Clostridial plasmids have been shown to carry antibiotic resistance determinants and virulence factors, resulting in the ability of these mobile genetic elements to contribute to pathogenesis. Despite many clostridial strains carrying plasmids, the role of plasmids in sporulation has not been studied. Using Clostridium 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 on C. sordellii strain ATCC9714 identified that the expression of most pCS1-1 genes were downregulated during spore formation, compared to vegetative cell growth; however, three genes were upregulated during sporulation. One of these genes, asrA, was insertionally inactivated and the resultant mutant characterised. The plasmid-encoded asrA gene influences the temporal progression of sporulation, with the mutant displaying a delayed sporulation phenotype.
A proteomic analysis of mature C. sordellii spores was also performed. Purified spores were cryomilled to disrupt their integrity, and mass spectrometry was used to identify the pCS1-1 proteins that contribute to the mature spore proteome. Five of the potential 89 proteins predicted to be encoded by pCS1-1 were found to be present in spores. The function of these plasmid-encoded proteins identified in our proteomics study is currently under investigation.
It has previously been assumed that while plasmids are important during vegetative growth, they are not active or relevant during the process of spore formation. Our results have shown, through a combination of transcriptomic and proteomic analyses, that plasmid-encoded elements are active during sporulation. In addition to this, we have demonstrated genetically that asrA on pCS1-1 contributes to the progression of 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.