Investigation into the mobile genetic elements of Clostridium difficile

Clostridium difficile is a pathogenic bacterium that can colonise both humans and various animals. Toxin production leads to clinical symptoms ranging from mild to severe diarrhoea and can result in potentially fatal pseudomembranous colitis. These symptoms are caused by the disruption of the cytoskeleton and tight junctions of gut epithelial cells by the toxins. Genomic sequencing of C. difficile has indicated the chromosome carries a number of mobile genetic elements including conjugative transposons, which can encode antibiotic resistance genes. Analysing the sequence of a number of C. difficile strains indicated that each genome carries at least one and often multiple conjugative transposons. For many of the genes on these elements, functions were predicted using various bioinformatic tools. The study of conjugative transposons in C. difficile has been limited by the lack of resistance genes encoded by the elements. Therefore, an antibiotic resistance gene was inserted into six of the elements in strains 630 and R20291 and filter-matings performed. Conjugative transfer was shown for all elements from strain 630 but not for Tn6103 from R20291. The study of transconjugants of these matings showed the pathogenicity locus, encoding the two major toxins of C. difficile, to transfer at a low frequency into a non-toxigenic recipient strain. Whole genome sequencing of transconjugants determined that the transfer is not limited to the pathogenicity locus but includes varying sizes of chromosomal DNA flanking the pathogenicity locus. RNA-seq was used for the comparison of mutants for transcriptional regulators of conjugative transposons CTn2 and CTn4, however no significant differential expression was detected. Furthermore, strain 630Δerm, a commonly used laboratory strain for the generation of knockout mutants, was compared to the wildtype strain 630. A predicted oxidative stress operon was upregulated in 630Δerm which raises the question of the biological impact of these results on the knockout model