Growth substrate limitation enhances anaerobic arsenic methylation by <i>Paraclostridium bifermentans</i> strain EML

Microbial arsenic methylation is established as a detoxification process under aerobic conditions (converting arsenite to monomethylated arsenate) but is proposed to be a microbial warfare strategy under anoxic conditions due to the toxicity of its main product, monomethylarsonous acid (MMAs(III)). Here we leveraged a paddy soil-derived anaerobic arsenic methylator, <i>Paraclostridium bifermentans</i> strain EML, to gain insights into this process. Strain EML was inoculated into a series of media involving systematic dilutions of Reinforced Clostridial Broth (RCB) with 25 µM arsenite to assess the impact of growth substrate concentration on arsenic methylation. Growth curves evidenced the sensitivity of strain EML to arsenite, and arsenic speciation analysis revealed the production of MMAs(III). Concentrations of MMAs(III) and arsenic methylation gene (<i>arsM</i>) transcription were found to be positively correlated with RCB dilution, suggesting that substrate limitation enhances <i>arsM</i> gene expression and associated anaerobic arsenic methylation. We propose that growth substrate competition among microorganisms may also contribute to an increase in anaerobic arsenic methylation. This hypothesis was further evaluated in an anaerobic co-culture system involving strain EML and either wild-type <i>Escherichia coli</i> K-12 MG1655 (WT) or <i>E. coli</i> expressing the MMAs(III)-resistance gene (<i>arsP</i>) (ArsP <i>E. coli</i>). We observed increased MMAs(III) production in the presence of <i>E. coli</i> than its absence and growth inhibition of WT <i>E. coli</i> to a greater extent than ArsP <i>E. coli</i>, presumably due to the MMAs(III) produced by strain EML. Collectively, our findings suggest an ecological role for anaerobic arsenic methylation, highlighting the significance of microbe-microbe competition and interaction in this process.IMPORTANCEMicrobial arsenic methylation is highly active in rice paddy fields under flooded conditions, leading to increased accumulation of methylated arsenic in rice grains. In contrast to the known detoxification process for aerobic arsenic methylation, the ecological role of anaerobic arsenic methylation remains elusive and is proposed to be an antibiotic-producing process involved in microbial warfare. In this study, we interrogated a rice paddy soil-derived anaerobic arsenic-methylating bacterium, <i>Paraclostridium bifermentans</i> strain EML, to explore the effect of growth substrate limitation on arsenic methylation in the context of the microbial warfare hypothesis. We provide direct evidence for the role of growth substrate competition in anaerobic arsenic methylation <i>via</i> anaerobic prey-predator co-culture experiments. Moreover, we demonstrate a feedback loop, in which a bacterium resistant to MMAs(III) enhances its production, presumably through enhanced expression of <i>arsM</i> resulting from substrate limitation. Our work uncovers the complex interactions between an anaerobic arsenic methylator and its potential competitors.