Peloplasma aerotolerans gen. nov., sp. nov., a Novel Anaerobic Free-Living Mollicute Isolated from a Terrestrial Mud Volcano.

A novel aerotolerant anaerobic bacterium (strain M4AhT) was isolated from a terrestrial mud volcano (Taman Peninsula, Russia). Cells were small, cell-wall-less, non-motile cocci, 0.32-0.65 µm in diameter. The isolate was a mesophilic, neutrophilic chemoorganoheterotroph, growing on carbohydrates (D-glucose, D-trehalose, D-ribose, D-mannose, D-xylose, D-maltose, D-lactose, D-cellobiose, D-galactose, D-fructose, and D-sucrose), proteinaceous compounds (yeast extract, tryptone), and pyruvate. Strain M4AhT tolerated 2% oxygen in the gas phase, was catalase-positive, and showed sustainable growth under microaerobic conditions. The dominant cellular fatty acids of strain M4AhT were C16:0 and C18:0. The G+C content of the genomic DNA was 32.42%. The closest phylogenetic relative of strain M4AhT was Mariniplasma anaerobium from the family Acholeplasmataceae (order Acholeplasmatales, class Mollicutes). Based on the polyphasic characterization of the isolate, strain M4AhT is considered to represent a novel species of a new genus, for which the name Peloplasma aerotolerans gen. nov., sp. nov. is proposed. The type strain of Peloplasma aerotolerans is M4AhT (=DSM 112561T = VKM B-3485T = UQM 41475T). This is the first representative of the order Acholeplasmatales, isolated from a mud volcano.

Phenotypic and genomic characterization of the first alkaliphilic aceticlastic methanogens and proposal of a novel genus Methanocrinis gen.nov. within the family Methanotrichaceae.

Highly purified cultures of alkaliphilic aceticlastic methanogens were collected for the first time using methanogenic enrichments with acetate from a soda lake and a terrestrial mud volcano. The cells of two strains were non-motile rods forming filaments. The mud volcano strain M04Ac was alkalitolerant, with the pH range for growth from 7.5 to 10.0 (optimum at 9.0), while the soda lake strain Mx was an obligate alkaliphile growing in the pH range 7.7-10.2 (optimum 9.3-9.5) in the presence of optimally 0.2-0.3 M total Na+. Genomes of both strains encoded all enzymes required for aceticlastic methanogenesis and different mechanisms of (halo)alkaline adaptations, including ectoine biosynthesis, which is the first evidence for the formation of this osmoprotectant in archaea. According to 16S rRNA gene phylogeny, the strains possessed 98.3-98.9% sequence identity and belonged to the obligately aceticlastic genus Methanothrix with M. harundinaceae as the most closely related species. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein-coding marker genes clearly indicated a polyphyletic origin of the species included in the genus Methanothrix. We propose to reclassify Methanothrix harrundinacea (type strain 8AcT) into a new genus, Methanocrinis gen. nov., with the type species Methanocrinis harrundinaceus comb. nov. We also propose under SeqCode the complete genome sequences of strain MxTs (GCA_029167045.1) and strain M04AcTs (GCA_029167205.1) as nomenclatural types of Methanocrinis natronophilus sp. nov. and Methanocrinis alkalitolerans sp. nov., respectively, which represent other species of the novel genus. This work demonstrates that the low energy aceticlastic methanogenesis may function at extreme conditions present in (halo)alkaline habitats.

Phenotypic and genomic characterization of Bathyarchaeum tardum gen. nov., sp. nov., a cultivated representative of the archaeal class Bathyarchaeia.

Bathyarchaeia are widespread in various anoxic ecosystems and are considered one of the most abundant microbial groups on the earth. There are only a few reports of laboratory cultivation of Bathyarchaeia, and none of the representatives of this class has been isolated in pure culture. Here, we report a sustainable cultivation of the Bathyarchaeia archaeon (strain M17CTs) enriched from anaerobic sediment of a coastal lake. The cells of strain M17CTs were small non-motile cocci, 0.4-0.7 µm in diameter. The cytoplasmic membrane was surrounded by an S-layer and covered with an outermost electron-dense sheath. Strain M17CTs is strictly anaerobic mesophile. It grows at 10-45°C (optimum 37°C), at pH 6.0-10.0 (optimum 8.0), and at NaCl concentrations of 0-60 g l-1 (optimum 20 g l-1). Growth occurred in the presence of methoxylated aromatic compounds (3,4-dimethoxybenzoate and vanillate) together with complex proteinaceous substrates. Dimethyl sulfoxide and nitrate stimulated growth. The phylogenomic analysis placed strain M17CTs to BIN-L-1 genus-level lineage from the BA1 family-level lineage and B26-1 order-level lineage (former subgroup-8) within the class Bathyarchaeia. The complete genome of strain M17CTs had a size of 2.15 Mb with a DNA G + C content of 38.1%. Based on phylogenomic position and phenotypic and genomic properties, we propose to assign strain M17CTs to a new species of a novel genus Bathyarchaeum tardum gen. nov., sp. nov. within the class Bathyarchaeia. This is the first sustainably cultivated representative of Bathyarchaeia. We propose under SeqCode the complete genome sequence of strain M17CTs (CP122380) as a nomenclatural type of Bathyarchaeum tardum, which should be considered as a type for the genus Bathyarchaeum, which is proposed as a type for the family Bathyarchaeaceae, order Bathyarchaeales, and of the class Bathyarchaeia.

The first cultivated representatives of the actinobacterial lineage OPB41 isolated from subsurface environments constitute a novel order Anaerosomatales.

The continental subsurface harbors microbial populations highly enriched in uncultured taxa. OPB41 is an uncultured order-level phylogenetic lineage within the actinobacterial class Coriobacteriia. OPB41 bacteria have a wide geographical distribution, but the physiology and metabolic traits of this cosmopolitan group remain elusive. From two contrasting subsurface environments, a terrestrial mud volcano and a deep subsurface aquifer, located in the central part of Eurasia, within the Caucasus petroleum region, we have isolated two pure cultures of anaerobic actinobacteria belonging to OPB41. The cells of both strains are small non-motile rods forming numerous pili-like appendages. Strain M08DHBT is mesophilic, while strain Es71-Z0120T is a true thermophile having a broad temperature range for growth (25-77°C). Strain M08DHBT anaerobically reduces sulfur compounds and utilizes an aromatic compound 3,4-dihydroxybenzoic acid. Strain Es71-Z0120T is an obligate dissimilatory Fe(III) reducer that is unable to utilize aromatic compounds. Both isolates grow lithotrophically and consume molecular hydrogen or formate using either thiosulfate, elemental sulfur, or Fe(III) as an electron acceptor. Genomes of the strains encode the putative reductive glycine pathway for autotrophic CO2 fixation, Ni-Fe hydrogenases, putative thiosulfate/polysulfide reductases, and multiheme c-type cytochromes presumably involved in dissimilatory Fe(III) reduction. We propose to assign the isolated strains to the novel taxa of the species-order levels and describe strain M08DHBT as Anaerosoma tenue gen. nov., sp. nov., and strain Es71-Z0120T as Parvivirga hydrogeniphila gen. nov., sp. nov., being members of Anaerosomatales ord. nov. This work expands the knowledge of the diversity, metabolic functions, and ecological role of the phylum Actinomycetota.

Alkalibacter mobilis sp. nov., an anaerobic bacterium isolated from a coastal lake.

A novel anaerobic chemoorganotrophic, facultatively alkaliphilic bacterium (strain M17 DMBT) was isolated from a coastal lake (Golubitsckoe, Taman Peninsula, Russia). Cells were motile rods, 1.6-2.1 µm long and 0.45 µm in diameter. The temperature range for growth was 14-42 °C, with an optimum at 30 °C. The pH range for growth was pH 5.5-10.0, with an optimum at pH 8.0-8.5. Growth of strain M17 DMBT was observed at NaCl concentrations of 1-12 % (w/v) with optimum growth at 1.5-2.0 %. Strain M17 MBTutilized glucose, fructose, sucrose, ribose, mannose, raffinose, arabinose, dextrin, yeast extract, peptone, carbon monoxide, vanillic acid and 3,4-dimethoxybenzoic acid. The end products from glucose fermentation were acetate and ethanol. The DNA G+C content of strain M17 DMBT was 39.1 mol%. The closest phylogenetic relative of strain M17 DMBT was Alkalibacter saccharofermentans with 97.8 % 16S rRNA gene sequence similarity. The OrthoANI value between M17 DMBT and A. saccharofermentans was 70.4 %. Based on the phenotypic, genotypic and phylogenetic characteristics of the isolate, strain M17 DMBT is considered to represent a novel species of the genus Alkalibacter for which the name Alkalibacter mobilis sp. nov. is proposed. The type strain of Alkalibacter mobilis is M17 DMBT (=KCTC 15920T=VKM B-3408T).

Characteristics of bacterial and yeast microbiomes in spontaneous and mixed-fermentation beer and cider.

The production of experimental beer and cider products has increased, worldwide. The complex microbiomes found in these beverages affect their organoleptic qualities and chemical compositions and can have diverse impacts on human health. The total diversity of a microbiome can be elucidated through the use of high-throughput sequencing and comprehensive data analysis tools. We analysed the bacterial and yeast microbiomes found in mixed and spontaneously fermented beers (n = 14) and unpasteurised apple ciders (n = 6), using high-throughput 16S rRNA and internal transcribed spacer (ITS) sequencing. The ratio of bacteria to yeast was measured using quantitative polymerase chain reaction (qPCR), and short-chain organic acids were analysed using high-performance liquid chromatography (HPLC). An upgraded version of the Knomics-Biota system was used to analyse the data. The microbiomes included both starter microorganisms and those that originate from the production environment and the raw materials. In addition to the common Saccharomyces and Brettanomyces, the yeast diversity included many non-conventional species. The bacterial community in beer was dominated by Lactobacillus species, whereas these communities were more diverse in cider. Lactobacillus acetotolerans was prevalent in wild ales, whereas Candida ethanolica was prevalent in cask-matured beverages. We observed complex patterns of subspecies-level yeast diversity across beer styles, breweries, and countries. Our study represents an exploratory analysis of non-conventional beer and cider microbiomes and metabolomes, which contributes information necessary to develop improved quality control processes and may drive innovative product development in experimental and artisanal brewing.

Respiratory Pathways Reconstructed by Multi-Omics Analysis in Melioribacter roseus, Residing in a Deep Thermal Aquifer of the West-Siberian Megabasin.

Melioribacter roseus, a representative of recently proposed Ignavibacteriae phylum, is a metabolically versatile thermophilic bacterium, inhabiting subsurface biosphere of the West-Siberian megabasin and capable of growing on various substrates and electron acceptors. Genomic analysis followed by inhibitor studies and membrane potential measurements of aerobically grown M. roseus cells revealed the activity of aerobic respiratory electron transfer chain comprised of respiratory complexes I and IV, and an alternative complex III. Phylogeny reconstruction revealed that oxygen reductases belonged to atypical cc(o/b)o3 -type and canonical cbb3 -type cytochrome oxidases. Also, two molybdoenzymes of M. roseus were affiliated either with Ttr or Psr/Phs clades, but not with typical respiratory arsenate reductases of the Arr clade. Expression profiling, both at transcripts and protein level, allowed us to assign the role of the terminal respiratory oxidase under atmospheric oxygen concentration for the cc(o/b)o3 cytochrome oxidase, previously proposed to serve for oxygen detoxification only. Transcriptomic analysis revealed the involvement of both molybdoenzymes of M. roseus in As(V) respiration, yet differences in the genomic context of their gene clusters allow to hypothesize about their distinct roles in arsenate metabolism with the 'Psr/Phs'-type molybdoenzyme being the most probable candidate respiratory arsenate reductase. Basing on multi-omics data, the pathways for aerobic and arsenate respiration were proposed. Our results start to bridge the vigorously increasing gap between homology-based predictions and experimentally verified metabolic processes, what is especially important for understudied microorganisms of novel lineages from deep subsurface environments of Eurasia, which remained separated from the rest of the biosphere for several geological periods.

A methylaspartate cycle in haloarchaea.

Access to novel ecological niches often requires adaptation of metabolic pathways to cope with new environments. For conversion to cellular building blocks, many substrates enter central carbon metabolism via acetyl-coenzyme A (acetyl-CoA). Until now, only two such pathways have been identified: the glyoxylate cycle and the ethylmalonyl-CoA pathway. Prokaryotes in the haloarchaea use a third pathway by which acetyl-CoA is oxidized to glyoxylate via the key intermediate methylaspartate. Glyoxylate condensation with another acetyl-CoA molecule yields malate, the final assimilation product. This cycle combines reactions that originally belonged to different metabolic processes in different groups of prokaryotes, which suggests lateral gene transfer and evolutionary tinkering of acetate assimilation. Moreover, it requires elevated intracellular glutamate concentrations, as well as coupling carbon assimilation with nitrogen metabolism.