The genome assembly and detection of biosynthetic gene clusters among four novel microbial isolates from deep subsurface rock biofilms

Tunnels in deep underground mines form a unique interface between surface and subsurface habitats, where surface microbes can enter through ventilation systems. These extreme conditions foster microbial adaptations, including the potential to produce novel natural products of biotechnological and medicinal importance. In this study, we characterized the genomes of four novel isolates from biofilms collected in a previously abandoned gold mine, used as a model underground system. Samples were obtained from thin, whitish, glistening biofilms (“cave silver”) on rock walls at 1,478 meters depth in SURF. From these samples, 10 GB of high-quality whole-genome sequences were generated, assembled, and annotated using multiple databases. The genomes were further analyzed for biosynthetic gene clusters (BGCs) encoding secondary metabolites using antiSMASH and NaPDoS. Draft assemblies revealed four genomes with diverse lengths and annotated features, including gene clusters associated with quorum sensing (QS). Several BGC-encoded compounds were identified, such as polyketides (PKS, PKS III, ketosynthase domain-KS), non-ribosomal peptides (NRPS, condensation domains), and terpenoids (terpenes I, polyenes type II). Enzyme analyses indicated both shared and unique activities among strains, highlighting potential cooperative and specialized functions within the biofilms. Additionally, CRISPR peptides were detected in three of the four strains. This work demonstrates how microbes colonizing extreme subsurface environments evolve specialized genomic features. Their biosynthetic capacity suggests the potential for producing natural products and secondary metabolites with applications in biotechnology and medicine.