Abstract Description

Bacteria of the phylum Actinomycetota are renowned for their potential to produce antimicrobial natural products and are widely distributed across diverse ecological environments. Actinomycetes associated with stromatolites have been underexplored, leaving a significant knowledge gap regarding their secondary metabolites and associated ecological roles. This study investigates the antimicrobial potential of actinobacteria isolated from stromatolite formations, leveraging bioassay-guided metabologenomics. From 108 actinobacterial isolates spanning 18 genera, eight demonstrated antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The whole genome phylogenomic analysis of these active strains revealed a novel Mycobacterium species, designated AMRU185. Biosynthetic gene cluster (BGC) analysis identified 36 putative BGCs, with non-ribosomal peptide synthetases, polyketides, and terpenes comprising the majority. BiG-SCAPE analysis confirmed that these clusters represented diverse BGC families, with some showing less than 40 % similarity to known clusters, indicating a high probability of novel metabolite production. Metabolomic profiling further highlighted distinct secondary metabolite profiles across isolates, with a member of the actinomycin family of compounds actinomycin D and collismycin A identified as major contributors to the observed antimicrobial effects. Stromatolite-derived actinobacteria as a rich source of novel antibacterial compounds, offering insights into the biosynthetic diversity of extremophilic actinobacteria and their potential applications in antimicrobial drug discovery.