Growth substrate limitation enhances anaerobic arsenic methylation by Paraclostridium bifermentans strain EML

Abstract

Microbial arsenic methylation is established as a detoxification process under aerobic conditions (converting arsenite to monomethylated arsenate) but 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 M arsenite to assess the impact of growth substrate concentration on arsenic methylation. Growth curves evidenced the sensitivity of strain EML to arsenite, and As 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 the RCB dilution, suggesting that substrate limitation enhances arsM gene expression and associated anaerobic arsenic methylation. We propose that growth substrate competition between microorganisms may also lead to an increase in anaerobic As methylation. This hypothesis was further evaluated in an anaerobic co-couture mode of strain EML with either wild-type Escherichia coli K-12 MG1655 (WT) or E. coli expressing the MMAs(III)-resistance gene (arsP), (ArsP E. coli). We found 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 MMAs(III) produced by strain EML. Taken together, our findings point to an ecological role for anaerobic arsenic methylation, highlighting the role of microbe-microbe competition/interaction in this process. IMPORTANCEAnaerobic arsenic methylation is enhanced in rice paddy soils under flooding conditions than that under drying conditions, leading to increased methylated arsenic accumulation in rice grains. Unlike the known detoxification role for aerobic arsenic methylation, the ecological role of anaerobic arsenic methylation remains elusive and is proposed to be an antibiotic-producing process involving in microbial warfare. In this study, we interrogated a rice paddy soil-derived anaerobic arsenic-methylating bacterium (Paraclostridium bifermentans strain EML) to investigate the effect of growth substrate limitation on arsenic methylation by strain EML in the context of the microbial warfare hypothesis. We provide direct evidence for the role of growth substrate competition in anaerobic arsenic methylation by strain EML. Furthermore, we evidence a feedback loop, by which a bacterium resistant to MMAs(III) enhances its production, presumably through enhanced arsM expression resulting from substrate limitation. Our work uncovers complex interactions between an anaerobic arsenic methylator and potential competitors.