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, Paraclostridium bifermentans 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 (arsM) transcription were found to be positively correlated with RCB dilution, suggesting that substrate limitation enhances arsM 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 Escherichia coli K-12 MG1655 (WT) or E. coli expressing the MMAs(III)-resistance gene (arsP) (ArsP E. coli). We observed increased MMAs(III) production in the presence of E. coli than its absence and growth inhibition of WT E. coli to a greater extent than ArsP E. coli, 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.