<p>Permafrost microorganisms have received increased attention due to their critical role in biogeochemical cycles and the potential biosafety risks associated with climate warming. However, knowledge regarding the depth-related community structure and function of permafrost microorganisms remains limited. In this study, we employed metagenomic methods to investigate microbial communities, functional genes, and their controlling factors in alpine permafrost of the Shule River headwaters on the northeastern margin of the Qinghai-Tibet Plateau. A total of 287 metagenome-assembled genomes were constructed, representing 20 bacterial phyla and 1 archaeal phylum. Additionally, we identified <styled-content style-type="number">2079</styled-content> viral contigs spanning more than 14 viral families, with approximately 67% constituting previously unknown taxa, forming a unique virome in alpine permafrost compared to other regions. Significant variations in bacterial and viral compositions, along with their metabolic potentials, were observed across vertical profiles from the active layer to the permafrost table layer. Viral diversity showed an initial increase followed by a decrease, reaching the maximum at the depth of 90-140 cm. We identified abundant genomic capabilities related to carbon, nitrogen, and sulfur cycling. Moreover, our analysis revealed 60 auxiliary metabolic genes in viruses and 7,000 putative biosynthetic gene clusters for secondary metabolites from 21 prokaryotic phyla. Soil temperature emerged as the most significant environmental variable influencing the composition of microbial communities and functional genes, as well as the diversity of microbial communities. These results offer valuable insights into the potential functional transformations and biosafety risks mediated by permafrost microorganisms under future warming.</p>
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