Abstract
Prokaryotes have developed multiple defence strategies against the foreign genetic elements that are constantly exerting pressures on them. CRISPR-Cas systems provide heritable, sequence-specific protection against these elements by acquiring short ‘spacer’ sequences into a CRISPR array, in a process termed adaptation. The CRISPR array consists of invader-derived spacer sequences interspaced by palindromic repeats and is expressed from a leader/promoter region as a single transcript. Spacers are expressed and processed to act as guides for cas ribonucleoprotein complexes which survey the cell to identify and degrade the complementary invading element. It was recently shown that the nucleoid-associated protein integration host factor (IHF) is essential for naïve adaptation by type I-E systems. IHF binds close to the leader-repeat border in a sequence-specific manner, allowing the recruitment of the Cas1(4)-Cas2(2) adaptation complex bound with a pre-spacer to simultaneously recognise the leader and an upstream recognition motif for integration. IHF is found in gram-negative bacteria and has a diverse range of physiological roles, including site-specific recombination, gene regulation and DNA replication. In this thesis, bioinformatic analysis revealed putative IHF binding sites in the promoter regions of the cas genes and the CRISPR arrays. I hypothesised that IHF, in addition to being required for adaptation by the type I-E and type I-F systems, may additionally regulate the CRISPR-Cas interference response. The Serratia IHF was expressed and purified to observe the specific binding of IHF to the promoter regions in vitro using electrophoretic mobility shift assays. This was validated further by in vivo analysis, and genetic knockouts of IHF were created. IHF was shown to be essential for primed adaptation and the successive clearance of a priming plasmid by the type I-E and type I-F systems. However, IHF did not definitively display a role in the regulation of the Serratia CRISPR-Cas systems transcriptionally or phenotypically.