Development of a novel genetic system for generation of markerless deletions in Clostridium difficile

C. difficile is an obligate anaerobic, Gram-positive, rodshaped and spore-forming bacterium. It is a well-recognised causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile has emerged as an important nosocomial pathogen in recent years, associated with considerable morbidity, mortality and economic burden. Despite its importance, functional genomic studies have been lagging behind in comparison to other enteric pathogens. This is attributed to the fact that C. difficile is difficult to manipulate genetically and the lack of robust, reproducible mutagenesis systems for many years. The ideal mutation for robust functional genomic studies is a markerless, in-frame deletion of the gene of interest. All systems developed for C. difficile, up to the start of this study, involve insertional inactivation of the gene of interest. This study describes the development of a novel genetic system for C. difficile, to create precise and markerless chromosomal deletions, using the meganuclease ISceI. For validation of the system, the addBA genes in C. difficile were deleted. The AddAB enzyme complex is important in the survival of many bacteria, since it maintains genome integrity, by the repair of double-strand breaks. Deletion of addBA in C. difficile did not significantly affect growth and viability, but the mutant strains were sensitive to DNA damaging agents. In addition, it was shown that C. difficile is capable of initiating the SOS response after DNA damage and that AddAB is not necessary for the induction of this response. The genetic system was further optimised to delete type IV pili (TFP)- associated genes, particularly pilT (CD3505) and pilA (CD3507), to investigate twitching motility. TFP are important in virulence and pathogenesis of many bacteria and twitching motility is often involved. TFP in C. difficile may be expressed in vivo during infection and may be involved in biofilm formation and colonization. To study potential TFP-mediated motility, a non-flagellated C. difficile strain was first constructed by deleting the fliC gene. The pilT gene, predicted to encode a protein involved in TFP retraction, was then deleted in the ΔfliC strain. A ΔpilT strain was also generated. Preliminary experimental work using these strains did not show any evidence for twitching motility and no difference between the ΔpilT strains and the parental strains. Examination of cells from the ΔfliC strain, under various conditions, did not reveal any pili, which indicates that TFP are regulated in C. difficile and that the TFP locus might be repressed at the transcriptional level. Preliminary work to investigate an intergenic region located upstream of the TFP locus in C. difficile, that might be involved in regulation, suggested that transcription is being initiated within a 500 bp region upstream of the CD3513 gene