Members of the genus Thermodesulfovibrio belong to the Nitrospirae phylum and all isolates characterized to date are neutrophiles. They have been isolated from terrestrial hot springs and thermophilic methanogenic anaerobic sludges. Their molecular signatures have, however, also been detected in deep subsurface. The purpose of this study was to characterize and analyze the genome of a newly isolated, facultatively alkaliphilic Thermodesulfovibrio from a 2 km deep aquifer system in Western Siberia, Russia. The new isolate, designated N1, grows optimally at pH 8.5 and at 65°C. It is able to reduce sulfate, thiosulfate or sulfite with a limited range of electron donors, such as formate, pyruvate, and lactate. Analysis of the 1.93 Mb draft genome of strain N1 revealed that it contains a set of genes for dissimilatory sulfate reduction, including sulfate adenyltransferase, adenosine-5′-phosphosulfate reductase AprAB, membrane-bound electron transfer complex QmoABC, dissimilatory sulfite reductase DsrABC, and sulfite reductase-associated electron transfer complex DsrMKJOP. Hydrogen turnover is enabled by soluble cytoplasmic, membrane-linked, and soluble periplasmic hydrogenases. The use of thiosulfate as an electron acceptor is enabled by a membrane-linked molybdopterin oxidoreductase. The N1 requirement for organic carbon sources corresponds to the lack of the autotrophic C1-fixation pathways. Comparative analysis of the genomes of Thermodesulfovibrio (T. yellowstonii, T. islandicus, T. àggregans, T. thiophilus, and strain N1) revealed a low overall genetic diversity and several adaptive traits. Consistent with an alkaliphilic lifestyle, a multisubunit Na+/H+ antiporter of the Mnh family is encoded in the Thermodesulfovibrio strain N1 genome. Nitrogenase genes were found in T. yellowstonii, T. aggregans, and T. islandicus, nitrate reductase in T. islandicus, and cellulose synthetase in T. aggregans and strain N1. Overall, our results provide genomic insights into metabolism of the Thermodesulfovibrio lineage in microbial communities of the deep subsurface biosphere.

We have sequenced metagenome of the microbial community of a deep subsurface thermal aquifer in the Tomsk Region of the Western Siberia, Russia. Our goal was the recovery of near-complete genomes of the community members to enable accurate reconstruction of metabolism and ecological roles of the microbial majority, including previously unstudied lineages. The water, obtained via a 2.6 km deep borehole 1-R, was anoxic, with a slightly alkaline pH, and a temperature around 45°C. Microbial community, as revealed by 16S rRNA gene profiling over 2 years, mostly consisted of sulfate-reducing Firmicutes and Deltaproteobacteria, and uncultured lineages of the phyla Chlorofexi, Ignavibacteriae and Aminicenantes (OP8). 25 composite genomes with more than 90% completeness were recovered from metagenome and used for metabolic reconstruction. Members of uncultured lineages of Chlorofexi and Ignavibacteriae are likely involved in degradation of carbohydrates by fermentation, and are also capable of aerobic and anaerobic respiration. The Chlorofexi bacterium has the Wood-Ljungdahl pathway of CO2 fixation. The recently identified candidate phylum Riflebacteria accounted for 5%-10% of microbial community. Metabolic reconstruction of a member of Riflebacteria predicted that it is an anaerobe capable to grow on carbohydrates by fermentation or dissimilatory Fe(III) reduction.

An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where it formed a single-species ecosystem fuelled by hydrogen from water radiolysis. It was believed that in situ conditions CDA relied on scarce energy supply and did not divide for hundreds to thousand years. We have isolated CDA strain BYF from a 2-km-deep aquifer in Western Siberia and obtained a laboratory culture growing with a doubling time of 28.5 h. BYF uses not only H 2 but also various organic electron donors for sulfate respiration. Growth required elemental iron, and ferrous iron did not substitute for it. A complex intracellular organization included gas vesicles, internal membranes, and electron-dense structures enriched in phosphorus, iron, and calcium. Genome comparison of BYF with the South African CDA revealed minimal differences mostly related to mobile elements and prophage insertions. Two genomes harbored <800 single-nucleotide polymorphisms and had nearly identical CRISPR loci. We suggest that spores with the gas vesicles may facilitate global distribution of CDA followed by colonization of suitable subsurface environments. Alternatively, a slow evolution rate in the deep subsurface could result in high genetic similarity of CDA populations at two sites spatially separated for hundreds of millions of years.

Microbial sulfate reduction in acid mine drainage is still considered to be confined to anoxic conditions, although several reports have shown that sulfate-reducing bacteria occur under microaerophilic or aerobic conditions. We have measured sulfate reduction rates of up to 60 nmol S cm(-3) day(-1) in oxidized layers of gold mine tailings in Kuzbass (SW Siberia). A novel, acidophilic, copper-tolerant Desulfosporosinus sp. I2 was isolated from the same sample and its genome was sequenced. The genomic analysis and physiological data indicate the involvement of transporters and additional mechanisms to tolerate metals, such as sequestration by polyphosphates. Desulfosporinus sp. I2 encodes systems for a metabolically versatile life style. The genome possessed a complete Embden-Meyerhof pathway for glycolysis and gluconeogenesis. Complete oxidation of organic substrates could be enabled by the complete TCA cycle. Genomic analysis found all major components of the electron transfer chain necessary for energy generation via oxidative phosphorylation. Autotrophic CO2 fixation could be performed through the Wood-Ljungdahl pathway. Multiple oxygen detoxification systems were identified in the genome. Taking into account the metabolic activity and genomic analysis, the traits of the novel isolate broaden our understanding of active sulfate reduction and associated metabolism beyond strictly anaerobic niches.

The goal of this work was to study the diversity of microorganisms inhabiting a deep subsurface aquifer system in order to understand their functional roles and interspecies relations formed in the course of buried organic matter degradation. A microbial community of a deep subsurface thermal aquifer in the Tomsk Region, Western Siberia was monitored over the course of 5 years via a 2.7 km deep borehole 3P, drilled down to a Palaeozoic basement. The borehole water discharges with a temperature of ca. 50°C. Its chemical composition varies, but it steadily contains acetate, propionate, and traces of hydrocarbons and gives rise to microbial mats along the surface flow. Community analysis by PCR-DGGE 16S rRNA genes profiling, repeatedly performed within 5 years, revealed several dominating phylotypes consistently found in the borehole water, and highly variable diversity of prokaryotes, brought to the surface with the borehole outflow. The major planktonic components of the microbial community were Desulfovirgula thermocuniculi and Methanothermobacter spp. The composition of the minor part of the community was unstable, and molecular analysis did not reveal any regularity in its variations, except some predominance of uncultured Firmicutes. Batch cultures with complex organic substrates inoculated with water samples were set in order to enrich prokaryotes from the variable part of the community. PCR-DGGE analysis of these enrichments yielded uncultured Firmicutes, Chloroflexi, and Ignavibacteriae. A continuous-flow microaerophilic enrichment culture with a water sample amended with acetate contained Hydrogenophilus thermoluteolus, which was previously detected in the microbial mat developing at the outflow of the borehole. Cultivation results allowed us to assume that variable components of the 3P well community are hydrolytic organotrophs, degrading buried biopolymers, while the constant planktonic components of the community degrade dissolved fermentation products to methane and CO2, possibly via interspecies hydrogen transfer. Occasional washout of minor community components capable of oxygen respiration leads to the development of microbial mats at the outflow of the borehole where residual dissolved fermentation products are aerobically oxidized. Long-term community analysis with the combination of molecular and cultivation techniques allowed us to characterize stable and variable parts of the community and propose their environmental roles.

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