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.

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.

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.

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.

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).

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.

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.

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.

The archaeal RNA chaperone TRAM0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis.

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5' untranslated regions (5'UTRs) of 20-60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5'UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5'UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

Hymenobacter caeli sp. nov., an airborne bacterium isolated from King George Island, Antarctica.

A rod-shaped and Gram-stain-negative bacterial strain 9AT, was isolated from an air sample collected at King George Island, maritime Antarctica. Phylogenetic analysis based on 16S rRNA gene sequence reveals that strain 9AT belongs to the genus Hymenobacter and shows the highest similarity to Hymenobacter coccineus CCM 8649T (96.8 %). The DNA G+C content based on the draft genome sequence is 64.9 mol%. Strain 9AT is strictly aerobic, psychrophilic, catalase-positive, oxidase-positive and non-motile. Growth is observed at 0-20 °C (optimum 10 °C), pH 6.0-8.0 (optimum pH 7.0), and in the absence of NaCl. The predominant menaquinone of strain 9AT is MK-7 and the major fatty acids comprise Summed Feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 25.2 %), iso-C15 : 0 (23.2 %), C16 : 1 ω5c (11.6 %), Summed Feature 4 (anteiso-C17 : 1 B/iso-C17 : 1 I) (9.6 %) and anteiso-C15 : 0 (9.6 %). The polar lipid profile consists of the major lipid phosphatidylethanolamine and moderate to minor amounts of phosphatidylserine, unidentified aminolipids, aminophospholipids, aminophosphoglycolipids, polar lipids lacking a functional group and an unidentified phospholipid and a glycolipid. In the polyamine pattern sym-homospermidine is predominant. On the basis of the results obtained, strain 9AT is proposed as a novel species of the genus Hymenobacter, for which the name Hymenobacter caeli sp. nov. is suggested. The type strain is 9AT (=CCM 8971T=LMG 32109T=DSM 111653T).

Frigoriflavimonas asaccharolytica gen. nov., sp. nov., a novel psychrophilic esterase and protease producing bacterium isolated from Antarctica.

The rod-shaped and Gram-stain-negative bacterial strain 16FT, isolated from an air sample collected at King George Island, maritime Antarctica, was investigated to determine its taxonomic status. Strain 16FT is strictly aerobic, catalase positive, oxidase positive and non-motile. Strain 16FT hydrolyses casein, lecithin, Tween 20, 60 and 80, but not aesculin, gelatin and starch. Growth of strain 16FT is observed at 0-20 °C (optimum 10 °C), pH 5.0-8.0 (optimum pH 6.0), and in the presence of 0-2.0% NaCl (optimum 0.5%). The predominant menaquinone is MK-6, and the major fatty acids comprise anteiso-C15:0 and iso-C15:0. The major polar lipids are phosphatidylethanolamine, ornithine lipid OL2, unidentified phospholipid PL1 and the unidentified lipids L3 and L6 lacking functional groups. The DNA G + C content based on the draft genome sequence is 32.3 mol%. Sequence analysis of the 16S rRNA gene indicates the highest similarity to Kaistella palustris 3A10T (95.4%), Kaistella chaponensis Sa 1147-06 T (95.2%), Kaistella antarctica AT1013T (95.1%), Kaistella carnis NCTC 13525 T (95.1%) and below 95.0% to other species with validly published names. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences places strain 16FT in a distinct branch, indicating a separate lineage within the family Weeksellaceae. Based on the data from our polyphasic approach, 16FT represents a novel species of a new genus, for which the name Frigoriflavimonas asaccharolytica gen. nov, sp. nov. is proposed. The type strain is 16FT (= CCM 8975 T = CGMCC No.1.16844 T).

Posttranslational Methylation of Arginine in Methyl Coenzyme M Reductase Has a Profound Impact on both Methanogenesis and Growth of Methanococcus maripaludis.

Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl coenzyme M reductase (Mcr) and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved posttranslational modifications (PTMs) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmpX (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the ΔmmpX mutant was complemented with the wild-type gene expressed by either a strong or a weak promoter, methylation was fully restored. Compared to the parental strain, maximal rates of methane formation by whole cells were reduced by 40 to 60% in the ΔmmpX mutant. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmpX resulted in a differential loss of arginine methylation among the mutants in vivo, suggesting that activities of Mmp10 directly modulated methylation. R275 was present in a highly conserved PXRR275(A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity.IMPORTANCE Mcr is the key enzyme in methanogenesis and a promising candidate for bioengineering the conversion of methane to liquid fuel. Our knowledge of Mcr is still limited. In terms of complexity, uniqueness, and environmental importance, Mcr is more comparable to photosynthetic reaction centers than conventional enzymes. PTMs have long been hypothesized to play key roles in modulating Mcr activity. Here, we directly link the mmpX gene to the arginine PTM of Mcr, demonstrate its association with methanogenesis activity, and offer insights into its substrate specificity and putative cofactor binding sites. This is also the first time that a PTM of McrA has been shown to have a substantial impact on both methanogenesis and growth in the absence of additional stressors.

The van Niel International Prize for Studies in Bacterial Systematics, awarded in 2020 to Tanja Woyke.

The Senate of The University of Queensland, on the recommendation of the Executive Board of the International Committee on Systematics of Prokaryotes, is pleased to present the van Niel International Prize for Studies in Bacterial Systematics for the triennium 2017-2020 to Dr Tanja Woyke in recognition of her contributions made to the field of bacterial systematics. The award, established in 1986 by Professor V. B. D. Skerman of The University of Queensland, honours the contribution of scholarship in the field of microbiology by Professor Cornelis Bernardus van Niel.

Reclassification of Sphingomonas aeria as a later heterotypic synonym of Sphingomonas carotinifaciens based on whole-genome sequence analysis.

The 16S rRNA gene sequences of Sphingomonas carotinifaciens L9-754T and Sphingomonas aeria B093034T possess 99.71 % sequence similarity. Further studies were undertaken to clarify the taxonomic assignments of these species. Whole-genome comparisons showed that S. aeria B093034Tand S. carotinifaciens L9-754T shared 96.9 % average nucleotide identity, 98.4 % average amino acid identity and 76.1 % digital DNA-DNA hybridization values. These values exceeded or approached the recommended species delineation threshold values. Furthermore, a phylogenetic tree based on 41 of the most conserved genes provided additional evidence that S. aeria B093034T and S. carotinifaciens L9-754T are very closely related. Based on this evidence we propose the reclassification of S. aeria Xue et al. 2018 as a later heterotypic synonym of S. carotinifaciens Madhaiyan et al. 2017.

Minutes of the International Committee on Systematics of Prokaryotes online discussion on the proposed use of gene sequences as type for naming of prokaryotes, and outcome of vote.

The International Committee on Systematics of Prokaryotes has held an electronic discussion on proposals to amend the International Code of Nomenclature of Prokaryotes in order to allow the use of gene sequence data as type. The scientific discussion is reported. Subsequently members of the International Committee on Systematics of Prokaryotes voted on these proposals, which were rejected.

Hymenobacter artigasi sp. nov., isolated from air sampling in maritime Antarctica.

A rod-shaped and Gram-stain-negative bacterial strain, 1BT, was isolated from an air sample collected at King George Island, maritime Antarctica. Strain 1BT is strictly aerobic, psychrophilic, catalase-positive, oxidase-positive and non-motile. Growth of strain 1BT is observed at 0-20 °C (optimum, 10 °C), pH 6.0-8.0 (optimum, pH 8.0) and in the presence of 0-1.0% NaCl (optimum, 0.5 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences places strain 1BT within the genus Hymenobacter and shows the highest similarity to Hymenobacter antarcticus VUG-A42aaT (97.5 %). The predominant menaquinone of strain 1BT is MK-7 and the major fatty acids (>10 %) comprise summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 32.5 %), iso-C15 : 0 (17.6 %) and anteiso C15 : 0 (12.3 %). The polar lipid profile consists of the major compounds phosphatidylethanolamine, phosphatidylserine, two unidentified aminolipids and one unidentified phospholipid. The DNA G+C content based on the draft genome sequence is 61.2 mol%. Based on the data from the current polyphasic study, 1BT represents a novel species of the genus Hymenobacter, for which the name Hymenobacter artigasi sp. nov. is suggested. The type strain is 1BT (=CCM 8970T=CGMCC 1.16843T).

Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities.

The class Deltaproteobacteria comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum Proteobacteria, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class Deltaproteobacteria encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the Oligoflexia. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes Deltaproteobacteria and Oligoflexia in the phylum Proteobacteria. Instead, the great majority of currently recognized members of the class Deltaproteobacteria are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class Oligoflexia represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum Thermodesulfobacteria, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the Thermodesulfobacteria rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.

Cupriavidus agavae sp. nov., a species isolated from Agave L. rhizosphere in northeast Mexico.

During the isolation of bacteria from the Agave L. rhizosphere in northeast Mexico, four strains with similar BOX-PCR patterns were collected. The 16S rRNA gene sequences of all four strains were very similar to each other and that of the type strains of Cupriavidus metallidurans CH34T (98.49 % sequence similarity) and Cupriavidus necator N-1T (98.35 %). The genome of strain ASC-9842T was sequenced and compared to those of other Cupriavidus species. ANIb and ANIm values with the most closely related species were lower than 95%, while the in silico DNA-DNA hybridization values were also much lower than 70 %, consistent with the proposal that they represent a novel species. This conclusion was supported by additional phenotypic and chemotaxonomic analyses. Therefore, the name Cupriavidus agavae sp. nov. is proposed with the type strain ASC-9842T (=LMG 26414T=CIP 110327T).

Phytobacter palmae sp. nov., a novel endophytic, N2 fixing, plant growth promoting Gammaproteobacterium isolated from oil palm (Elaeis guineensis Jacq.).

A novel strain S29T with high nitrogen fixing ability was isolated from surface-sterilized leaf tissues of oil palm (Elaeis guineensis) growing in Science Park II, Singapore. On the basis of 16S rRNA gene analysis and multilocus sequence typing with the rpoB, gyrB, infB and atpD genes, strain S29T was a member of the genus Phytobacter, with Phytobacter ursingii ATCC 27989T and Phytobacter diazotrophicus LS 8T as its closest relatives. Unique biochemical features that differentiated strain S29T from its closest relatives were the ability to utilize melibiose, α-cyclodextrin, glycogen, adonital, d-arabitol, m-inositol and xylitol. The major fatty acids were C14 : 0, C16 : 0, C17 : 0, C16 : 1 ω5c and summed feature 2 containing C16 : 1 ω7c and/or C16 : 1 ω6c. The polar fatty acid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and aminolipids. The draft genome of strain S29T comprised 5, 284, 330 bp with a G + C content of 52.6 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain S29T and the phylogenetically related Enterobacterales species were lower than 95 % and 70 %, respectively. Thus, the polyphasic evidences generated through the phenotypic, chemotaxonomic and genomic methods confirmed that strain S29T represents a novel species of the genus Phytobacter, for which we propose the name Phytobacter palmae sp. nov. with the type strain of S29T (=DSM 27342T=KACC 17598T).

Chitinasiproducens palmae gen. nov., sp. nov., a new member of the family Burkholderiaceae isolated from leaf tissues of oil palm (Elaeis guineensis Jacq.).

A Gram-stain-negative, aerobic, rod-shaped, leaf-associated bacterium, designated JS23T, was isolated from surface-sterilized leaf tissue of an oil palm grown in Singapore and was investigated by polyphasic taxonomy. Phylogenetic analyses based on 16S rRNA gene sequences and 180 conserved genes in the genome of several members of Burkholderiaceae revealed that strain JS23T formed a distinct evolutionary lineage independent of other taxa within the family Burkholderiaceae. The predominant ubiquinone was Q-8. The primary polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminophospholipid. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c /C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c). The size of the genome is 5.36 Mbp with a DNA G+C content of 66.2 mol%. Genomic relatedness measurements such as average nucleotide identity, genome-to-genome distance and digital DNA-DNA hybridization clearly distinguished strain JS23T from the closely related genera Burkholderia, Caballeronia, Mycetohabitans, Mycoavidus, Pandoraea, Paraburkholderia, Robbsia and Trinickia. Furthermore, average amino acid identity values and the percentages of conserved proteins, 56.0-68.4 and 28.2-45.5, respectively, were well below threshold values for genus delineation and supported the assignment of JS23T to a novel genus. On the basis of the phylogenetic, biochemical, chemotaxonomic and phylogenomic evidence, strain JS23T is proposed to represent a novel species of a new genus within the family Burkholderiaceae, for which the name Chitinasiproducens palmae gen. nov., sp. nov., is proposed with the type strain of JS23T (= DSM 27307T=KACC 17592T).