Terrestrial hydrothermal environments are formed by the release of high-temperature groundwater to the earth's surface. Thermal water sources can be either natural or anthropogenic, e. g. arising from deep well drilling. Sites of geothermal water discharge are commonly characterized by the development of microbial mats and sediment deposition (Slobodkin and Slobodkina, 2014). Thermophilic microorganisms inhabiting such ecosystems have important evolutionary and biotechnological significance. Their ability to thrive at elevated temperatures offers insights into early life evolution and the molecular adaptations that enabled survival under extreme conditions similar to those of the primordial Earth. Moreover, these organisms produce thermostable enzymes and metabolites that are valuable for biofuel production, pharmaceutical applications, and the food and paper industries (Wagner and Wiegel, 2008; DeCastro et al., 2016).Culture-independent analysis of thermophilic microbial communities from continental hot springs has revealed a high phylogenetic diversity and the presence of numerous uncultured taxa (Mardanov et al., 2011; Menzel et al., 2015; Merkel et al., 2017; Podar et al., 2020; Toshchakov et al., 2021; Malygina et al., 2023; George et al., 2023).

Exploring the structure and metabolic potential of thermophilic microbial communities in new geographic locations expand our understanding of the diversity and functioning of these extreme ecotopes. In the Republic of Uzbekistan, natural mineral springs with different temperature are known in several regions, including Jizzakh, Kashkadarya, and Surkhandarya. These environments have been studied primarily by geologists in the context of geothermal anomalies and for their potential as sources of drinking and therapeutic mineral water (Zui et al., 2020; Tashpulatova et al., 2024). Hot waters also emerge from man-made artesian wells. One of these well is located in the Navoi region, where it discharges high thermal groundwater from Paleozoic deposits at the depth 150–300 m. The outflow forms a free-flowing surface stream covered by microbial and algal mats; the stream is used by the local population for domestic needs. To date, no culture-independent microbiological surveys of thermal environments in Uzbekistan have been reported.

Numerous uncultivated high-rank taxa of prokaryotes have been described based solely on data of metagenome-assembled genomes (MAGs) (Brown et al., 2015; Chuvochina et al., 2023). Among these, there are several classes of Desulfobacterota, a phylum with a broad range of metabolic capabilities (Murphy et al., 2021). According to the Genome Taxonomy Database (GTDB release 10-RS226) Desulfobacterota comprises 14 classes with proposed names, as well as 22 unclassified class-level lineages (Parks et al., 2021). Although the physiological characteristics of microorganisms cannot be unambiguously inferred from genomic data alone, some cultured members of Desulfobacterota are thermophiles, and the classes Thermodesulfobacteria and Dissulfuribacteria contain only thermophilic species.

Another example of a high-ranking bacterial taxon is the candidate phylum WOR-3, as classified in GTDB (release 10-RS226). The name WOR-3 (White Oak River group) was introduced by Baker et al. (2015) for a candidate phylum of uncultivated bacteria, based on MAGs recovered from estuarine sediments of the White Oak River (North Carolina, USA) and 16S rRNA gene sequences mainly from marine and estuarine environments. In 2017, MAGs related to WOR-3 group were identified in Guaymas Basin sediments and the name ‘Candidatus Stahlbacteria’ was proposed for this lineage (Dombrowski et al., 2017). That same year, MAGs related to EM3 bacterial lineage originally discovered in Octopus Spring (Yellowstone National Park, USA), were identified in subsurface hydrothermal fluids from the Juan de Fuca Ridge flank and the name Hydrothermae was proposed to EM3 group (Reysenbach et al., 1994; Jungbluth et al., 2017). The phylogenetic tree presented in the study of Jungbluth et al. (2017) shows that EM3 and WOR-3 groups form two adjacent branches within the same cluster. EM3 related MAGs recovered from Yellowstone National Park thermal springs were earlier assigned to ‘Ca. Caldipriscus’ and ‘Ca. Thermoproauctor’. These taxa formed a distinct clade initially named ‘Ca. Pyropristinus’ (Colman et al., 2016), which was later corrected to ‘Ca. Caldipriscota’ in accordance with the International Code of Nomenclature of Prokaryotes, (Oren and Göker, 2023).

Currently, three names are associated with this phylum: ‘Candidtus Stahliibacteriota’ (without a designated type genus), ‘Candidatus Caldipriscota’ (with the type genus ‘Ca. Caldipriscus’), and ‘Candidatus. Hydrothermota’ (including ‘Ca. Hydrothermus pacificus’) (Chuvochina et al., 2019; Oren and Göker, 2023). In the Genome Taxonomy Database (GTDB release R226), the phylum remains designated as WOR-3. It is divided into seven classes, including WOR-3 sensu stricto and ‘Ca. Hydrothermia’, which encompasses ‘Ca. Caldipriscus’ and related taxa.

The phylum WOR-3 currently includes more than 380 MAGs that have been detected in a variety of environments, although detailed physiological insights have remained limited. Until recently, only three species represented by MAGs had been formally described with Candidatus status: ‘Ca. Caldipriscus sp. T1’, ‘Ca. Thermoproauctor sp. T2”, and ‘Ca. Hydrothermus pacificus' (Colman et al., 2016; Chuvochina et al., 2019). The recent isolation in pure culture of strain sy37, as the first cultivable WOR-3 representative (Mori et al., 2025), has significantly advanced our understanding of the physiology and ecological role of this lineage. Based on metagenome-assembled genomes, WOR-3 taxa were predicted to be heterotrophic, with potential for aerobic respiration (Baker et al., 2015; Colman et al., 2016; Dombrowski et al., 2017; Chuvochina et al., 2019). These predictions were confirmed by the metabolic properties of strain sy37, which is a thermophilic, microaerophilic, chemoheterotrophic rod capable of both aerobic and anaerobic respiration using elemental sulfur as terminal electron acceptors.

Here, we present results of the metagenomic study of microbial communities from a geothermal environment in Uzbekistan and taxonomically assign two of the recovered MAGs to a novel class within Desulfobacterota and a novel class within candidate phylum WOR-3 (GTDB taxonomy).