Lipophilic Redox Mediators Reprogram Intracellular Electron Flow to Promote Arsenic Methylation in Soil Fermentative Bacteria

Abstract

Anaerobic arsenic (As) methylation by fermentative microorganisms, particularly Clostridium-related populations, controls As speciation, mobility, and volatilization in flooded paddy soils, yet the mechanisms promoting this process remain poorly understood. Here, we reveal that phenazine- and phenoxazine-like redox mediators can enhance anaerobic As methylation via reshaping the intracellular electron allocation in a representative As-methylating fermentative bacterium. We integrated As speciation analysis, metabolite profiling, proteomics, and metabolic-flux modeling to determine how resazurin, as a represenative lipophilic redox mediator, reshapes fermentative metabolism and methylated As (MeAs) formation. The membrane-permeable redox mediator resazurin increased the apparent rate constant of MeAs formation by up to fourfold relative to the hydrophilic mediator anthraquinone-2,6-disulfonic acid (AQDS). Unlike AQDS, resazurin enhanced dimethylarsine formation while establishing a more reducing intracellular environment by diverting electrons from hydrogen evolution, indicating altered intracellular electron disposal. At the proteomic level, resazurin induced coordinated increases in As(III) methyltransferase (ArsM) and proteins involved in S-adenosylmethionine (SAM) biosynthesis, representing the primary molecular responses associated with enhanced As methylation. Proteins involved in amino-acid-fueled energy conservation and redox homeostasis were also enriched, potentially creating favorable cellular conditions for SAM-dependent As methylation. In a paddy-soil-derived fermentative consortium, resazurin likewise enhanced MeAs production and increased transcript levels of Clostridium-associated arsM and the SAM synthetase gene (metK). These findings identify lipophilicity-dependent, redox-compound-mediated intracellular electron flow reprogramming as a previously underappreciated control on anaerobic As methylation and provide a mechanistic basis for predicting MeAs formation in flooded soil environments.