Genome Taxonomy Database Research Articles

Luteimonas flava sp. nov. and Aquilutibacter rugosus gen. nov., sp. nov., isolated from freshwater environments in China and re-examining the taxonomic status of genera Luteimonas and Lysobacter

Three Gram-stain-negative, aerobic, short rod-shaped and motile strains (FXH3WT, SHGZ20W and SMYT11WT) were isolated from freshwater environments in China. Comparisons based on the 16S rRNA gene sequences indicated that strains FXH3WT and SHGZ20W showed the highest 16S rRNA gene sequence similarity of about 99.6% to ‘Luteimonas cellulosilyticus’ MIC 1.5, and strain SMYT11WT showed the highest 16S rRNA gene sequence similarity of 99.8% to Luteimonas fraxinea D4P002T, respectively. Observing the phylogenetic trees reconstructed based on the 16S rRNA gene sequences, the species of genera Luteimonas and Lysobacter were not monophyletic and often mixed together. The further reconstructed phylogenomic tree and Genome Taxonomy Database also showed that the species of both genera were polyphyletic, implying that the current taxonomic status of the species of both genera was questionable. The calculated OrthoANIu, digital DNA–DNA hybridization and average amino acid sequence identity (AAI) values supported that strains FXH3WT and SHGZ20W should belong to the same novel species and strain SMYT11WT should also represent an independent novel species. Combining the AAI values and phylogenomic analyses, the species of genera Luteimonas and Lysobacter should be reassigned to 12 genera (Luteimonas, Lysobacter, Cognatiluteimonas, Noviluteimonas, Pseudoluteimonas, Solilutibacter, Agrilutibacter, Cognatilysobacter, Marilutibacter, Novilysobacter, Montanilutibacter and Aerolutibacter). The AAI values 69.5–76.0% were also proposed as the Lysobacteraceae-specific thresholds for genus delineation. Strain SMYT11WT should represent a novel species of the genus Luteimonas, for which the name Luteimonas flava sp. nov. (type strains SMYT11WT=GDMCC 1.4275T=KCTC 8304T) is proposed. Strains FXH3WT and SHGZ20W should represent a novel species of a new genus Aquilutibacter rugosus gen. nov., sp. nov. The type strain of the type species is FXH3WT (=GDMCC 1.4096T=KCTC 8154T).

The mOTUs online database provides web-accessible genomic context to taxonomic profiling of microbial communities.

Determining the taxonomic composition (taxonomic profiling) is a fundamental task in studying environmental and host-associated microbial communities. However, genome-resolved microbial diversity on Earth remains undersampled, and accessing the genomic context of taxa detected during taxonomic profiling remains a challenging task. Here, we present the mOTUs online database (mOTUs-db), which is consistent with and interfaces with the mOTUs taxonomic profiling tool. It comprises 2.83 million metagenome-assembled genomes (MAGs) and 919090 single-cell and isolate genomes from 124295 species-level taxonomic units. In addition to being one of the largest prokaryotic genome resources to date, all MAGs in the mOTUs-db were reconstructed de novo in 117902 individual samples by abundance correlation of scaffolds across multiple samples for improved quality metrics. The database complements the Genome Taxonomy Database, with over 50% of its species-level taxonomic groups being unique. It also offers interactive querying, enabling users to explore and download genomes at various taxonomic levels. The mOTUs-db is accessible at https://motus-db.org.

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data 1 . Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function 2-4 . We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB) 5 . GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which overlapping triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB- derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

The dynamic history of prokaryotic phyla: discovery, diversity and division.

Here, I review the dynamic history of prokaryotic phyla. Following leads set by Darwin, Haeckel and Woese, the concept of phylum has evolved from a group sharing common phenotypes to a set of organisms sharing a common ancestry, with modern taxonomy based on phylogenetic classifications drawn from macromolecular sequences. Phyla came as surprising latecomers to the formalities of prokaryotic nomenclature in 2021. Since then names have been validly published for 46 prokaryotic phyla, replacing some established names with neologisms, prompting criticism and debate within the scientific community. Molecular barcoding enabled phylogenetic analysis of microbial ecosystems without cultivation, leading to the identification of candidate divisions (or phyla) from diverse environments. The introduction of metagenome-assembled genomes marked a significant advance in identifying and classifying uncultured microbial phyla. The lumper-splitter dichotomy has led to disagreements, with experts cautioning against the pressure to create a profusion of new phyla and prominent databases adopting a conservative stance. The Candidatus designation has been widely used to provide provisional status to uncultured prokaryotic taxa, with phyla named under this convention now clearly surpassing those with validly published names. The Genome Taxonomy Database (GTDB) has offered a stable, standardized prokaryotic taxonomy with normalized taxonomic ranks, which has led to both lumping and splitting of pre-existing phyla. The GTDB framework introduced unwieldy alphanumeric placeholder labels, prompting recent publication of over 100 user-friendly Latinate names for unnamed prokaryotic phyla. Most candidate phyla remain 'known unknowns', with limited knowledge of their genomic diversity, ecological roles, or environments. Whether phyla still reflect significant evolutionary and ecological partitions across prokaryotic life remains an area of active debate. However, phyla remain of practical importance for microbiome analyses, particularly in clinical research. Despite potential diminishing returns in discovery of biodiversity, prokaryotic phyla offer extensive research opportunities for microbiologists for the foreseeable future.

Full-length 16S rRNA gene sequencing combined with adequate database selection improves the description of Arctic marine prokaryotic communities

BackgroundHigh-throughput sequencing of the full-length 16S rRNA gene has improved the taxonomic classification of prokaryotes found in natural environments. However, sequencing of shorter regions from the same gene, like the V4-V5 region, can provide more cost-effective high throughput. It is unclear which approach best describes prokaryotic communities from underexplored environments. In this study, we hypothesize that high-throughput full-length 16S rRNA gene sequencing combined with adequate taxonomic databases improves the taxonomic description of prokaryotic communities from underexplored environments in comparison with high-throughput sequencing of a short region of the 16S rRNA gene.ResultsTo test our hypothesis, we compared taxonomic profiles of seawater samples from the Arctic Ocean using: full-length and V4-V5 16S rRNA gene sequencing in combination with either the Genome Taxonomy Database (GTDB) or the Silva taxonomy database. Our results show that all combinations of sequencing strategies and taxonomic databases present similar results at higher taxonomic levels. However, at lower taxonomic levels, namely family, genus, and most notably species level, the full-length approach led to higher proportions of Amplicon Sequence Variants (ASVs) assigned to formally valid taxa. Hence, the best taxonomic description was obtained by the full-length and GTDB combination, which in some cases allowed for the identification of intraspecific diversity of ASVs.ConclusionsWe conclude that coupling high-throughput full-length 16S rRNA gene sequencing with GTDB improves the description of microbiome profiling at lower taxonomic ranks. The improvements reported here provide more context for scientists to discuss microbial community dynamics within a solid taxonomic framework in environments like the Arctic Ocean with still underrepresented microbiome sequences in public databases.

A genome-based investigation of the Priestia species isolated from anthrax endemic regions in Kruger National Park

Priestia is a genus that was renamed from the genus Bacillus based on the conserved signature indels (CSIs) in protein sequences that separate Priestia species from Bacillus, with the latter only including species closely related to B. subtilis and B. cereus. Diagnosis of anthrax, a zoonotic disease, is implicated by tripartite anthrax virulence genes (lef, pagA, and cya) and poly-γ-D-glutamic acid capsular genes cap-ABCDE of Bacillus anthracis. Due to the amplification of anthrax virulence genes in Priestia isolates, the search for homologous anthrax virulence genes within the Priestia genomes (n = 9) isolated from animal blood smears was embarked upon through whole genome sequencing. In silico taxonomic identification of the isolates was conducted using genome taxonomy database (GTDB), average nucleotide identity (ANI), and multi-locus sequence typing (MLST), which identified the genomes as P. aryabhattai (n = 5), P. endophytica (n = 2) and P. megaterium (n = 2). A pan-genome analysis was further conducted on the Priestia genomes, including the screening of virulence, antibiotic resistance genes and mobile genetic elements on the sequenced genomes. The oligoribonuclease NrnB protein sequences showed that Priestia spp. possess a unique CSI that is absent in other Bacillus species. Furthermore, the CSI in P. endophytica is unique from other Priestia spp. Pan-genomic analysis indicates that P. endophytica clusters separately from P. aryabhattai and P. megaterium. In silico BLASTn genome analysis using the SYBR primers, Taqman probes and primers that target the chromosomal marker (Ba-1), protective antigen (pagA), and lethal factor (lef) on B. anthracis, showed partial binding to Priestia regions encoding for hypothetical proteins, pyridoxine biosynthesis, hydrolase, and inhibitory proteins. The antibiotic resistance genes (ARG) profile of Priestia spp. showed that the genomes contained no more than two ARGs. This included genes conferring resistance to rifamycin and fosfomycin on P. endophytica, as well as clindamycin on P. aryabhattai and P. megaterium. Priestia genomes lacked B. anthracis plasmids and consisted of plasmid replicon types with unknown functions. Furthermore, the amplification of Priestia strains may result in false positives when qPCR is used to detect the virulence genes of B. anthracis in soil, blood smears, and/or environmental samples.

Genomic analysis of the class Phycisphaerae reveals a versatile group of complex carbon-degrading bacteria

Bacteria of the phylum Planctomycetota have received much attention over the years due to their unique cell biology and potential for biotechnological application. Within the phylum, bacteria of the class Phycisphaerae have been found in a multitude of environmental datasets. However, only a few species have been brought into culture so far and even enrichments are scarce. Therefore, very little is known about their lifestyle, which has hindered efforts to estimate their environmental relevance. Here, we analysed all medium- and high-quality Phycisphaerae genomes represented in the genome taxonomy database to learn more about their physiology. We combined automatic and manual annotation efforts to provide a bird’s eye view of their diverse energy metabolisms. Contrasting previous reports, we did not find indications for the presence of genes for anaerobic ammonium oxidation in any Phycisphaerae genome. Instead, we found that many members of this class are adapted to a facultative anaerobic or strictly fermentative lifestyle and may be specialized in the breakdown of carbon compounds produced by other organisms. Based on these findings, we provide a practical overview of organic carbon substrates predicted to be utilized by Phycisphaerae families.

Metagenomic insights into Heimdallarchaeia clades from the deep-sea cold seep and hydrothermal vent

Heimdallarchaeia is a class of the Asgardarchaeota, are the most probable candidates for the archaeal protoeukaryote ancestor that have been identified to date. However, little is known about their life habits regardless of their ubiquitous distribution in diverse habitats, which is especially true for Heimdallarchaeia from deep-sea environments. In this study, we obtained 13 metagenome-assembled genomes (MAGs) of Heimdallarchaeia from the deep-sea cold seep and hydrothermal vent. These MAGs belonged to orders o_Heimdallarchaeales and o_JABLTI01, and most of them (9 MAGs) come from the family f_Heimdallarchaeaceae according to genome taxonomy database (GTDB). These are enriched for common eukaryote-specific signatures. Our results show that these Heimdallarchaeia have the metabolic potential to reduce sulfate (assimilatory) and nitrate (dissimilatory) to sulfide and ammonia, respectively, suggesting a previously unappreciated role in biogeochemical cycling. Furthermore, we find that they could perform both TCA and rTCA pathways coupled with pyruvate metabolism for energy conservation, fix CO2 and generate organic compounds through an atypical Wood-Ljungdahl pathway. In addition, many genes closely associated with bacteriochlorophyll and carotenoid biosynthesis, and oxygen-dependent metabolic pathways are identified in these Heimdallarchaeia MAGs, suggesting a potential light-utilization by pigments and microoxic lifestyle. Taken together, our results indicate that Heimdallarchaeia possess a mixotrophic lifestyle, which may give them more flexibility to adapt to the harsh deep-sea conditions.

Phylogeny and Metabolic Potential of New Giant Sulfur Bacteria of the Family Beggiatoaceae from Coastal-Marine Sulfur Mats of the White Sea.

The family Beggiatoaceae is currently represented by 25 genera in the Genome Taxonomy Database, of which only 6 have a definite taxonomic status. Two metagenome-assembled genomes (MAGs), WS_Bin1 and WS_Bin3, were assembled from metagenomes of the sulfur mats coating laminaria remnants in the White Sea. Using the obtained MAGs, we first applied phylogenetic analysis based on whole-genome sequences to address the systematics of Beggiatoaceae, which clarify the taxonomy of this family. According to the average nucleotide identity (ANI) and average amino acid identity (AAI) values, MAG WS_Bin3 was assigned to a new genus and a new species in the family Beggiatoaceae, namely, 'Candidatus Albibeggiatoa psychrophila' gen. nov., sp. nov., thus providing the revised taxonomic status of the candidate genus 'BB20'. Analysis of 16S rRNA gene homology allowed us to identify MAG WS_Bin1 as the only currently described species of the genus 'Candidatus Parabeggiatoa', namely, 'Candidatus Parabeggiatoa communis', and consequently assign the candidate genus 'UBA10656', including four new species, to the genus 'Ca. Parabeggiatoa'. Using comparative whole-genome analysis of the members of the genera 'Candidatus Albibeggiatoa' and 'Ca. Parabeggiatoa', we expanded information on the central pathways of carbon, sulfur and nitrogen metabolism in the family Beggiatoaceae.

Putative genome contamination has minimal impact on the GTDB taxonomy.

The Genome Taxonomy Database (GTDB) provides a species to domain classification of publicly available genomes based on average nucleotide identity (ANI) (for species) and a concatenated gene phylogeny normalized by evolutionary rates (for genus to phylum), which has been widely adopted by the scientific community. Here, we use the Genome UNClutterer (GUNC) software to identify putatively contaminated genomes in GTDB release 07-RS207. We found that GUNC reported 35,723 genomes as putatively contaminated, comprising 11.25 % of the 317,542 genomes in GTDB release 07-RS207. To assess the impact of this high level of inferred contamination on the delineation of taxa, we created 'clean' versions of the 34,846 putatively contaminated bacterial genomes by removing the most contaminated half. For each clean half, we re-calculated the ANI and concatenated gene phylogeny and found that only 77 (0.22 %) of the genomes were not consistent with their original classification. We conclude that the delineation of taxa in GTDB is robust to the putative contamination detected by GUNC.

Phylogenomic analyses and reclassification of the Mesorhizobium complex: proposal for 9 novel genera and reclassification of 15 species.

The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40-80.91% and 83.98-86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.

Genomic comparison of deep-sea hydrothermal genera related to Aeropyrum, Thermodiscus and Caldisphaera, and proposed emended description of the family Acidilobaceae

Deep-sea hydrothermal vents host archaeal and bacterial thermophilic communities, including taxonomically and functionally diverse Thermoproteota. Despite their prevalence in high-temperature submarine communities, Thermoproteota are chronically under-represented in genomic databases and issues have emerged regarding their nomenclature, particularly within the Aeropyrum–Thermodiscus–Caldisphaera. To resolve some of these problems, we identified 47 metagenome-assembled genomes (MAGs) within this clade, from 20 previously published deep-sea hydrothermal vent and submarine volcano metagenomes, and 24 MAGs from public databases. Using phylogenomic analysis, Genome Taxonomy Database Toolkit (GTDB-Tk) taxonomic assessment, 16S rRNA gene phylogeny, average amino acid identity (AAI) and functional gene patterns, we re-evaluated of the taxonomy of the Aeropyrum–Thermodiscus–Caldisphaera. At least nine genus-level clades were identified with two or more MAGs. In accordance with SeqCode requirements and recommendations, we propose names for three novel genera, viz. Tiamatella incendiivivens, Hestiella acidicharens and Calypsonella navitae. A fourth genus was also identified related to Thermodiscus maritimus, for which no available sequenced genome exists. We propose the novel species Thermodiscus eudorianus to describe our high-quality Thermodiscus MAG, which represents the type genome for the genus. All three novel genera and T. eudorianus are likely anaerobic heterotrophs, capable of fermenting protein-rich carbon sources, while some Tiamatella, Calypsonella and T. eudorianus may also reduce polysulfides, thiosulfate, sulfur and/or selenite, and the likely acidophile, Hestiella, may reduce nitrate and/or perchlorate. Based on phylogenomic evidence, we also propose the family Acidilobaceae be amended to include Caldisphaera, Aeropyrum, Thermodiscus and Stetteria and the novel genera described here.

Integrating taxonomic signals from MAGs and contigs improves read annotation and taxonomic profiling of metagenomes.

Metagenomic analysis typically includes read-based taxonomic profiling, assembly, and binning of metagenome-assembled genomes (MAGs). Here we integrate these steps in Read Annotation Tool (RAT), which uses robust taxonomic signals from MAGs and contigs to enhance read annotation. RAT reconstructs taxonomic profiles with high precision and sensitivity, outperforming other state-of-the-art tools. In high-diversity groundwater samples, RAT annotates a large fraction of the metagenomic reads, calling novel taxa at the appropriate, sometimes high taxonomic ranks. Thus, RAT integrative profiling provides an accurate and comprehensive view of the microbiome from shotgun metagenomics data. The package of Contig Annotation Tool (CAT), Bin Annotation Tool (BAT), and RAT is available at https://github.com/MGXlab/CAT_pack (from CATpack v6.0). The CAT pack now also supports Genome Taxonomy Database (GTDB) annotations.

Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool.

The Interactive Tree Of Life (https://itol.embl.de) is an online tool for the management, display, annotation and manipulation of phylogenetic and other trees. It is freely available and open to everyone. iTOL version 6 introduces a modernized and completely rewritten user interface, together with numerous new features. A new dataset type has been introduced (colored/labeled ranges), greatly upgrading the functionality of the previous simple colored range annotation function. Additional annotation options have been implemented for several existing dataset types. Dataset template files now support simple assignment of annotations to multiple tree nodes through substring matching, including full regular expression support. Node metadata handling has been greatly extended with novel display and exporting options, and it can now be edited interactively or bulk updated through annotation files. Tree labels can be displayed using multiple simultaneous font styles, with precise positioning, sizing and styling of each individual label part. Various bulk label editing functions have been implemented, simplifying large scale changes of all tree node labels. iTOL's automatic taxonomy assignment functions now support trees based on the Genome Taxonomy Database (GTDB), in addition to the NCBI taxonomy. The functionality of the optional user account pages has been expanded, simplifying the management, navigation and sharing of projects and trees. iTOL currently handles more than one and a half million trees from >130000 individual user accounts.

AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life. Our server offers users the ability to identify, compare, and visualize gene neighborhoods of interest from 30 238 bacterial genomes and 1672 archaeal genomes, through integration with the comprehensive Genome Taxonomy Database and AnnoTree databases. Identified gene neighborhoods can be visualized using pre-computed functional annotations from different sources such as KEGG, Pfam and TIGRFAM, or clustered based on similarity. Alternatively, users can upload and explore their own custom genomic datasets in GBK, GFF or CSV format, or use AnnoView as a genome browser for relatively small genomes (e.g. viruses and plasmids). Ultimately, we anticipate that AnnoView will catalyze biological discovery by enabling user-friendly search, comparison, and visualization of genomic data. AnnoView is available at http://annoview.uwaterloo.ca.

Circular metagenome-assembled genome of Candidatus Patescibacteria recovered from anaerobic digestion sludge.

A single-contig, circular metagenome-assembled genome (cMAG) of Candidatus (Ca.) Patescibacteria was reconstructed from a mesophilic full-scale food waste treatment plant in Japan. The genome is of small size and lacks fundamental biosynthetic pathways. Taxonomic analysis using the Genome Taxonomy Database revealed that this cMAG belonged to the genus JAEZRQ01 (Ca. Parcubacteria).

Phylogenomic and molecular markers based studies on clarifying the evolutionary relationships among Peptoniphilus species. Identification of several Genus-Level clades of Peptoniphilus species and transfer of some Peptoniphilus species to the genus Aedoeadaptatus

To clarify the evolutionary relationships among Peptoniphilus species, whose members show association with increased risk for prostate cancer, detailed phylogenomic and comparative analyses were conducted on their genome sequences. In phylogenetic trees based on core genome proteins and 16S rRNA gene sequences, Peptoniphilus species formed eight distinct clades, with Aedoeadaptatus and Anaerosphaera species branching between them. The observed clades designated as Peptoniphilus sensu stricto (encompassing its type species), Harei, Lacrimalis, Duerdenii, Mikwangii, Stercorisuis, Catoniae and Aedoeadaptatus, show genus level divergence based on 16S rRNA similarity and average amino acid identity (AAI). The Genome Taxonomy Database also assigns most of these clades to distinct taxa. Several Peptoniphilus species (viz. P. coxii, P. ivorii, P. nemausensis and some non-validly published species) grouped reliably with the type species of Aedoeadaptatus (A. acetigenes) and are affiliated to this genus based on 16S rRNA similarity, AAI, and multiple uniquely shared molecular signatures. Hence, we are proposing the transfer of these species into the emended genus Aedoeadaptatus. Our analyses on protein sequences from Peptoniphilus genomes have also identified 54 novel molecular markers consisting of conserved signature indels (CSIs), which are specific for different Peptoniphilus species clades and provide reliable means for their demarcation in molecular terms. Lastly, we also show that based on the shared presence of these CSIs in the genomes of uncharacterized Peptoniphilus spp. (cultured and uncultured), their affiliations to the specific Peptoniphilus clades can be accurately predicted. These results should prove useful in understanding the potential involvement of Peptoniphilus-related species in diseases.

Metagenome-assembled genomes of three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis.

The digestive organs of terrestrial isopods harbour bacteria of the recently proposed mollicute family Hepatoplasmataceae. The only complete genome available so far for Hepatoplasmataceae is that of 'Candidatus Hepatoplasma crinochetorum'. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose genomospecies names for three MAGs that show substantial sequence divergence from any previously known Hepatoplamsataceae members: 'Candidatus Tyloplasma litorale' identified from the semiterrestrial isopod Tylos granuliferus, 'Candidatus Hepatoplasma vulgare' identified from the common pill bug Armadillidium vulgare, and 'Candidatus Hepatoplasma scabrum' identified from the common rough woodlouse Porcellio scaber. Phylogenomic analysis of 155 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. The 16S ribosomal RNA gene sequences and phylogenomic analysis showed that 'Candidatus Tyloplasma litorale' and other semiterrestrial isopod-associated mollicutes represent the placeholder genus 'g_Bg2' in the r214 release of the Genome Taxonomy Database, warranting their assignment to a novel genus. Our analysis also revealed that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery. Although the localization of the Hepatoplasmatacae members have not been verified microscopically in this study, these genomic characteristics are compatible with the idea that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host, as has been demonstrated for other members of the family. We could not find evidence that Hepatoplasmataceae encode polysaccharide-degrading enzymes that aid host digestion. If they are to provide nutritional benefits, it may be through extra-copy nucleases, peptidases, and a patatin-like lipase. Exploration of potential host-symbiont interaction-associated genes revealed large, repetitive open reading frames harbouring beta-sandwich domains, possibly involved with host cell adhesion. Overall, genomic analyses suggest that isopod-mollicute symbiosis is not characterized by carbohydrate degradation, and we speculate on their potential role as defensive symbionts through spatial competition with pathogens to prevent infection.

GSR-DB: a manually curated and optimized taxonomical database for 16S rRNA amplicon analysis.

Amplicon-based 16S ribosomal RNA sequencing remains a widely used method to profile microbial communities, especially in low biomass samples, due to its cost-effectiveness and low-complexity approach. Reference databases are a mainstay for taxonomic assignments, which typically rely on popular databases such as SILVA, Greengenes, Genome Taxonomy Database (GTDB), or Ribosomal Database Project (RDP). However, the inconsistency of the nomenclature across databases and the presence of shortcomings in the annotation of these databases are limiting the resolution of the analysis. To overcome these limitations, we created the GSR database (Greengenes, SILVA, and RDP database), an integrated and manually curated database for bacterial and archaeal 16S amplicon taxonomy analysis. Unlike previous integration approaches, this database creation pipeline includes a taxonomy unification step to ensure consistency in taxonomical annotations. The database was validated with three mock communities, two real data sets, and a 10-fold cross-validation method and compared with existing 16S databases such as Greengenes, Greengenes 2, GTDB, ITGDB, SILVA, RDP, and MetaSquare. Results showed that the GSR database enhances taxonomical annotations of 16S sequences, outperforming current 16S databases at the species level, based on the evaluation of the mock communities. This was confirmed by the 10-fold cross-validation, except for Greengenes 2. The GSR database is available for full-length 16S sequences and the most commonly used hypervariable regions: V4, V1-V3, V3-V4, and V3-V5.IMPORTANCETaxonomic assignments of microorganisms have long been hindered by inconsistent nomenclature and annotation issues in existing databases like SILVA, Greengenes, Greengenes2, Genome Taxonomy Database, or Ribosomal Database Project. To overcome these issues, we created Greengenes-SILVA-RDP database (GSR-DB), accurate and comprehensive taxonomic annotations of 16S amplicon data. Unlike previous approaches, our innovative pipeline includes a unique taxonomy unification step, ensuring consistent and reliable annotations. Our evaluation analyses showed that GSR-DB outperforms existing databases in providing species-level resolution, especially based on mock-community analysis, making it a game-changer for microbiome studies. Moreover, GSR-DB is designed to be accessible to researchers with limited computational resources, making it a powerful tool for scientists across the board. Available for full-length 16S sequences and commonly used hypervariable regions, including V4, V1-V3, V3-V4, and V3-V5, GSR-DB is a go-to database for robust and accurate microbial taxonomy analysis.

Multiple independent losses of sporulation and peptidoglycan in the Mycoplasmatales and related orders of the class Bacilli.

Many peptidoglycan-deficient bacteria such as the Mycoplasmatales are known host-associated lineages, lacking the environmental resistance mechanisms and metabolic capabilities necessary for a free-living lifestyle. Several peptidoglycan-deficient and non-sporulating orders of interest are thought to be descended from Gram-positive sporulating Bacilli through reductive evolution. Here we annotate 2650 genomes belonging to the class Bacilli, according to the Genome Taxonomy Database, to predict the peptidoglycan and sporulation phenotypes of three novel orders, RFN20, RF39 and ML615J-28, known only through environmental sequence surveys. These lineages are interspersed between peptidoglycan-deficient non-sporulating orders including the Mycoplasmatales and Acholeplasmatales, and more typical Gram-positive orders such as the Erysipelotrichales and Staphylococcales. We use the extant genotypes to perform ancestral state reconstructions. The novel orders are predicted to have small genomes with minimal metabolic capabilities and to comprise a mix of peptidoglycan-deficient and/or non-sporulating species. In contrast to expectations based on cultured representatives, the order Erysipelotrichales lacks many of the genes involved in peptidoglycan and endospore formation. The reconstructed evolutionary history of these traits suggests multiple independent whole-genome reductions and loss of phenotype via intermediate transition states that continue into the present. We suggest that the evolutionary history of the reduced-genome lineages within the class Bacilli is one driven by multiple independent transitions to host-associated lifestyles, with the degree of reduction in environmental resistance and metabolic capabilities correlated with degree of host association.