Ramesh, Amritha (2020) Investigation of molecular mechanisms of biofilm dispersal and attempts to create an inducible gene expression system in 'Campylobacter'. (PhD thesis), Kingston University, .
Abstract
Campylobacter jejuni is the leading cause of bacterial foodborne diseases worldwide. C. jejuni and Campylobacter coli are the most predominant species of the genus that are responsible for gastrointestinal diseases in humans (Epps et al., 2013). C. jejuni 11168H strain can form biofilms, whose cells are more resistant to antimicrobials and disinfectants, perhaps due to the fact that the bacterial cells are secured in an extra cellular polymeric matrix consisting of eDNA (extracellular DNA), proteins, and polysaccharides (Brown et al., 2015b). Bacterial dispersion takes place following the biofilm maturation step. The dispersed cells demonstrate greater colonizing properties than their sessile counterparts (Guilhen et al., 2017). Therefore, studying the factors governing the dispersal process is vital as it would give a better understanding of the pathogen and may lead to the development of novel antimicrobial treatments, which in turn may help prevent future cases of campylobacteriosis. In this study, dispersal of C. jejuni 11168H biofilms was observed. The role of the cj0979 gene in biofilm formation was investigated and it was concluded that this gene alone was not responsible for the regulation of C. jejuni 11168H biofilm dispersal. Purified Cj0979 exhibited DNase activity and could degrade lambda DNA. In addition, purified Cj0979 reduced C. jejuni 11168H biofilm development, when added to the initial stages of biofilm formation. Importantly, these findings provide insights into the dispersal process of C. jejuni biofilms, which has not been reported elsewhere. A development and validation of an arabinoseinducible gene expression system was undertaken in thisresearch to aid in the study of essential genes and nuclease encoding genes involved in biofilm dispersal. As a primary step, arabinose transporter genes araE and lacYA177C were introduced into C. jejuni since the transporters required for arabinose uptake are not present within these bacteria. Arabinose is essential for promoter function. The integration constructs carrying the arabinose transporter genes, were verified by genome sequencing, confirming that they were free of deletions and pointmutations. The gene cassettes carrying the araE and modified lacY genes were successfully integrated into the C. jejuni 11168H chromosome. Despite successful introduction of araE and modified lacY genes into C. jejuni 11168H induction of gene expression from PBAD promoter could not be achieved. Possible reasons for this finding are discussed.
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