Decoding the adaptive strategies of versatile diazotrophs to multi-metal(loid) stress in mercury-mining impacted farmland soils

Diazotrophs are crucial for Earth's nitrogen cycle via biological nitrogen fixation, while also modulating other elemental cycles and exhibiting bioremediation potential. However, their responses to co-occurring heavy metal(loid) (HM) contaminants in polluted soils remain poorly understood. Using combined nifH (encoding nitrogenase) amplicon and metagenomic sequencing, we characterized the taxonomic structure and metabolic potential of diazotrophic community across multi-HM contamination gradients in mercury-mining impacted farmlands (paddy vs. upland). Results identified selenium (upland soils: 0-3.08 mg kg⁻¹) and arsenic (paddy soils: 5.38-17.1 mg kg⁻¹) as the primary HMs shaping diazotrophic diversity, whereas mercury (0.067-99.6 mg kg⁻¹) showed a significant but weak correlation. Selenium and mercury correlated positively with diversity in upland soils (arsenic negatively), whereas all three HMs correlated negatively in paddy soils. Diazotrophic indicator taxa varied by HM type, yet certain taxa tolerated all three HMs simultaneously-notably Chromatiaceae/Pseudomonadaceae in upland soils and Xanthobacteraceae in paddy soils. Moreover, diazotrophs in upland soils exhibited synergistic associations with functional guilds involved in HM resistance and element cycling (e.g., carbon fixation and hydrogen metabolism), contrasting with the negative correlations in paddy soils. Metagenomic binning indicated that dominant diazotrophs were primarily aerobic heterotrophs with versatile metabolic potentials, including multi-HM resistance (e.g., arsenic/mercury reduction, efflux, and antioxidation) and energy acquisition via trace gas (CO, H₂), manganese, and sulfide oxidation. These findings provide novel insights into diazotrophic adaptive strategies under multi-HM stress, advancing our understanding of their ecological and environmental functions.