Comparative genomics of the Nap2-2B clade reveals substrate partitioning, niche diversification, and reciprocal cofactor auxotrophies among uncultured hydrocarbon-degrading Peptococcaceae

Uncultured Peptococcaceae of the Nap2-2B clade are frequently detected in methanogenic hydrocarbon-degrading environments, yet their metabolic diversity remains poorly understood. Here, we analyse 34 metagenome-assembled genomes spanning four genera within this clade. Phylogenomic analysis of 741 Desulfotomaculales genomes places Nap2-2B as a monophyletic family-level lineage. 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 sulfate reduction. Pangenome-level pathway reconstruction identifies complementary cofactor biosynthetic potential, notably in cobalamin and pantothenate biosynthesis, consistent with possible cofactor complementation. Genome-scale metabolic modeling further indicates that the alkane-degrading lineage can support syntrophic hexane degradation, whereas the aromatic lineage cannot under the modeled conditions because it lacks pyruvate:ferredoxin oxidoreductase. Together, these data support a tightly integrated syntrophic guild in which substrate partitioning, possible cofactor complementation, and distinct electron-disposal strategies may structure community assembly, shape carbon and electron flow, and influence methanogenic hydrocarbon attenuation in anoxic tailings environments.