Specific anrA primers uncover microbial antimony reduction and community dynamics in contaminated environments

Antimony (Sb) pollution poses serious environmental and health threats due to its toxicity and persistence. Microbial Sb(V) reduction is a key detoxification process, yet the ecological roles of Sb(V)-reducing bacteria (SbRBs) remain poorly understood, largely due to the absence of specific molecular detection tools. Here, we address this gap by developing and validating two primer sets targeting the putative Sb(V) reductase gene, anrA. Phylogenetic analysis of anrA homologs revealed four major evolutionary clades, guiding the rational design of primers F2R2 and F3R3. These primers were tested both in silico and experimentally, successfully amplifying anrA genes from diverse Sb-contaminated soils in Anhui, Hunan, and Guizhou Provinces, China. Sb(V) reduction assays confirmed that anrA-positive samples exhibited active Sb(V) reduction. Quantitative PCR with F2R2 revealed anrA gene abundances ranging from 8.86 × 10⁴ copies/g to 1.72 × 10⁸ copies/g, strongly correlating with total soil Sb levels (Pearson's correlation coefficient = 0.9289; p < 0.001), suggesting that anrA abundance may serve as a proxy for Sb contamination. High-throughput sequencing using F3R3 uncovered extensive anrA diversity and region-specific phylogenetic patterns. Our work provides a reliable molecular tools for detecting and profiling anrA-harboring SbRBs in environmental samples, enabling improved monitoring and offering a foundation for future Sb bioremediation strategies.