Divergent evolutionary trajectories and mechanistic insights into arsenite and antimonite adaptation in the bacterium Achromobacter sp. As-55

Microbes in mining areas face multifaceted stress from arsenic (As) or antimony (Sb) exposure, yet their distinct adaptations to high levels of arsenite (As(III)) or antimonite (Sb(III)) are not well understood. In this study, we subjected the As(III)-oxidizing bacterium Achromobacter sp. As-55 to laboratory evolution under As(III) or Sb(III) stress, yielding strains capable of oxidizing As(III) under very high levels of As(III)/Sb(III) and displaying increased dual metalloid resistance. Our results showed that Sb(III)-evolved populations achieved hyper-resistance to Sb(III) and antibiotics, a phenotype linked to a significantly higher mutational load (26-254) compared to As(III)-evolved lineages (9-28). Under prolonged exposure to high concentrations of As(III) or Sb(III), the As-55 strain evolved a streamlined and optimized resistance network through adaptive genetic changes. Adaptation to As(III) converged on a single mutation in the acr3 efflux pump across all 12 populations, potentially reflecting a strategy that limits arsenic influx. In contrast, Sb(III) resistance may be associated with extensive genetic alterations. Mutations potentially relieve repression of the arsAB operon, enhancing efflux activity, and remodeling transcriptional networks and energy metabolism, which could contribute to high-level Sb(III) resistance. Crucially, arsO was inactivated by nonsense or frameshift mutations in both As(III)- and Sb(III)-evolved populations. This may reflect an adaptive loss-of-function and suggests a model in which As(III)/Sb(III) evolutionary pressure alters cellular metabolic priorities. Our findings thereby elucidate the mechanistic basis of metalloid-specific adaptation and guide rational genetic modification of strains for environmental bioremediation.