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A motile, rod-shaped and yellow-coloured bacterium, designated strain SYSU D60001

Carbon fixation by chemoautotrophic microbes such as homoacetogens had a major impact on the transition from the inorganic to the organic world. Recent reports have shown the presence of genes for key enzymes associated with the Wood–Ljungdahl pathway (WLP) in the phylum Actinobacteria, which adds to the diversity of potential autotrophs. Here, we compiled 42 actinobacterial metagenome-assembled genomes (MAGs) from new and existing metagenomic datasets and propose three novel classes, Ca. Aquicultoria, Ca. Geothermincolia and Ca. Humimicrobiia. Most members of these classes contain genes coding for acetogenesis through the WLP, as well as a variety of hydrogenases (NiFe groups 1a and 3b–3d; FeFe group C; NiFe group 4-related hydrogenases). We show that the three classes acquired the hydrogenases independently, yet the carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS) was apparently present in their last common ancestor and was inherited vertically. Furthermore, the Actinobacteria likely donated genes for CODH/ACS to multiple lineages within Nitrospirae, Deltaproteobacteria (Desulfobacterota), and Thermodesulfobacteria through multiple horizontal gene transfer events. Finally, we show the apparent growth of Ca. Geothermincolia and H(2)-dependent acetate production in hot spring enrichment cultures with or without the methanogenesis inhibitor 2-bromoethanesulfonate, which is consistent with the proposed homoacetogenic metabolism.

Two anaerobic bacteria, designated strains SYSU GA16112T and SYSU GA16107, were isolated from a hot spring in Tengchong County, Yunnan Province, south-west PR China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SYSU GA16112T and SYSU GA16107 belong to the family Dysgonamonadaceae. Cells of strains SYSU GA16112T and SYSU GA16107 were Gram-stain-negative, rod-shaped and non-motile. The major fatty acids (>10 %) of strains SYSU GA16112T and SYSU GA16107 were identified as anteiso-C15 : 0 and anteiso-C17 : 0 3OH. The polar lipid profile of strain SYSU GA16112T was found to consist of phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phosphoglycolipids, two unidentified aminolipids and one unidentified polar lipid, while that of strain SYSU GA16107 consisted of phosphatidylethanolamine, two unidentified polar lipids, three unidentified aminophospholipids, two unidentified phosphoglycolipids and one unidentified aminolipid. The genomic DNA G+C contents of strains SYSU GA16112T and SYSU GA16107 were determined to be 41.90 and 41.89 %, respectively, and the average nucleotide identity value between them was 99.99 %. Based on their morphological and physiological properties, and results of phylogenetic analyses, strains SYSU GA16112T and SYSU GA16107 are considered to represent a novel species of a novel genus, for which the name Seramator thermalis gen. nov., sp. nov. (type strain SYSU GA16112T=CGMCC 1.5281T=KCTC 15753T) is proposed.

An alkaliphilic actinobacterial strain, designated Hz 6-5T, was isolated from saline-alkaline soil from Songnen Plain in north-eastern China. The isolate formed light yellow-colored colonies and its cells were Gram-staining positive, non-motile, and non-spore-forming short rods. The strain was aerobic with optimal growth at 33 °C, pH 9.0, and in the presence of 0.5% (w/v) NaCl or 3% (w/v) KCl. It was catalase-positive and oxidase-negative. The isolate had highest 16S rRNA gene sequence similarities to the type strains of the species Nesternkonia natronophila M8T (98.2%), N. salmonea GY074T (98.1%), and N. sphaerica GY239T (97.4%), and the isolate formed a subclade with the type strains of these species in the neighbor-joining tree based on the 16S rRNA gene sequences. The phylogenetic tree based on the phylogenomic analysis also showed the same results. The DNA‒DNA relatedness (DDH) values of isolate Hz 6-5T with N. natronophila M8T, N. halophila DSM 16378T, and N. halobia CGMCC 1.2323T were 21.2%, 36.5%, and 32.0%, respectively. The characteristic diamino acid of strain Hz 6-5T was found to be lysine. The respiratory quinones were MK-9, MK-8, MK-7(H4), MK-7(H2) and MK-7 and the major cellular fatty acids (> 10%) were anteiso-C15:0, anteiso-C17:0 and iso-C16:0. The polar lipids detected for strain Hz 6-5T were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, an unidentified glycolipid, and two unidentified phospholipids. The DNA G + C content of isolate Hz 6-5T was 60.8%. Based on the results of phylogenetic analysis supported by morphological, physiological, chemotaxonomic, and other differentiating phenotypic evidence, strain Hz 6-5T is considered to represent a novel species of the genus Nesterenkonia, for which the name Nesterenkonia haasae sp. nov. is proposed. The type strain is Hz 6-5T (=CPCC 205100T=NBRC 113521T).

The taxonomic status of a Nocardiopsis strain, designated H13T, isolated from a high altitude Atacama Desert soil, was established by using a polyphasic approach. The strain was found to have chemotaxonomic, cultural and morphological characteristics consistent with its classification within the genus Nocardiopsis and formed a well-supported clade in the Nocardiopsis phylogenomic tree together with the type strains of Nocardiopsis alborubida , Nocardiopsis dassonvillei and Nocardiopsis synnematoformans. Strain H13T was distinguished from its closest relatives by low average nucleotide identity (93.2–94.9 %) and in silico DNA–DNA hybridization (52.5–62.4 %) values calculated from draft genome assemblies and by a range of phenotypic properties. On the basis of these results, it is proposed that the isolate be assigned to the genus Nocardiopsis as Nocardiopsis deserti sp. nov. with isolate H13T (=CGMCC 4.7585T=KCTC 49249T) as the type strain.

A coccobacilli-shaped proteobacterium, designated strain SYSU XM001T, was isolated from an activated sludge sample collected from JiMei sewage treatment plant, Xiamen, PR China. Cells were Gram-stain-negative, catalase-positive and oxidase-negative. The 16S rRNA gene sequence of strain SYSU XM001T shared less than 92 % sequence identities with members of the class Alphaproteobacteria , with highest sequence similarity to Aquidulcibacter paucihalophilus TH1-2T (91.6 %; family Hyphomonadaceae , order Rhodobacterales ). The strain exhibited growth at 25–37 °C, pH 7.0–9.0 and in the presence of up to 1 % (w/v) NaCl. Its chemotaxonomic features included ubiquinone-10 as the respiratory isoprenologue, iso-C16 : 0, 10-methyl C16 : 0 TSBA and anteiso-C17 : 0 as major cellular fatty acids and monoglycosyldiglyceride, glucuronopyranosyldiglyceride and two unidentified glycolipids as the main polar lipids. The DNA G+C content was determined to be 62.9 % (draft genome). Analyses of the phylogenetic data and differences in the chemotaxonomic and biochemical features from related genera in the family Hyphomonadaceae indicated that strain SYSU XM001T merits representation of a novel species of a novel genus, for which the name Vitreimonas flagellata gen. nov., sp. nov. is proposed. The type strain of Vitreimonas flagellata is SYSU XM001T (=CGMCC 1.16661T=KCTC 62915T).

A thermophilic bacterium, designated SYSU G03088T, was isolated from Moincer hot spring, Tibet, PR China. Polyphasic taxonomic analyses and whole-genome sequencing were used to determine the taxonomic position and genomic profiles of the strain. Phylogenetic analysis using 16S rRNA gene sequence indicated that SYSU G03088T showed highest sequence similarity to Rhodobacter blasticus CGMCC 1.3365T (96.0 % sequence identity). The strain could be differentiated from most recognized species of the genus Rhodobacter by its slightly purple colony colour, distinct phenotypic characters and low ANI values. Cells were Gram-staining negative, and oval-to-rod shaped. Poly-β-hydroxybutyrate and vesicular intracytoplasmic membrane structures were formed inside cells. Growth occurred optimally at 45 °C and pH 7.0. Ubiquinone 10 was the only respiratory quinone. The major fatty acids (>10 %) were C18 : 0, C18 : 1ω7c 11-methyl and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The detected polar lipids of SYSU G03088T included diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmethylethanolamine. The DNA G+C content of SYSU G03088T was 67.7 % (genome). On the basis of the differences in the phenotypic characteristics and phylogenetic analyses, SYSU G03088T is considered to represent a novel species of the genus Rhodobacter , for which the name Rhodobacter flagellatus sp. nov. is proposed. The type strain is SYSU G03088T (=CGMCC 1.16876T=KCTC 72354T).

Genome analysis is one of the main criteria for description of new taxa. Availability of genome sequences for all the actinobacteria with a valid nomenclature will, however, require another decade’s works of sequencing. This paper describes the rearrangement of the higher taxonomic ranks of the members of the phylum ‘ Actinobacteria ’, using the phylogeny of 16S rRNA gene sequences and supported by the phylogeny of the available genome sequences. Based on the refined phylogeny of the 16S rRNA gene sequences, we could arrange all the members of the 425 genera of the phylum ‘ Actinobacteria ’ with validly published names currently in use into six classes, 46 orders and 79 families, including 16 new orders and 10 new families. The order Micrococcales Prévot 1940 (Approved Lists 1980) emend. Nouioui et al. 2018 is now split into 11 monophyletic orders: the emended order Micrococcales and ten proposed new orders Aquipuribacterales , Beutenbergiales , Bogoriellales , Brevibacteriales , Cellulomonadales , Demequinales , Dermabacterales , Dermatophilales , Microbacteriales and Ruaniales . Further, the class ‘ Actinobacteria ’ Stackebrandt et al. 1997 emend. Nouioui et al. 2018 was described without any nomenclature type, and therefore the name ‘ Actinobacteria ’ is deemed illegitimate. In accordance to Rule 8 of the International Code of Nomenclature of Prokaryotes, Parker et al. 2019, we proposed the name Actinomycetia which is formed by using the stem of the name Actinomycetales Buchanan 1917 (Approved Lists 1980) emend. Zhi et al. 2009, to replace the name ‘ Actinobacteria ’. The nomenclature type of the proposed new class Actinomycetia is the order Actinomycetales Buchanan 1917 (Approved Lists 1980) emend. Zhi et al. 2009.

A genome-based phylogeny of the publicly available genomes of the genus Nocardiopsis reveals that Nocardiopsis baichengensis YIM 90130T is closely related to Nocardiopsis halophila DSM 44494T. Overall genome relatedness indices between the genomes of the two species were above the threshold values necessary for defining a new species (dDDH, 82.9 %; ANIb, 97.8 %; ANIm, 98.3 %; AAI, 97.7 %). Both species showed a similar G+C content of 73.6 mol% in their genomes. Considering the high similarity between the genomes of the two species, we propose that Nocardiopsis baichengesis Li et al. 2006 should be reclassified as a later heterotypic synonym of Nocardiopsis halophila Al-Tai and Ruan 1994.

Two Francisella -like bacteria, designated strains SYSU SYW-5T and SYSU SYW-6T, were isolated from coastal seawater sampled at Huizhou Double-Moon Bay, Guangdong Province, PR China. Cells were found to be Gram-stain-negative, non-motile, non-spore-forming and of pleomorphic shape (coccobacilli or rod). Chemotaxonomic analysis of the plasma membrane revealed ubiquinone-8 as the respiratory quinone, and saturated branched-chain (anteiso-C15 : 0) and saturated straight-chain (C18 : 0) fatty acids as major components (>8 % of total fatty acid). The major polar lipids comprised diphosphatidylglycerol (cardiolipin), phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine, as well as an unidentified aminophospholipid and an unidentified phospholipid in SYSU SYW-5T, and two unknown polar lipids in SYSU SYW-6T. Comparison of 16S rRNA gene sequences showed that SYSU SYW-5T had maximum similarity to Fastidiosibacter lacustris SYSU HZH-2T (93.4 %), while SYSU SYW-6Thad maximum similarity to Cysteiniphilum litorale SYSU D3-2T (97.5 %). Phylogenetic dendrograms based on 16S rRNA genes and 31 concatenated protein markers revealed that the two novel strains represent a distinct lineage within the family Fastidiosibacteraceae . The average nucleotide identity and in silico DNA–DNA hybridization values between the two isolates and their related species were well below the threshold limit for species delineation (

Phylogenomic analyses were performed on the nine species of the genus Meiothermus and four species of the genus Calidithermus. Phylogenetic analysis, low values of genomic relatedness indices and functional diversity analysis indicated that Meiothermus silvanus should not be classified within the clades for Meiothermus and Calidithermus but instead be reclassified as a new genus, for which we propose the name Allomeiothermus gen. nov., with Allomeiothermus silvanus comb. nov. as type species. In addition, the species Meiothermus cateniformans Zhang et al. (Int J Syst Evol Microbial 60:840-844, 2010) should also be reclassified as a later heterotypic synonym of Meiothermus taiwanensis Chen et al. (Int J Syst Evol Microbiol 52:1647-1654, 2002) emend. Raposo et al. (2019). This reclassification is based on the high genomic relatedness indices (98.8% ANI; 90.2% dDDH; 99% AAI) that are above the threshold values necessary for defining a new species, as well as on the observation of overlapping functions on Principal Coordinate Analysis plot generated from Clusters of Orthologous Genes.

A moderately thermophilic, aerobic, Gram-stain-negative, non-spore-forming, rod-shaped and yellow-pigmented bacterium, designated strain SYSU G00007T, was isolated from a hot spring slurry sample. Optimum growth was observed at 37–45 °C and pH 7. Pairwise comparison of the 16S rRNA gene sequence of strain SYSU G00007T and other Novosphingobium species showed sequence similarities ranging from 93.7 to 97.9 %. Strain SYSU G00007T showed highest sequence identity to Novosphingobium subterraneum DSM 12447T (97.9 %). The average nucleotide identities and digital DNA–DNA hybridization values between strain SYSU G00007T and its closely related phylogenetic neighbours were below 81 and 31 %, respectively, indicating that strain SYSU G00007T represented a novel species of the genus Novosphingobium . The DNA G+C content of strain SYSU G00007T was 64.3 % (genome). The major respiratory quinone was ubiquinone Q-10. The polar lipid profile included diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two sphingoglycolipids, two unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. Spermidine was the only polyamine detected. The major fatty acids were C19 : 0cyclo ω8c, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The results obtained from phylogenetic, chemotaxonomic and phenotypic analyses support the conclusion that strain SYSU G00007T represents a novel species of the genus Novosphingobium , for which we proposed the name Novosphingobium meiothermophilum sp. nov. The type strain is SYSU G00007T (=KCTC 52672T=CCTCC AB2017010T).

Strain SYSU D60016T, a rod-shaped bacterium that tends to form clusters, was isolated from a soil sample collected from the Gurbantunggut desert, China. Cells were Gram-stain-negative, catalase-negative and oxidase-positive. The 16S rRNA gene sequence of strain SYSU D60016T shared less than 94 % sequence identity with members of the family Hymenobacteraceae . The strain exhibited growth at 15–50 °C, pH 6–8 and in the presence of up to 3 % (w/v) NaCl. Its chemotaxonomic features included MK-7 as the respiratory menaquinone, iso-C15 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B) as major cellular fatty acids and phosphatidylethanolamine as the main polar lipid. The DNA G+C content was determined to be 50.1 % (genome). Analyses of the phylogenetic data and differences in the chemotaxonomic and biochemical features from related genera in the family Hymenobacteraceae indicated that strain SYSU D60016T merits representation of a novel species of a novel genus in the family Hymenobacteraceae . The name Botryobacter ruber gen. nov., sp. nov. is, therefore, proposed to represent the phylogenetic position of strain SYSU D60016T in the family Hymenobacteraceae . The type strain of the proposed new taxon is SYSU D60016T (=KCTC 52794T=NBRC 112957T).

A Gram-stain-negative, aerobic, non-spore-forming and rod-shaped bacterium, designated YIM 730274T, was isolated from a sediment sample collected from a hot spring located in Tibet, PR China, and was characterized by using a polyphasic taxonomy approach. Cells were motile by means of a polar flagellum. The strain was oxidase- and catalase-positive, and contained polyalkanoates and polyphosphate as storage polymers. Growth occurred at 25–50 °C, at pH 6.0–8.5 and with 0.5–1.0 % NaCl. The major fatty acids (>10 %) were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The known polar lipids comprised of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The isoprenoid quinone was Q-8. The G+C content of genomic DNA was 70.7 mol%. The results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the strain forms a monophyletic branch at the periphery of the evolutionary radiation occupied by the genus Aquabacterium in the class Betaproteobacteria . The most closely related phylogenetic neighbours were Aquabacterium limnoticum ABP-4T (97.8 % 16S rRNA gene sequence identity) and Aquabacterium commune B8T (97.2 % 16SrRNA gene sequence identity). DNA–DNA relatedness values between YIM 730274T and A. limnoticum KCTC 23306T (46.4±0.4 %) and A. commune DSM 11901T (42.2±1.2 %) were well below the 70 % limit for species identification. YIM 730274T was distinguishable from other members of the genus Aquabacterium by the differences in phenotypic, chemotaxonomic and genotypic characteristics. YIM 730274T merits recognition as a representative of a novel species of the genus Aquabacterium . It is proposed that the isolate should be classified in the genus Aquabacterium as representing a novel species, Aquabacterium tepidiphilum sp. nov. The type strain is YIM 730274T (=KCTC 52716T=CCTCC AB 2016295T).

A Gram-stain-negative, non-motile, short rod and aerobic bacterium, designated strain SYSU M10001T, was isolated from a water sample collected from the coastal region of Pearl River Estuary, Guangdong Province, PR China. Strain SYSU M10001T showed optimal growth at 28 °C, pH 7.0 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and concatenation of 20 protein markers revealed a distinct lineage for strain SYSU M10001T in the order Rhizobiales . Strain SYSU M10001T showed highest 16S rRNA gene sequence similarities to Hyphomicrobium nitrativorans NL23T (91.1 %) and Hyphomicrobium hollandicum IFAM KB-677T (91.1 %). The respiratory ubiquinone was Q-8. The polar lipids of the strain comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, two unidentified phospholipids and three unidentified lipids. The predominant cellular fatty acids identified were C19 : 0cyclo ω8c, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The G+C content was determined to be 65.5 % (genome). On the basis of differences in the phenotypic, physiological and biochemical characteristics, and results of the phylogenetic analyses, strain SYSU M10001T is proposed to represent a novel species in a novel genus for which the name Aestuariivirga litoralis gen. nov., sp. nov. The type strain of the type species Aestuariivirga litoralis is SYSU M10001T (=NBRC 112960T=KCTC 52945T). Besides, the distinct phylogenetic lineage and the distinct chemotaxonomic profile among the families in the order Rhizobiales indicated that strain SYSU M10001T should represent a new family for which the name Aestuariivirgaceae fam. nov. is proposed.

A motile, rod-shaped and yellow coloured proteobacterium, designated strain SYSU D60017T, was isolated from a desert soil sample. The bacterium was found to be an obligately aerobic, mesophilic and neutrophilic chemo-heterotroph. Cells were observed to be Gram-stain negative, catalase positive and oxidase positive. The major cellular fatty acids were identified as C19:0ω8c cyclo and Summed Feature 8 (C18:1ω7c and/or C18:1ω6c). The main respiratory quinone identified was ubiquinone-10. The DNA G + C content was determined to be 63.8% based on draft genome sequence data. The polar lipids detected were identified as diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and five unidentified polar lipids. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SYSU D60017T is a member of the order Rhizobiales, but forms a distinct phylogenetic lineage. The differences in the phenotypic characteristics from members of related genera and its distinct phylogenetic position suggested that the isolate SYSU D60017T represents a novel species of a novel genus within the order Rhizobiales, for which the name Flaviflagellibacter deserti gen. nov., sp. nov. is proposed. The type strain of the new taxon is SYSU D60017T (= CGMCC 1.16444T = NBRC 112958T).

Microbes are the most abundant and diverse cellular life forms on Earth and colonize a wide range of environmental niches. However, more than 99% of bacterial and archaeal species have not been obtained in pure culture [1] and we have only glimpsed the surface of this mysterious microbial world. This is so-called Microbial Dark Matter (MDM): the enormous diversity of yet-uncultivated microbes that microbiologists can only study by using cultivation-independent techniques. Recently, a number of international projects have dramatically increased our understanding of the extent and distribution of microbial diversity, including the Global Catalogue of Microorganisms (GCM), the Genomic Encyclopedia of Bacteria and Archaea (GEBA), the Earth Microbiome Project (EMP), the Genomic Encyclopedia of Bacteria and Archaea-Microbial Dark Matter (GEBA-MDM) and several primate microbiome projects; however, the functional diversity of MDM is still mysterious. This perspective addresses why MDM deserves scientific effort and illustrates challenges and opportunities in the future study of these enigmas.

Earth is dominated by a myriad of microbial communities, but the majority fails to grow under in situ laboratory conditions. The basic cause of unculturability is that bacteria dominantly occur as biofilms in natural environments. Earlier improvements in the culture techniques are mostly done by optimizing media components. However, with technological advancement particularly in the field of genome sequencing and cell imagining techniques, new tools have become available to understand the ecophysiology of microbial communities. Hence, it becomes easier to mimic environmental conditions in the culture plate. Other methods include co-culturing, emendation of growth factors, and cultivation after physical cell sorting. Most recently, techniques have been proposed for bacterial cultivation by employing genomic data to understand either microbial interactions (network-directed targeted bacterial isolation) or ecosystem engineering (reverse genomics). Hopefully, these techniques may be applied to almost all environmental samples, and help fill the gaps between the cultured and uncultured microbial communities.

The perplexity of the complex multispecies community interactions is one of the many reasons why majority of the microorganisms are still uncultivated. We analyzed the entire co-occurrence networks between the OTUs of Tibet and Yunnan hot spring samples, and found that less abundant OTUs such as genus Tepidimonas (relative abundant Chloroflexus (relative abundant, 13.9%) formed the peripheral vertexes. A preliminary growth-promotion assay determined that Tepidimonas sp. strain SYSU G00190W enhanced the growth of Chloroflexus sp. SYSU G00190R. Exploiting this result, an ameliorated isolation medium containing 10% spent-culture supernatant of Tepidimonas sp. strain SYSU G00190W was prepared for targeted isolation of Chloroflexi in the Tibet and Yunnan hot spring samples. 16S rRNA gene fingerprinting characterized majority of the colonies isolated from these media as previously uncultivated Chloroflexi, of which 36 are potential novel species (16S rRNA sequence identity