Karnachuk, Olga V.

The class Limnochordia harbors a single cultivated member, the mesophilic Limnochorda pilosa, which was isolated from a meromictic lake. Despite numerous molecular signatures reported in various ecosystems, the ecophysiological versatility of this deeply branched lineage of Firmicutes (Bacillota) remains poorly understood. The objective of this study was to use targeted cultivation, based on metagenome-assembled genomes from a deep terrestrial aquifer in Western Siberia, to isolate two new thermophilic members of the class. These isolates, described as Geochorda subterranea gen. nov. sp. nov. and Carboxydochorda subterranea gen. nov. sp. nov. within the Geochordaceae fam. nov., were capable of both anaerobic and aerobic respiration using fumarate and O2, respectively, with simple sugars as electron donors. The cultivated Geochordaceae have demonstrated fermentative growth and degradation of various polymers, including starch, maltose, maltodextrin, xylan, and chitin. The carboxydotrophic C. subterranea sp. nov. exhibited autotrophic growth via the Calvin–Benson–Bassham cycle, using CO, H2, and formate as electron donors and O2 as an electron acceptor, adding metabolic flexibility to the bacterium in the nutrient-depleted “deep biosphere” and supporting the possibility of aerobic metabolism in the deep subsurface. The broad physiological potential deciphered from physiological experiments and comparative genomic data explains the widespread distribution of uncultivated members of the class Limnochordia in various ecosystems, where they can oxidize complex organic substrates through both aerobic and anaerobic respiration, as well as pursue a chemolithotrophic lifestyle through the oxidation of H2 or CO.

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and “Candidatus Thiothrix anitrata” sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.

The candidate phyla radiation is a large monophyletic lineage comprising unculturable bacterial taxa with small cell and genome sizes, mostly known from genomes obtained from environmental sources without cultivation. Here, we present the closed complete genome of a member of the superphylum Microgenomates obtained from the metagenome of a deep subsurface thermal aquifer. Phylogenetic analysis indicates that the new bacterium, designated Ch65, represents a novel phylum-level lineage within the Microgenomates group, sibling to the candidate phylum Collierbacteria. The Ch65 genome has a highly unusual nucleotide composition with one strand of highly enriched in cytosine versus guanine throughout the whole length. Such nucleotide composition asymmetry, also detected in the members of Ca. Collierbacteria and Ca. Beckwithbacteria, suggests that most of the Ch65 chromosome is replicated in one direction. A genome analysis predicted that the Ch65 bacterium has fermentative metabolism and could produce acetate and lactate. It lacks respiratory capacity, as well as complete pathways for the biosynthesis of lipids, amino acids, and nucleotides. The Embden–Meyerhof glycolytic pathway and nonoxidative pentose phosphate pathway are mostly complete, although glucokinase, 6-phosphofructokinase, and transaldolase were not found. The Ch65 bacterium lacks secreted glycoside hydrolases and conventional transporters for importing sugars and amino acids. Overall, the metabolic predictions imply that Ch65 adopts the lifestyle of a symbiont/parasite, or a scavenger, obtaining resources from the lysed microbial biomass. We propose the provisional taxonomic assignment ‘Candidatus Chazhemtobacterium aquaticus’, genus ‘Chazhemtobacterium‘, family ‘Chazhemtobacteraceae‘ in the Microgenomates group.

AbstractThermal ecosystems associated with underground coal combustion sites are rare and less studied than geothermal features. Here we analysed microbial communities of near-surface ground layer and bituminous substance in an open quarry heated by subsurface coal fire by metagenomic DNA sequencing. Taxonomic classification revealed dominance of only a few groups of Firmicutes. Near-complete genomes of three most abundant species, ‘Candidatus Carbobacillus altaicus’ AL32, Brockia lithotrophica AL31, and Hydrogenibacillus schlegelii AL33, were assembled. According to the genomic data, Ca. Carbobacillus altaicus AL32 is an aerobic heterotroph, while B. lithotrophica AL31 is a chemolithotrophic anaerobe assimilating CO2 via the Calvin cycle. H. schlegelii AL33 is an aerobe capable of both growth on organic compounds and carrying out CO2 fixation via the Calvin cycle. Phylogenetic analysis of the large subunit of RuBisCO of B. lithotrophica AL31 and H. schlegelii AL33 showed that it belongs to the type 1-E. All three Firmicutes species can gain energy from aerobic or anaerobic oxidation of molecular hydrogen, produced as a result of underground coal combustion along with other coal gases. We propose that thermophilic Firmicutes, whose spores can spread from their original geothermal habitats over long distances, are the first colonizers of this recently formed thermal ecosystem.

Summary A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M ‐2 T was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46° C ) coming out of a 2775‐m‐deep oil exploration well ( T omsk region, R ussia). Strain P3M ‐2 T is a moderate thermophile and facultative anaerobe growing on mono‐, di‐ or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, F e( III ), As ( V )]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is I gnavibacterium album , the only chemoorganotrophic member of the phylum C hlorobi . New genus and species M elioribacter roseus are proposed for isolate P3M ‐2 T . Together with I . album , the new organism represents the class I gnavibacteria assigned to the phylum Chlorobi . The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1 . Phylogenetic analysis of M . roseus and I . album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum‐level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes – C hlorobi group with a sole class I gnavibacteria , two families I gnavibacteriaceae and M elioribacteraceae and two species I . album and M . roseus . This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of C hlorobi . The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically.