Characterisation of Agr quorum sensing in Clostridium autoethanogenum

The Gram-positive, anaerobic, acetogenic bacterium Clostridium autoethanogenum is regarded as an important biocatalyst in the current advancement of industrial gas fermentation. The ever-widening diversity of industrially-relevant acetogenic bacteria has inspired a rational approach into utilising industrial waste gases as a viable feedstock, with goals of mitigating greenhouse gas emissions, and supporting an alternative means of fuel and high-value chemical production. Combined with this effort, is the expanding repertoire of gene editing tools that have allowed for the improvement of gas fermentation processes and increased spectrum of fermentative products. Despite these advances, there remain many pertinent questions, which addressed, can further the understanding of metabolism and physiology in acetogenic bacteria. This includes cell-to-cell communication and signalling, Quorum Sensing. In this project, these questions are addressed through the study of the Agr QS system in C. autoethanogenum. Signalling peptide genes, agrD1 and agrD2 were disabled separately and more importantly, in tandem, which effectively abolished Agr signalling. Phenotypic characterisation of the double agrD mutants revealed a significant increase in ethanol at the expense of acetate output. Further observations exhibited a complete utilisation of the fructose carbon source, and the inability to fully re-assimilate CO2. These findings markedly contrasted with the wild type, and both single knock-out agrD mutants. Proteomics and enzyme activity analysis of the double AgrD mutant revealed a marked down-regulation of Wood-Ljungdahl pathway genes that included the CO2-assimilating, carbon monoxide dehydrogenase / acetyl-CoA synthase complex subunits and hydrogenases. An up-regulation of alcohol dehydrogenases was observed explaining ethanol increases, alongside an unexpected upregulation of bacterial micro-compartment clusters. These findings led to the hypothesis that the C. autoethanogenum Agr system influences the ancient Wood-Ljungdahl pathway, primarily as a means of survival by managing carbon-source utilisation and regulation.