Abstract
Prokaryotes persist in a variety of ecological niches; each imposing diverse selective pressures. Among the regular threats that must be endured is invasion by foreign genetic elements, including bacteriophages (phages) and plasmids, which intimately shape the evolutionary trajectory of their hosts. To better protect themselves against these extrachromosomal sequences, bacteria and archaea have amassed a variety of defence systems. CRISPR-Cas adaptive immunity is one such mechanism and provides a ‘genetic-memory bank’ of prior exposure to coordinate the sequence-specific degradation of nucleic acids. Given the far-reaching implications that CRISPR technology has for medical and industrial applications, significant research has been directed at elucidating its functional aspects. In contrast, our understanding of how bacteria regulate the expression of these systems in native environments remains in its infancy. By determining the stimulatory cues which drive CRISPR-Cas activity, we gain insight into their evolutionary origin and plausibly attain tools to counter pathogenic strains harbouring this immunity. The aim of this current study was to characterise regulatory networks controlling CRISPR-Cas activity in two separate species – Pectobacterium atrosepticum and Serratia sp. ATCC39006 – which between them harbour systems belonging to three separate subtypes (I-E, I-F and III-A). Here, I demonstrate that the induction of CRISPR-Cas immunity is intimately linked to nutritional sensors within the host organism. Specially, signalling through CRP-cAMP universally activates expression across all systems in both species. In addition, a previously unlinked regulatory pathway, involving homologues of the E. coli carbon storage regulator (Csr) system, has been identified in Serratia which activates type I-E expression and represses that of the III-A system. Finally, I demonstrate that CRISPR-Cas induction is dependent on chemical communication (quorum sensing) in Serratia, which conveys population density signals to ‘pre-emptively’ activate immunity as the probability of infection increases during localised growth. Overall, this thesis provides significant insight into the previously under-investigated area of CRISPR-Cas regulation and offers effective methodology for the future identification of regulatory mechanisms.