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Abstract The great majority of microorganisms are as‐yet‐uncultivated, mostly found in extreme environments. High‐throughput sequencing provides data‐rich genomes from single‐cell and metagenomic techniques, which has enabled researchers to obtain a glimpse of the unexpected genetic diversity of “microbial dark matter.” However, cultivating microorganisms from extreme environments remains essential for dissecting and utilizing the functions of extremophiles. Here, we provide a straightforward protocol for efficiently isolating prokaryotic microorganisms from different extreme habitats (thermal, xeric, saline, alkaline, acidic, and cryogenic environments), which was established through previous successful work and our long‐term experience in extremophile resource mining. We propose common processes for extremophile isolation at first and then summarize multiple cultivation strategies for recovering prokaryotic microorganisms from extreme environments and meanwhile provide specific isolation tips that are always overlooked but important. Furthermore, we propose the use of multi‐omics‐guided microbial cultivation approaches for culturing these as‐yet‐uncultivated microorganisms and two examples are provided to introduce how these approaches work. In summary, the protocol allows researchers to significantly improve the isolation efficiency of pure cultures and novel taxa, which therefore paves the way for the protection and utilization of microbial resources from extreme environments.

Abstract Biological denitrification is a crucial process in the nitrogen biogeochemical cycle, and Thermus has been reported to be a significant heterotrophic denitrifier in terrestrial geothermal environments. However, neither the denitrification potential nor the evolutionary history of denitrification genes in the genus Thermus or phylum Deinococcota is well understood. Here, we performed a comparative analysis of 23 Thermus genomes and identified denitrification genes in 15 Thermus strains. We confirmed that Thermus harbors an incomplete denitrification pathway as none of the strains contain the nosZ gene. Ancestral character state reconstructions and phylogenetic analyses showed that narG, nirS, and norB genes were acquired by the last common ancestor of Thermales and were inherited vertically. In contrast, nirK of Thermales was acquired via two distinct horizontal gene transfers from Proteobacteria to the genus Caldithermus and from an unknown donor to the common ancestor of all known Thermus species except Thermus filiformis. This study expands our understanding of the genomic potential for incomplete denitrification in Thermus, revealing a largely vertical evolutionary history of the denitrification pathway in the Thermaceae, and supporting the important role for Thermus as an important heterotrophic denitrifier in geothermal environments.