCD genes in prokaryotes and subsequent massive horizontal gene transfer coinciding with the divergence of archaea and bacteria. Previous arguments for the origin of PCD were philosophical in nature with two popular propositions being the "addiction" and 'original sin' hypotheses. Our data support the 'original sin' hypothesis, which argues for a pleiotropic origin of the PCD toolkit with pro-life and pro-death functions tracing back to the emergence of cellular life-the Last Universal Common Ancestor State.

Prokaryotic Genome Annotation.

In the last decade, the high-throughput and relatively low cost of short-read sequencing technologies have revolutionized prokaryotic genomics. This has led to an exponential increase in the number of bacterial and archaeal genome sequences available, as well as corresponding increase of genome assembly and annotation tools developed. Together, these hardware and software technologies have given scientists unprecedented options to study their chosen microbial systems without the need for large teams of bioinformaticists or supercomputing facilities. While these analysis tools largely fall into only a few categories, each may have different requirements, caveats and file formats, and some may be rarely updated or even abandoned. And so, despite the apparent ease in sequencing and analyzing a prokaryotic genome, it is no wonder that the budding genomicist may quickly find oneself overwhelmed. Here, we aim to provide the reader with an overview of genome annotation and its most important considerations, as well as an easy-to-follow protocol to get started with annotating a prokaryotic genome.

A catalogue of 1,167 genomes from the human gut archaeome.

The human gut microbiome plays an important role in health, but its archaeal diversity remains largely unexplored. In the present study, we report the analysis of 1,167 nonredundant archaeal genomes (608 high-quality genomes) recovered from human gastrointestinal tract, sampled across 24 countries and rural and urban populations. We identified previously undescribed taxa including 3 genera, 15 species and 52 strains. Based on distinct genomic features, we justify the split of the Methanobrevibacter smithii clade into two separate species, with one represented by the previously undescribed 'Candidatus Methanobrevibacter intestini'. Patterns derived from 28,581 protein clusters showed significant associations with sociodemographic characteristics such as age groups and lifestyle. We additionally show that archaea are characterized by specific genomic and functional adaptations to the host and carry a complex virome. Our work expands our current understanding of the human archaeome and provides a large genome catalogue for future analyses to decipher its impact on human physiology.

Evaluating de Novo Assembly and Binning Strategies for Time Series Drinking Water Metagenomes.

Reconstructing microbial genomes from metagenomic short-read data can be challenging due to the unknown and uneven complexity of microbial communities. This complexity encompasses highly diverse populations, which often includes strain variants. Reconstructing high-quality genomes is a crucial part of the metagenomic workflow, as subsequent ecological and metabolic inferences depend on their accuracy, quality, and completeness. In contrast to microbial communities in other ecosystems, there has been no systematic assessment of genome-centric metagenomic workflows for drinking water microbiomes. In this study, we assessed the performance of a combination of assembly and binning strategies for time series drinking water metagenomes that were collected over 6 months. The goal of this study was to identify the combination of assembly and binning approaches that result in high-quality and -quantity metagenome-assembled genomes (MAGs), representing most of the sequenced metagenome. Our findings suggest that the metaSPAdes coassembly strategies had the best performance, as they resulted in larger and less fragmented assemblies, with at least 85% of the sequence data mapping to contigs greater than 1 kbp. Furthermore, a combination of metaSPAdes coassembly strategies and MetaBAT2 produced the highest number of medium-quality MAGs while capturing at least 70% of the metagenomes based on read recruitment. Utilizing different assembly/binning approaches also assists in the reconstruction of unique MAGs from closely related species that would have otherwise collapsed into a single MAG using a single workflow. Overall, our study suggests that leveraging multiple binning approaches with different metaSPAdes coassembly strategies may be required to maximize the recovery of good-quality MAGs. IMPORTANCE Drinking water contains phylogenetic diverse groups of bacteria, archaea, and eukarya that affect the esthetic quality of water, water infrastructure, and public health. Taxonomic, metabolic, and ecological inferences of the drinking water microbiome depend on the accuracy, quality, and completeness of genomes that are reconstructed through the application of genome-resolved metagenomics. Using time series metagenomic data, we present reproducible genome-centric metagenomic workflows that result in high-quality and -quantity genomes, which more accurately signifies the sequenced drinking water microbiome. These genome-centric metagenomic workflows will allow for improved taxonomic and functional potential analysis that offers enhanced insights into the stability and dynamics of drinking water microbial communities.

DiSCo: a sequence-based type-specific predictor of Dsr-dependent dissimilatory sulphur metabolism in microbial data.

Current methods in comparative genomic analyses for metabolic potential prediction of proteins involved in, or associated with the Dsr (dissimilatory sulphite reductase)-dependent dissimilatory sulphur metabolism are both time-intensive and computationally challenging, especially when considering metagenomic data. We developed DiSCo, a Dsr-dependent dissimilatory sulphur metabolism classification tool, which automatically identifies and classifies the protein type from sequence data. It takes user-supplied protein sequences and lists the identified proteins and their classification in terms of protein family and predicted type. It can also extract the sequence data from user-input to serve as basis for additional downstream analyses. DiSCo provides the metabolic functional prediction of proteins involved in Dsr-dependent dissimilatory sulphur metabolism with high levels of accuracy in a fast manner. We ran DiSCo against a dataset composed of over 190 thousand (meta)genomic records and efficiently mapped Dsr-dependent dissimilatory sulphur proteins in 1798 lineages across both prokaryotic domains. This allowed the identification of new micro-organisms belonging to Thaumarchaeota and Spirochaetes lineages with the metabolic potential to use the Dsr-pathway for energy conservation. DiSCo is implemented in Perl 5 and freely available under the GNU GPLv3 at https://github.com/Genome-Evolution-and-Ecology-Group-GEEG/DiSCo.

Identical sequences found in distant genomes reveal frequent horizontal transfer across the bacterial domain.

Horizontal gene transfer (HGT) is an essential force in microbial evolution. Despite detailed studies on a variety of systems, a global picture of HGT in the microbial world is still missing. Here, we exploit that HGT creates long identical DNA sequences in the genomes of distant species, which can be found efficiently using alignment-free methods. Our pairwise analysis of 93,481 bacterial genomes identified 138,273 HGT events. We developed a model to explain their statistical properties as well as estimate the transfer rate between pairs of taxa. This reveals that long-distance HGT is frequent: our results indicate that HGT between species from different phyla has occurred in at least 8% of the species. Finally, our results confirm that the function of sequences strongly impacts their transfer rate, which varies by more than three orders of magnitude between different functional categories. Overall, we provide a comprehensive view of HGT, illuminating a fundamental process driving bacterial evolution.

Brockarchaeota, a novel archaeal phylum with unique and versatile carbon cycling pathways.

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, 'Brockarchaeota', named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

Two Archaeal Metagenome-Assembled Genomes from El Tatio Provide New Insights into the Crenarchaeota Phylum.

A phylogenomic and functional analysis of the first two Crenarchaeota MAGs belonging to El Tatio geysers fields in Chile is reported. A soil sample contiguous to a geothermal activity exposed lagoon of El Tatio was used for shotgun sequencing. Afterwards, contigs were binned into individual population-specific genomes data. A phylogenetic placement was carried out for both MAG 9-5TAT and MAG 47-5TAT. Then functional comparisons and metabolic reconstruction were carried out. Results showed that both MAG 9-5TAT and MAG 47-5TAT likely represent new species in the genus Thermoproteus and the genus Sulfolobus, respectively. These findings provide new insights into the phylogenetic and genomic diversity for archaea species that inhabit the El Tatio geysers field and expand the understanding of the Crenarchaeota phylum diversity.

Natronorubrum halalkaliphilum sp. nov., a haloalkaliphilic archaeon isolated from soda lake in Inner Mongolia Autonomous Region, China.

A haloalkaliphilic strain JWXQ-INN-674T was isolated from the water sample of a soda lake in Inner Mongolia Autonomous Region, China. Cells of the strain were coccoid, motile, and strictly aerobic. The strain was able to grow in presence of 2.6-5.4 M NaCl (optimum concentration is 3.4 M) at 30-50 °C (optimum temperature is 42 °C) and pH 7-9.5 (optimum pH is 9.0). The 16S rRNA gene sequence of strain JWXQ-INN-674T showed 95.3-96.6% similarity to members of the genus Natronorubrum of the family Natrialbaceae. The whole genome sequencing of strain JWXQ-INN-674T revealed a genome size of 4.56 M bp and a DNA G + C content of 62.5 mol%. Genome relatedness of strain JWXQ-INN-674T and other species in the genus Natronorubrum was analyzed by average nucleotide identity and digital DNA-DNA hybridization with the values of 76.8-90.6 and 23.1-39.3%, respectively. The strain possessed the polar lipids phosphatidylglycerol and methylated phosphatidylglycerol phosphate lipid. No glycolipids were detected. Based on phylogenetic analysis, phenotypic characteristics, chemotaxonomic properties and genome relatedness, the isolate was proposed as the type strain of a novel species of genus Natronorubrum, Natronorubrum halalkaliphilum sp. nov. (type strain JWXQ-INN-674T = CGMCC 1.17283T = JCM 34245T).

Complex Evolution of the Mismatch Repair System in Eukaryotes is Illuminated by Novel Archaeal Genomes.

Repairing DNA damage is one of the most important functions of the 'housekeeping' proteins, as DNA molecules are constantly subject to different kinds of damage. An important mechanism of DNA repair is the mismatch repair system (MMR). In eukaryotes, it is more complex than it is in bacteria or Archaea due to an inflated number of paralogues produced as a result of an extensive process of gene duplication and further specialization upon the evolution of the first eukaryotes, including an important part of the meiotic machinery. Recently, the discovery and sequencing of Asgard Archaea allowed us to revisit the MMR system evolution with the addition of new data from a group that is closely related to the eukaryotic ancestor. This new analysis provided evidence for a complex evolutionary history of eukaryotic MMR: an archaeal origin for the nuclear MMR system in eukaryotes, with subsequent acquisitions of other MMR systems from organelles.

Investigation into archaeal extremophilic lifestyles through comparative proteogenomic analysis.

Archaea are a group of primary life forms on Earth and could thrive in many unique environments. Their successful colonization of extreme niches requires corresponding adaptations at proteogenomic level in order to maintain stable cellular structures and active physiological functions. Although some studies have already investigated the extremophilic lifestyles of archaeal species based on genomic features and protein structures, there is a lack of comparative proteogenomic analysis in a large scale. In this study, we explored 686 high-quality archaeal genomes (proteomes) sourced from the Pathosystems Resource Integration Center (PATRIC) database. General patterns of genomic features such as genome size, coding capacity (coding genes and non-coding regions), and G + C contents were re-confirmed. Protein domain distribution patterns were then identified across archaeal species. Domains with unknown functions (DUFs) and mini proteins were investigated in terms of their distributions due to their importance in archaeal physiological functions. In addition, physicochemical properties of protein sequences, such as stability, hydrophobicity, isoelectric point, aromaticity and amino acid compositions in corresponding archaeal groups were compared. Unique features associated with extremophilic lifestyles were observed, which suggested that evolutionary adaptations to different extreme environments had intrinsic impacts on archaeal protein features. Taken together, this systematic study facilitates a better understanding of the mechanisms behind the extremophilic lifestyles of archaeal species, which will further contribute to the evolutionary explorations of archaeal adaptations both experimentally and theoretically in the future studies.Communicated by Ramaswamy H. Sarma.

Biological functions, genetic and biochemical characterization, and NMR structure determination of the small zinc finger protein HVO_2753 from Haloferax volcanii.

The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.

Genomic analysis of facultatively oligotrophic haloarchaea of the genera Halarchaeum, Halorubrum, and Halolamina, isolated from solar salt.

Extremely halophilic archaea (haloarchaea) belonging to the phylum Euryarchaeota have been found in high-salinity environments. In this study, Halarchaeum sp. CBA1220, Halorubrum sp. CBA1229, and Halolamina sp. CBA1230, which are facultatively oligotrophic haloarchaea, were isolated from solar salt by culture under oligotrophic culture conditions. The complete genomes of strains CBA1220, CBA1229, and CBA1230 were sequenced and were found to contain 3,175,875, 3,582,278, and 3,465,332 bp, with a G + C content of 68.25, 67.66, and 66.75 mol %, respectively. In total, 60, 36, and 33 carbohydrate-active enzyme genes were determined in the respective strains. The strains harbored various genes encoding stress-tolerance proteins, including universal stress proteins, cold-shock proteins, and rubrerythrin and rubrerythrin-related proteins. The genome data produced in this study will facilitate further research to improve our understanding of other halophilic strains and promote their industrial application.

RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains nearly 200 000 bacterial and archaeal genomes and 150 million proteins with up-to-date annotation. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP) since 2018 have resulted in a substantial reduction in spurious annotation. The hierarchical collection of protein family models (PFMs) used by PGAP as evidence for structural and functional annotation was expanded to over 35 000 protein profile hidden Markov models (HMMs), 12 300 BlastRules and 36 000 curated CDD architectures. As a result, >122 million or 79% of RefSeq proteins are now named based on a match to a curated PFM. Gene symbols, Enzyme Commission numbers or supporting publication attributes are available on over 40% of the PFMs and are inherited by the proteins and features they name, facilitating multi-genome analyses and connections to the literature. In adherence with the principles of FAIR (findable, accessible, interoperable, reusable), the PFMs are available in the Protein Family Models Entrez database to any user. Finally, the reference and representative genome set, a taxonomically diverse subset of RefSeq prokaryotic genomes, is now recalculated regularly and available for download and homology searches with BLAST. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

CRISPR Arrays Away from cas Genes.

CRISPR-Cas systems typically consist of a CRISPR array and cas genes that are organized in one or more operons. However, a substantial fraction of CRISPR arrays are not adjacent to cas genes. Definitive identification of such isolated CRISPR arrays runs into the problem of false-positives, with unrelated types of repetitive sequences mimicking CRISPR. We developed a computational pipeline to eliminate false CRISPR predictions and found that up to 25% of the CRISPR arrays in complete bacterial and archaeal genomes are located away from cas genes. Most of the repeats in these isolated arrays are identical to repeats in cas-adjacent CRISPR arrays in the same or closely related genomes, indicating an evolutionary relationship between isolated arrays and arrays in typical CRISPR-cas loci. The spacers in isolated CRISPR arrays show nearly as many matches to viral genomes as spacers from complete CRISPR-cas loci, suggesting that the isolated arrays were either functionally active recently or continue to function. Reconstruction of evolutionary events in closely related bacterial genomes suggests three routes of evolution of isolated CRISPR arrays: (1) loss of cas genes in a CRISPR-cas locus, (2) de novo generation of arrays from off-target spacer integration into sequences resembling the corresponding repeats, and (3) transfer by mobile genetic elements. Both combination of de novo emerging arrays with cas genes and regain of cas genes by isolated arrays via recombination likely contribute to functional diversification in CRISPR-Cas evolution.

Bacterial-like nitric oxide synthase in the haloalkaliphilic archaeon Natronomonas pharaonis.

Bacterial nitric oxide (NO) synthases (bNOS) play diverse and important roles in microbial physiology, stress resistance, and virulence. Although bacterial and mammalian NOS enzymes have been well-characterized, comparatively little is known about the prevalence and function of NOS enzymes in Archaea. Analysis of archaeal genomes revealed that highly conserved bNOS homologs were restricted to members of the Halobacteria. Of these, Natronomonas pharaonis NOS (npNOS) was chosen for further characterization. NO production was confirmed in heterologously expressed His-tagged npNOS by coupling nitrite production from N-hydroxy-L-arginine in an H2O2-supported reaction. Additionally, the nos gene was successfully targeted and disrupted to create a Nmn. pharaonis nos mutant by adapting an established Natrialba magadii transformation protocol. Genome re-sequencing of this mutant revealed an additional frameshift in a putative cation-acetate symporter gene, which could contribute to altered acetate metabolism in the nos mutant. Inactivation of Nmn. pharaonis nos was also associated with several phenotypes congruent with bacterial nos mutants (altered growth, increased oxygen consumption, increased pigment, increased UV susceptibility), suggesting that NOS function may be conserved between bacteria and archaea. These studies are the first to describe genetic inactivation and characterization of a Nmn. pharaonis gene and provides enhanced tools for probing its physiology.