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Deep-subsurface hot brines in northwest Poland, extracted through boreholes reaching 1.6 and 2.6 km below the ground surface, were microbiologically investigated using culture-independent and culture-dependent methods. The high-throughput sequencing of 16S rRNA gene amplicons showed a very low diversity of bacterial communities, which were dominated by phyla Proteobacteria and Firmicutes Bacterial genera potentially involved in sulfur oxidation and nitrate reduction ( Halothiobacillus and Methylobacterium ) prevailed in both waters over the sulfate reducers (" Candidatus Desulforudis" and Desulfotomaculum ). Only one archaeal taxon, affiliated with the order Thermoplasmatales, was detected in analyzed samples. Bacterial isolates obtained from these deep hot brines were closely related to Bacillus paralicheniformis based on the 16S rRNA sequence similarity. However, genomic and physiological analyses made for one of the isolates, Bacillus paralicheniformis strain TS6, revealed the existence of more diverse metabolic pathways than those of its moderate-temperature counterpart. These specific traits may be associated with the ecological adaptations to the extreme habitat, which suggest that some lineages of B. paralicheniformis are halothermophilic. IMPORTANCE Deep-subsurface aquifers, buried thousands of meters down the Earth's crust, belong to the most underexplored microbial habitats. Although a few studies revealed the existence of microbial life at the depths, the knowledge about the microbial life in the deep hydrosphere is still scarce due to the limited access to such environments. Studying the subsurface microbiome provides unique information on microbial diversity, community structure, and geomicrobiological processes occurring under extreme conditions of the deep subsurface. Our study shows that low-diversity microbial assemblages in subsurface hot brines were dominated by the bacteria involved in biogeochemical cycles of sulfur and nitrogen. Based on genomic and physiological analyses, we found that the Bacillus paralicheniformis isolate obtained from the brine under study differed from the mesophilic species in the presence of specific adaptations to harsh environmental conditions. We indicate that some lineages of B. paralicheniformis are halothermophilic, which was not previously reported., (Copyright © 2020 Kalwasińska et al.)