The Bacterial Antimonite Oxidase AnoA: Unexpected Diversity and Environmental Widespread Occurrence

The growing contamination of urban areas by antimony (Sb) has sparked interest in microbial processes that modulate Sb speciation in ecosystems. The bacterial antimonite oxidase AnoA is the only oxidase known so far whose gene expression is specifically induced by Sb(III), but its annotation in public databases is currently lacking. Here, the computational search for AnoA orthologs predicted an unexpected phylogenetic distribution across the Pseudomonadota and Actinomycetota. Putative orthologs were identified in both known Sb(III)-oxidisers (e.g., Shinella, Hydrogenophaga, Bosea, Cupriavidus and Pseudomonas) and taxa not previously linked to the Sb cycle (e.g., Bradyrhizobium, Mesorhizobium, Methylobacterium and Paraburkholderia). The anoA gene is single-copy in most Proteobacterial genomes, but is often detected in multiple copies in the Actinomycetota. Furthermore, sequence evolutionary distances suggest that it is mainly inherited vertically, with horizontal transfer events, in particular towards the Gammaproteobacteria. Using the constructed database, new PCR primers were designed and outperformed existing strain-specific primers in amplifying the anoA gene from samples with varying Sb levels and microbial profiles. Sequencing and quantification of PCR amplicons revealed a diverse range of sequences in sediments and natural biofilms, indicating that the oxidase is more environmentally diverse and widespread than previously thought and may play a significant role in Sb(III) detoxification.