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bioRxiv · 10.1101/2025.09.13.675994

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

Agany, D. D. M.·Zylla, J. L.·Aryal, S.·Gorbatenko, O.·Bergmann, D.·Gadhamshetty, V.·Gnimpieba, E. Z.
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Abstract

Tunnels in deep underground mines provide a unique interface between the surface and deep subsurface habitats. There, microbes from the surface may enter the deep tunnels as surface air is drawn into the deep mine tunnels to provide ventilation. This extreme hosting environment provides a condition for microbes to develop novel capabilities, such as the production of natural products of biotechnology or medicinal importance. This study characterized the genomes of four novel isolates from deep subsurface biofilms of the previously abandoned gold mine, which was used as a model for the underground study. Here the microbiome samples were obtained from thin, whitish, glistening biofilm samples naturally formed on the rock walls (1478 meters deep at SURF). These samples are herein referred to as "cave silver" biofilms. The samples provide 10 GB of high-quality whole genome sequences that were assembled into contigs/scaffolds and structurally and functionally annotated against various databases. Subsequently, the genomes were analyzed for biosynthetic gene clusters (BGCs) encoding secondary metabolites of biotechnology and medical importance using the antiSMASH and the NaPDoS web servers, respectively. In brief, the assemblies produced four drafted genomes of different lengths and annotated features for each strains genome, including gene clusters involved in the quorum sensing (QS) pathway. Several BGC-encoded secondary metabolites of natural products or compounds such as polyketides (PKS, PKS III, ketosynthase domain-KS), non-ribosomal peptides (condensation domain, NRPS), and terpenoids (terpenes I, polyenes type II), were identified. Furthermore, many overexpressed enzymes, most of which are shared among the strains and some unique to individual bacteria strains, were identified, revealing possible shared and individualized strain activities in the biofilms during the sample collection. CRISPR peptides were also detected in three of the four bacteria strains. In this study, we examine the genomes of microbes colonizing extreme subsurface environments and how these conditions promote the development of microbial systems that are potentially capable of producing medicinally natural products and secondary metabolites that may be used to enhance advancements in biotechnology products. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/675994v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@17a82f0org.highwire.dtl.DTLVardef@2a73f0org.highwire.dtl.DTLVardef@121c3e9org.highwire.dtl.DTLVardef@1c51f9b_HPS_FORMAT_FIGEXP M_FIG C_FIG ImportanceDeep subsurface environments host unique microbial communities with specialized adaptations. By characterizing genomes from "cave silver" biofilms, this study reveals biosynthetic gene clusters and pathways for secondary metabolite production, including polyketides, peptides, and terpenoids. These findings highlight the potential of extreme-environment microbes as a novel source of biotechnologically and medically valuable natural products.

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Agany, D. D. M., Zylla, J. L., Aryal, S., Gorbatenko, O., Bergmann, D., Gadhamshetty, V., Gnimpieba, E. Z.. 2025-09-14. The genome assembly and detection of biosynthetic gene clusters among four novel microbial isolates from deep subsurface rock biofilms. https://doi.org/10.1101/2025.09.13.675994

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