Comparative genomics of the Nap2-2B clade reveals substrate partitioning and niche diversification among uncultured hydrocarbon-degrading Desulfotomaculales.

Abstract

Uncultured Nap2-2B bacteria (order Desulfotomaculales; formerly family Peptococcaceae) are frequently detected in methanogenic hydrocarbon-degrading environments, yet their metabolic diversity remains poorly understood. Here, we analysed 17 GTDB r232 metagenome-assembled genomes (MAGs) from four genera within this clade. A bac120 phylogeny places Nap2-2B as a monophyletic family-level lineage within Desulfotomaculales. Glycyl radical enzyme phylogeny and operon context reveal strict substrate partitioning: SCADC1-2-3 encodes alkylsuccinate synthase for aliphatic hydrocarbon activation, 46-80 and UBA4053 encode benzylsuccinate synthase for aromatic activation, and JAIMBK01 lacks hydrocarbon activation genes but retains complete dissimilatory sulfate reduction pathway genes. Pangenome-level pathway reconstruction identifies complementary cofactor biosynthetic potential, notably in cobalamin and pantothenate biosynthesis, consistent with possible cofactor complementation. Genome-scale metabolic modeling suggests that the alkane-degrading SCADC1-2-3 lineage can support syntrophic hexane degradation, whereas the aromatic lineage cannot grow on the alkane FBA test because it lacks AssA and PFOR. A parallel aromatic-substrate FBA for 46-80 MAGs did not yield growth under minimal curation, reflecting the greater complexity of the downstream benzoyl-CoA pathway. Together, these data support a syntrophic guild structured by substrate partitioning, putative cofactor complementation, and distinct electron-disposal strategies that may shape methanogenic hydrocarbon attenuation in anoxic tailings environments.