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

ABSTRACT Soils harbor immense biosynthetic gene cluster (BGC) diversity that mediates microbial interactions, yet this potential remains unevenly mapped and poorly characterized across diverse bacterial lineages. Ktedonobacteria (phylum Chloroflexota ) are an actinomycete-like lineage widely distributed in terrestrial soils, including oligotrophic volcanic deposits; however, their secondary metabolism and genome architecture remain poorly characterized. Here, we integrate targeted cultivation from volcanic soils at Mount Zao (Japan) with genome-resolved metagenomics and comparative analysis of public genomes to examine biosynthetic potential across 183 ktedonobacterial genomes. We identified 1,546 BGCs and grouped them into 1,162 non-redundant gene-cluster families (GCFs) using antiSMASH and BiG-SLiCE. Nearly one quarter of genomes encoded ≥10 distinct GCFs, and several family-level clades exhibited high GCF richness that approached that of Streptomyces within our data set, highlighting a putatively biosynthetically rich yet underexplored soil bacterial lineage. Most ktedonobacterial BGCs were highly divergent from current reference collections and exhibited unusually low intra-genomic redundancy, suggesting broad putative chemical diversity. Long-read assemblies from 10 cultured strains revealed recurrent 1.6–3.5 Mb ECE-like contigs with chromid-like features, but distinct maintenance features. These replicons were consistently enriched in BGCs and mobility-associated genes, with mobility loci concentrated near BGC boundaries. Collectively, our results expand the phylogenetic landscape of soil biosynthetic diversity and highlight ECE-like contigs as major genomic reservoirs for secondary metabolism in Ktedonobacteria . IMPORTANCE Soil bacteria produce many of the small molecules that become medicines and help microbes interact with each other. Yet most of this chemical diversity remains unexplored because many soil lineages are difficult to cultivate and remain genomically underrepresented. Much of what we know comes from well-studied groups such as actinomycetes, leaving many soil lineages largely unexplored. We analyzed 183 genomes from Ktedonobacteria , an actinomycete-like group within the phylum Chloroflexota that is widespread in terrestrial soils, including nutrient-poor volcanic deposits. We uncovered a large and diverse set of gene clusters predicted to produce secondary metabolites, many of which lack close counterparts in current reference collections. We also show that these clusters are concentrated on large ECE-like contigs with chromid-like features, pointing to a dedicated genomic reservoir that can accumulate and reshuffle biosynthetic traits. Our results expand the known sources of soil biosynthetic diversity and provide a foundation for future cultivation and functional characterization of Ktedonobacteria metabolites.