Contrasting gene gain and loss shape metabolic diversification in the Ralstonia solanacearum species complex

Metabolic repertoires are continually remodeled by gene acquisition and loss, but how these contrasting processes jointly generate phenotypic diversity within bacterial species complexes remains poorly understood. Here, we elucidated the genetic and evolutionary basis of the classical biovar phenotypes of the Ralstonia solanacearum species complex (RSSC), which are defined by differential utilization of three hexitols (sorbitol, mannitol, and dulcitol) and three disaccharides (lactose, maltose, and cellobiose). Although this classification has been used for over half a century, the genetic determinants and evolutionary origins of these phenotypes have not been systematically resolved. Machine-learning analysis of genomic data from 250 RSSC strains with documented biovar phenotypes, combined with genome-wide association analysis and gene deletion, identified the smoEFGK gene cluster and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (FADGDH) as the major determinants of hexitol and disaccharide utilization, respectively. Comparative phylogenomics revealed fundamentally different evolutionary histories of these loci. An ancestral smoEFGK system was lost early in RSSC evolution and subsequently reacquired by the ancestor of phylotype I through horizontal transfer from a Burkholderia-related lineage, whereas vertically inherited FADGDH underwent recurrent, independent losses across multiple RSSC lineages. Extending the analysis to 1,082 RSSC strains further showed that disaccharide-utilization capacity was significantly depleted among monocot-associated strains after accounting for phylogenetic relatedness and geographic origin. Together, these findings reveal how contrasting trajectories of gene gain and loss can reshape bacterial metabolic repertoires, generate lineage-associated phenotypic diversity, and potentially contribute to ecological differentiation.