Abstract
In the environment, genetic dissemination between prokaryotes can result in beneficial but also deleterious outcomes for the recipient. Therefore, bacteria and archaea possess many resistance mechanisms, classified as innate or adaptive, to control their exposure to foreign genetic elements. CRISPR-Cas systems represent the only known prokaryotic adaptive immune response, during which ‘memories’ of past infections facilitate subsequent immunity against the same foreign genetic elements. Little is known about how CRISPR-Cas systems are controlled within native hosts, so that defense may be enhanced while potential fitness costs are reduced. Identifying regulatory networks controlling CRISPR-Cas immunity is crucial to understanding when adaptive defense is favored or dispensable.
Due to a lack of high-throughput tools to identify regulators, we developed a customizable technique to comprehensively identify regulators of target gene expression. The SorTn-seq method employs fluorescence activated cell sorting (FACS) to enrich mutants with altered fluorescent reporter activity from within a saturated transposon mutant pool. Sorted cells are deep sequenced to identify transposon insertion sites, and insertion numbers within genomic features are compared amongst the sorted fractions to identify potential regulators or regulatory regions. This technique proved robust in identifying regulators of type III-A CRISPR-Cas activity in Serratia sp. ATCC 39006, an enterobacterium containing three active CRISPR-Cas systems. Expression changes were experimentally validated for 19 predicted regulators, which influence important aspects of cell physiology, such as resource utilization, motility, and stress response.
We further demonstrated that activation of the regulator of capsular polysaccharide synthesis (Rcs) pathway repressed expression of all three CRISPR-Cas subtypes in Serratia. Likewise, Rcs activation led to an increase in plasmid transfer and maintenance. Simultaneously, activation of the Rcs stress response enhanced resistance against multiple bacteriophages via surface modifications. Our results suggest that cell stress differentially influences bacterial immune strategies, which has important consequences for horizontal gene transfer. Overall, this thesis enhances our understanding of CRISPR-Cas regulation and contributes to the broader field of bacterial genetics by describing a novel approach for the study of gene regulation.