Clostridial plasmids often encode antibiotic resistance determinants and virulence factors. As such, these mobile genetic elements are important etiological agents of pathogenesis. Despite many clostridial strains carrying plasmids, the role of plasmids in sporulation has not been studied. Using Paraclostridium sordellii as our model spore-forming organism, we have focussed on the toxin-encoding conjugative plasmid, pCS1-1, to investigate if plasmids contribute to, or are influenced by, spore formation.

RNA-Seq analysis identified that the expression of most pCS1-1 genes was downregulated during spore formation, compared to vegetative cell growth. However, three genes were upregulated during sporulation, notably one encoding a putative anaerobic sulphate reductase, AsrA. Further characterisation determined the plasmid-encoded AsrA influences the temporal progression of sporulation, with the mutant displaying a delayed sporulation phenotype and a reduced total spore titre.

Additionally, during early sporulation the asrA mutant demonstrated a significant reduction in the expression of key sporulation regulators in comparison to wild-type. Metabolomic approaches determined that wild-type and asrA mutant cells had vastly different metabolic profiles during vegetative growth and early sporulation. We theorise that these metabolic differences influence the initiation and progression of the highly metabolically dependent process of sporulation, resulting in the altered sporulation phenotypes observed.

It has been assumed that while plasmids are important during vegetative growth, they play a passive role during the process of spore formation. Our results have shown that plasmid-encoded elements are active during sporulation. Aditionally, we have demonstrated that asrAon pCS1-1 contributes to the progression of sporulation, likely promoting sporogenesis directly or indirectly through manipulation of metabolic processes key to sporulation. This is the first time that a plasmid-encoded gene has been shown to modulate the sporulation process in any spore-forming bacterium. This work supports the need for further research into the role of plasmids throughout all life stages of the clostridia.