Advancements in prokaryotic systematics and the role of Bergey's International Society for Microbial Systematicsin addressing challenges in the meta-data era.

Prokaryotes are ubiquitous in the biosphere, important for human health and drive diverse biological and environmental processes. Systematics of prokaryotes, whose origins can be traced to the discovery of microorganisms in the 17th century, has transitioned from a phenotype-based classification to a more comprehensive polyphasic taxonomy and eventually to the current genome-based taxonomic approach. This transition aligns with a foundational shift from studies focused on phenotypic traits that have limited comparative value to those using genome sequences. In this context, Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB) and Bergey's International Society for Microbial Systematics (BISMiS) play a pivotal role in guiding prokaryotic systematics. This review focuses on the historical development of prokaryotic systematics with a focus on the roles of BMSAB and BISMiS. We also explore significant contributions and achievements by microbiologists, highlight the latest progress in the field and anticipate challenges and opportunities within prokaryotic systematics. Additionally, we outline five focal points of BISMiS that are aimed at addressing these challenges. In conclusion, our collaborative effort seeks to enhance ongoing advancements in prokaryotic systematics, ensuring its continued relevance and innovative characters in the contemporary landscape of genomics and bioinformatics.

Commentary on the proposed Section 10 amendments to the International Code of Nomenclature of Prokaryotes regarding Candidatus names.

Amendments were proposed to the International Code of Nomenclature of Prokaryotes (ICNP) in January [Arahal et al. (2024) Int. J Syst. Evol. Microbiol. 74: 006188] that would cause major changes in the treatment of Candidatus names. The amendments introduce Section 10 to name taxa whose names cannot be validly published under the ICNP because of the absence of type strains. This section creates a parallel 'pro-nomenclature' and formalizes alternative material which could serve as nomenclatural types. When conspecific isolates of taxa with Candidatus names are deposited in culture collections as type strains, the names can be validly published, and it is required that the same Candidatus name be used. While the amendments are promoted to provide stable names and rules of nomenclature for uncultivated taxa, the system is deeply flawed. It removes the permanent association between names and types, which will make the meaning of names imprecise and ambiguous. It creates 'pro-nomenclature', which is confusing and unnecessary. Since many taxa which cannot be validly named under the ICNP can already be named under the SeqCode, it duplicates and creates overlap with an established nomenclatural system without providing tangible benefits. As the SeqCode recognizes names formed under the ICNP, the ICNP should recognize names formed under the SeqCode as they have done for the Cyanobacteria named under the International Code of Nomenclature for algae, fungi and plants (ICN). For these reasons, we urge the members of the International Committee of Systematics of Prokaryotes (ICSP) to reject these amendments.

Why and how to use the SeqCode.

The SeqCode, formally called the Code of Nomenclature of Prokaryotes Described from Sequence Data, is a new code of nomenclature in which genome sequences are the nomenclatural types for the names of prokaryotic species. While similar to the International Code of Nomenclature of Prokaryotes (ICNP) in structure and rules of priority, it does not require the deposition of type strains in international culture collections. Thus, it allows for the formation of permanent names for uncultured prokaryotes whose nearly complete genome sequences have been obtained directly from environmental DNA as well as other prokaryotes that cannot be deposited in culture collections. Because the diversity of uncultured prokaryotes greatly exceeds that of readily culturable prokaryotes, the SeqCode is the only code suitable for naming the majority of prokaryotic species. The start date of the SeqCode was January 1, 2022, and the online Registry (https://seqco.de/) was created to ensure valid publication of names. The SeqCode recognizes all names validly published under the ICNP before 2022. After that date, names validly published under the SeqCode compete with ICNP names for priority. As a result, species can have only one name, either from the SeqCode or ICNP, enabling effective communication and the creation of unified taxonomies of uncultured and cultured prokaryotes. The SeqCode is administered by the SeqCode Committee, which is comprised of the SeqCode Community and elected administrative components. Anyone with an interest in the systematics of prokaryotes is encouraged to join the SeqCode Community and participate in the development of this resource.

Quis custodiet ipsos custodes? A call for community participation in the governance of the SeqCode.

Codes of nomenclature that provide well-regulated and stable frameworks for the naming of taxa are a fundamental underpinning of biological research. These Codes themselves require systems that govern their administration, interpretation and emendment. Here we review the provisions that have been made for the governance of the recently introduced Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which provides a nomenclatural framework for the valid publication of names of Archaea and Bacteria using isolate genome, metagenome-assembled genome or single-amplified genome sequences as type material. The administrative structures supporting the SeqCode are designed to be open and inclusive. Direction is provided by the SeqCode Community, which we encourage those with an interest in prokaryotic systematics to join.

Antiquaquibacter oligotrophicus gen. nov., sp. nov., a novel oligotrophic bacterium from groundwater.

In this study, a Gram-stain-positive, non-motile, oxidase- and catalase-negative, rod-shaped, bacterial strain (SG_E_30_P1T) that formed light yellow colonies was isolated from a groundwater sample of Sztaravoda spring, Hungary. Based on 16S rRNA phylogenetic and phylogenomic analyses, the strain was found to form a distinct linage within the family Microbacteriaceae. Its closest relatives in terms of near full-length 16S rRNA gene sequences are Salinibacterium hongtaonis MH299814 (97.72 % sequence similarity) and Leifsonia psychrotolerans GQ406810 (97.57 %). The novel strain grows optimally at 20-28 °C, at neutral pH and in the presence of NaCl (1-2 w/v%). Strain SG_E_30_P1T contains MK-7 and B-type peptidoglycan with diaminobutyrate as the diagnostic amino acid. The major cellular fatty acids are anteiso-C15 : 0, iso-C16 : 0 and iso-C14 : 0, and the polar lipid profile is composed of diphosphatidylglycerol and phosphatidylglycerol, as well as an unidentified aminoglycolipid, aminophospholipid and some unidentified phospholipids. The assembled draft genome is a contig with a total length of 2 897 968 bp and a DNA G+C content of 65.5 mol%. Amino acid identity values with it closest relatives with sequenced genomes of <62.54 %, as well as other genome distance results, indicate that this bacterium represents a novel genus within the family Microbacteriaceae. We suggest that SG_E_30_P1T (=DSM 111415T=NCAIM B.02656T) represents the type strain of a novel genus and species for which the name Antiquaquibacter oligotrophicus gen. nov., sp. nov. is proposed.

Phylogenomics-based reclassifications in the genus Psychrobacter including emended descriptions of Psychrobacter pacificensis, Psychrobacter proteolyticus and Psychrobacter submarinus.

The taxonomic status of 43 Psychrobacter species was examined based upon the genome sequences of their type strains. Three groups of type strains were found to be conspecific, Psychrobacter salsus Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) and Psychrobacter submarinus Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291); Psychrobacter oceani Matsuyama et al. (Int J Syst Evol Microbiol 65:1450-1455, 2015. 10.1099/ijs.0.000118) and Psychrobacter pacificensis Maruyama et al. (Int J Syst Evol Microbiol 50:835-846, 2000. 10.1099/00207713-50-2-835); and Psychrobacter proteolyticus Denner et al. (Syst Appl Microbiol 24:44-53, 2001. 10.1078/0723-2020-00006), Psychrobacter marincola Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291) and Psychrobacter adeliensis Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956). For all three groups, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values are > 97.69% and > 80.2%, respectively. This conclusion is supported by similarities in morphology, growth properties, and fatty acid compositions. Based on this evidence, we propose the reclassification of Psychrobacter salsus Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) as a later heterotypic synonym of Psychrobacter submarinus Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291); Psychrobacter oceani Matsuyama et al. (Int J Syst Evol Microbiol 65:1450-1455, 2015. 10.1099/ijs.0.000118) as a later heterotypic synonym of Psychrobacter pacificensis Maruyama et al. (Int J Syst Evol Microbiol 50:835-846, 2000. 10.1099/00207713-50-2-835), and Psychrobacter marincola Romanenko et al. (Int J Syst Evol Microbiol 52:1291-1297, 2002. 10.1099/00207713-52-4-1291) and Psychrobacter adeliensis Shivaji et al. (Syst Appl Microbiol 27:628-635, 2004. 10.1078/0723202042369956) as later heterotypic synonyms of Psychrobacter proteolyticus Denner et al. (Syst Appl Microbiol 24:44-53, 2001. 10.1078/0723-2020-00006).

Model Organisms To Study Methanogenesis, a Uniquely Archaeal Metabolism.

Methanogenic archaea are the only organisms that produce CH4 as part of their energy-generating metabolism. They are ubiquitous in oxidant-depleted, anoxic environments such as aquatic sediments, anaerobic digesters, inundated agricultural fields, the rumen of cattle, and the hindgut of termites, where they catalyze the terminal reactions in the degradation of organic matter. Methanogenesis is the only metabolism that is restricted to members of the domain Archaea. Here, we discuss the importance of model organisms in the history of methanogen research, including their role in the discovery of the archaea and in the biochemical and genetic characterization of methanogenesis. We also discuss outstanding questions in the field and newly emerging model systems that will expand our understanding of this uniquely archaeal metabolism.

Internal transcription termination widely regulates differential expression of operon-organized genes including ribosomal protein and RNA polymerase genes in an archaeon.

Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCR and western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.

Proposed minimal standards for description of methanogenic archaea.

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA-DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

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.

Oxidative Stress Regulates a Pivotal Metabolic Switch in Dimethylsulfoniopropionate Degradation by the Marine Bacterium Ruegeria pomeroyi.

Dimethylsulfoniopropionate (DMSP) is an abundant organic compound in marine surface water and source of dimethyl sulfide (DMS), the largest natural sulfur source to the upper atmosphere. Marine bacteria either mineralize DMSP through the demethylation pathway or transform it to DMS through the cleavage pathway. Factors that regulate which pathway is utilized are not fully understood. In chemostat experiments, the marine Roseobacter Ruegeria pomeroyi DSS-3 was exposed to oxidative stress either during growth with H2O2 or by mutation of the gene encoding catalase. Oxidative stress reduced expression of the genes in the demethylation pathway and increased expression of those encoding the cleavage pathway. These results are contrary to the sulfur demand hypothesis, which theorizes that DMSP metabolism is driven by sulfur requirements of bacterial cells. Instead, we find strong evidence consistent with oxidative stress control over the switch in DMSP metabolism from demethylation to DMS production in an ecologically relevant marine bacterium. IMPORTANCE Dimethylsulfoniopropionate (DMSP) is the most abundant low-molecular-weight organic compound in marine surface water and source of dimethyl sulfide (DMS), a climatically active gas that connects the marine and terrestrial sulfur cycles. Marine bacteria are the major DMSP consumers, either generating DMS or consuming DMSP as a source of reduced carbon and sulfur. However, the factors regulating the DMSP catabolism in bacteria are not well understood. Marine bacteria are also exposed to oxidative stress. RNA sequencing (RNA-seq) experiments showed that oxidative stress induced in the laboratory reduced expression of the genes encoding the consumption of DMSP via the demethylation pathway and increased the expression of genes encoding DMS production via the cleavage pathway in the marine bacterium Ruegeria pomeroyi. These results support a model where DMS production in the ocean is regulated in part by oxidative stress.

Genome Sequencing Suggests Diverse Secondary Metabolism in Coral-Associated Aquimarina megaterium.

We report here the genome sequences of three Aquimarina megaterium strains isolated from the octocoral Eunicella labiata. We reveal a coding potential for versatile carbon metabolism and biosynthesis of natural products belonging to the polyketide, nonribosomal peptide, and terpene compound classes.

Expression of divergent methyl/alkyl coenzyme M reductases from uncultured archaea.

Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences.

SeqCode: a nomenclatural code for prokaryotes described from sequence data.

Most prokaryotes are not available as pure cultures and therefore ineligible for naming under the rules and recommendations of the International Code of Nomenclature of Prokaryotes (ICNP). Here we summarize the development of the SeqCode, a code of nomenclature under which genome sequences serve as nomenclatural types. This code enables valid publication of names of prokaryotes based upon isolate genome, metagenome-assembled genome or single-amplified genome sequences. Otherwise, it is similar to the ICNP with regard to the formation of names and rules of priority. It operates through the SeqCode Registry ( https://seqco.de/ ), a registration portal through which names and nomenclatural types are registered, validated and linked to metadata. We describe the two paths currently available within SeqCode to register and validate names, including Candidatus names, and provide examples for both. Recommendations on minimal standards for DNA sequences are provided. Thus, the SeqCode provides a reproducible and objective framework for the nomenclature of all prokaryotes regardless of cultivability and facilitates communication across microbiological disciplines.

Zwartia hollandica gen. nov., sp. nov., Jezberella montanilacus gen. nov., sp. nov. and Sheuella amnicola gen. nov., comb. nov., representing the environmental GKS98 (betIII) cluster.

We present two strains affiliated with the GKS98 cluster. This phylogenetically defined cluster is representing abundant, mainly uncultured freshwater bacteria, which were observed by many cultivation-independent studies on the diversity of bacteria in various freshwater lakes and streams. Bacteria affiliated with the GKS98 cluster were detected by cultivation-independent methods in freshwater systems located in Europe, Asia, Africa and the Americas. The two strains, LF4-65T (=CCUG 56422T=DSM 107630T) and MWH-P2sevCIIIbT (=CCUG 56420T=DSM 107629T), are aerobic chemoorganotrophs, both with genome sizes of 3.2 Mbp and G+C values of 52.4 and 51.0 mol%, respectively. Phylogenomic analyses based on concatenated amino acid sequences of 120 proteins suggest an affiliation of the two strains with the family Alcaligenaceae and revealed Orrella amnicola and Orrella marina (= Algicoccus marinus) as being the closest related, previously described species. However, the calculated phylogenomic trees clearly suggest that the current genus Orrella represents a polyphyletic taxon. Based on the branching order in the phylogenomic trees, as well as the revealed phylogenetic distances and chemotaxonomic traits, we propose to establish the new genus Zwartia gen. nov. and the new species Z. hollandica sp. nov. to harbour strain LF4-65T and the new genus Jezberella gen. nov. and the new species J. montanilacus sp. nov. to harbour strain MWH-P2sevCIIIbT. Furthermore, we propose the reclassification of the species Orrella amnicola in the new genus Sheuella gen. nov. The new genera Zwartia, Jezberella and Sheuella together represent taxonomically the GKS98 cluster.

Development of the SeqCode: A proposed nomenclatural code for uncultivated prokaryotes with DNA sequences as type.

Over the last fifteen years, genomics has become fully integrated into prokaryotic systematics. The genomes of most type strains have been sequenced, genome sequence similarity is widely used for delineation of species, and phylogenomic methods are commonly used for classification of higher taxonomic ranks. Additionally, environmental genomics has revealed a vast diversity of as-yet-uncultivated taxa. In response to these developments, a new code of nomenclature, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), has been developed over the last two years to allow naming of Archaea and Bacteria using DNA sequences as the nomenclatural types. The SeqCode also allows naming of cultured organisms, including fastidious prokaryotes that cannot be deposited into culture collections. Several simplifications relative to the International Code of Nomenclature of Prokaryotes (ICNP) are implemented to make nomenclature more accessible, easier to apply and more readily communicated. By simplifying nomenclature with the goal of a unified classification, inclusive of both cultured and uncultured taxa, the SeqCode will facilitate the naming of taxa in every biome on Earth, encourage the isolation and characterization of as-yet-uncultivated taxa, and promote synergies between the ecological, environmental, physiological, biochemical, and molecular biological disciplines to more fully describe prokaryotes.

Faunimonas pinastri gen. nov., sp. nov., an endophyte from a pine tree of the family Pleomorphomonadaceae, class Alphaproteobacteria.

Bacterial strain A52C2T was isolated from the endophytic microbial community of a Pinus pinaster tree trunk and characterized. Strain A52C2T stained Gram-negative and formed rod-shaped cells that grew optimally at 30 °C and at pH 6.0-7.0. The G+C content of the DNA was 65.1 mol %. The respiratory quinone was ubiquinone 10, and the major fatty acids were cyclo-C19:0 ω8c and C18:0, representing 70.1 % of the total fatty acids. Phylogenetic analyses based on the 16S rRNA gene sequences placed strain A52C2T in a distinct lineage within the order Hyphomicrobiales, family Pleomorphomonadaceae. The 16S rRNA gene sequence similarities of A52C2T to that of Mongoliimonas terrestris and Oharaeibacter diazotrophicus were 93.15 and 93.2 %, respectively. The draft genome sequence of strain A52C2T comprises 4 196 045 bases with a 195-fold mapped coverage of the genome. The assembled genome consists of 43 contigs of more than 1 000 bp (N50 contig size was 209 720 bp). The genome encodes 4033 putative coding sequences. The phylogenetic, phenotypic and chemotaxonomic data showed that strain A52C2T (=UCCCB 130T=CECT 8949T=LMG 29042T) represents the type of a novel species and genus, for which we propose the name Faunimonas pinastri gen. nov., sp. nov.

Substrate Specificity of the 3-Methylmercaptopropionyl Coenzyme A Dehydrogenase (DmdC1) from Ruegeria pomeroyi DSS-3.

The acyl-coenzyme A (CoA) dehydrogenase family enzyme DmdC catalyzes the third step in the dimethylsulfoniopropionate (DMSP) demethylation pathway, the oxidation of 3-methylmercaptopropionyl-CoA (MMPA-CoA) to 3-methylthioacryloyl-CoA (MTA-CoA). To study its substrate specificity, the recombinant DmdC1 from Ruegeria pomeroyi was characterized. In addition to MMPA-CoA, the enzyme was highly active with short-chain acyl-CoAs, with Km values for MMPA-CoA, butyryl-CoA, valeryl-CoA, caproyl-CoA, heptanoyl-CoA, caprylyl-CoA, and isobutyryl-CoA of 36, 19, 7, 11, 14, 10, and 149 µM, respectively, and kcat values of 1.48, 0.40, 0.48, 0.73, 0.46, 0.23, and 0.01 s-1, respectively. Among these compounds, MMPA-CoA was the best substrate. The high affinity of DmdC1 for its substrate supports the model for kinetic regulation of the demethylation pathway. In contrast to DmdB, which catalyzes the formation of MMPA-CoA from MMPA, CoA, and ATP, DmdC1 was not affected by physiological concentrations of potential effectors, such as DMSP, MMPA, ATP, and ADP. Thus, compared to the other enzymes of the DMSP demethylation pathway, DmdC1 has only minimal adaptations for DMSP metabolism compared to other enzymes in the same family with similar substrates, supporting the hypothesis that it evolved relatively recently from a short-chain acyl-CoA dehydrogenase involved in fatty acid oxidation. IMPORTANCE We report the kinetic properties of DmdC1 from the model organism R. pomeroyi and close an important gap in the literature. While the crystal structure of this enzyme was recently solved and its mechanism of action described (X. Shao, H. Y. Cao, F. Zhao, M. Peng, et al., Mol Microbiol 111:1057-1073, 2019, https://doi.org/10.1111/mmi.14211), its substrate specificity and sensitivity to potential effectors was never examined. We show that DmdC1 has a high affinity for other short-chain acyl-CoAs in addition to MMPA-CoA, which is the natural substrate in DMSP metabolism and is not affected by the potential effectors tested. This evidence supports the hypothesis that DmdC1 possesses few adaptations to DMSP metabolism and likely evolved relatively recently from a short-chain acyl-CoA dehydrogenase involved in fatty acid oxidation. This work is important because it expands our understanding of the adaptation of marine bacteria to the increased availability of DMSP about 250 million years ago.