Chromid-like secondary replicons as key sites of biosynthetic gene clusters in Ktedonobacteria

Abstract Soils harbour immense biosynthetic gene cluster (BGC) diversity that can mediate microbial interactions, yet this potential is still mapped unevenly across the tree of life. Ktedonobacteria —a class of actinomycete-like bacteria within phylum Chloroflexota —are widespread in terrestrial environments and repeatedly dominate pioneer communities in extremely oligotrophic volcanic bare-ground soils; however, their secondary metabolism and genome architecture remain poorly characterised. Here, we integrate targeted cultivation using volcanic soils from Mount Zao with genome-resolved metagenomics and public genomes to analyse 183 ktedonobacterial genomes. Using antiSMASH and BiG-SLiCE, we identified 1,546 BGCs comprising 1,162 non-redundant gene-cluster families (GCFs). In our dataset, nearly one quarter of genomes encode ≥10 distinct GCFs, and several family-level clades show mean GCF counts comparable to those in genus Streptomyces . Most ktedonobacterial BGCs are highly divergent from reference collections and exhibit unusually low intra-genomic redundancy, suggesting broad, underexplored chemotypes. Long-read assemblies from ten strains reveal recurrent 1.6–3.5 Mb chromid-like secondary replicons with chromosome-like composition but distinct maintenance signatures. These replicons are consistently enriched in BGCs and mobility-associated genes, with mobility loci concentrated near BGC boundaries. Collectively, our results expand the current knowledge of the phylogenetic landscape of soil biosynthetic diversity and highlight chromid-like secondary replicons as major genomic reservoirs for specialised metabolism in Ktedonobacteria .