Comparative genomics of the Smithellaceae: reductive evolution and a mobile alkane-activation module in Smithella.

Smithella are repeatedly implicated as primary alkane activators in methanogenic hydrocarbon-degrading communities, yet the propionate-oxidizing type species and many relatives cannot activate alkanes, and the genus's diversity and functional differentiation remain poorly resolved. Here, we present genus-scale comparative genomics of Smithella across 108 Smithellaceae metagenome-assembled genomes from three genera. The 74 Smithella genomes resolve into 23 species, 15 of them unnamed. Alkylsuccinate synthase occurs as two paralogous families, A and B. They differ by a third of their residues and coexist in the same genome. Both reject the species topology, family B decisively. assA has been gained and lost independently in several lineages. The families differ in mobility: family A is single-copy, confined to two species and in a conserved neighbourhood, whereas family B duplicates within genomes, spans four species and occupies a far less conserved context, most often beside a 2-hydroxyisobutanoyl-CoA mutase. Reductive specialization is pervasive, including genus-wide loss of flagellar motility, dissimilatory sulfite reduction, and de novo cobamide (vitamin B₁₂) biosynthesis. In consortium models, both lineages behaved equivalently on propionate, the substrate they share, and a hydrogenotrophic partner was obligatory for alkane oxidation, for which H₂ and formate are the only electron acceptors. The genus appears to favour propionate dismutation, thermodynamically preferred when hydrogen removal is incomplete; both lineages have largely lost the methylmalonyl-CoA epimerase most sister genomes retain. Alkane activation is therefore a mobile accessory on an otherwise conserved syntrophic core. Acquiring it widens the substrate range while tightening the requirement for a hydrogenotrophic partner.