An Investigation of Bacteriophage Anti-CRISPR and Anti-CRISPR Associated Proteins

Phage infection poses a major threat to bacterial survival. To protect themselves, bacteria have evolved numerous defence mechanisms. The best known of these systems is CRISPR-Cas, which provides sequence-specific protection against phage infection. In turn, phages have evolved inhibitors of CRISPR-Cas systems known as anti-CRISPRS. Here, I investigated different aspects of anti-CRISPR biology. It was previously established that anti-CRISPRs must function during the process of infection to protect phages targeted by the P. aeruginosa CRISPR-Cas system; however, it was not known how they manifest during this process. I determined that anti-CRISPR genes are robustly expressed prior to any significant degradation of phage DNA by CRISPR-Cas machinery. This expression was tied to a powerful promoter found in association with anti-CRISPR loci. While the anti-CRISPR sequences are quite diverse, the organization of their locus is well conserved. Phages were found to carry one to three anti-CRISPR genes followed by a highly conserved helix-turn-helix motif encoding gene, known as anti-CRISPR associated 1 (aca1). I determined that aca1 encodes an essential repressor of anti-CRISPR transcription, thereby explaining its conservation. Additionally, I determined that other helix-turn-helix motif containing genes found in association with anti-CRISPR loci in diverse bacterial species also act as repressors of anti-CRISPR transcription. To date, the mechanism of action of less than half of the described anti-CRISPR protein families have been studied in detail. I have demonstrated that AcrIE3 from phage DMS3 is dual function anti-CRISPR with activities against the type I-E and type I-F CRISPR-Cas systems of P. aeruginosa. Additionally, AcrIE3 was found to inhibit both CRISPR-Cas systems by preventing target DNA binding. More work is needed to determine how AcrIE3 inhibits DNA binding. Together, these studies provide insight into the spread of anti-CRISPR operons in mobile genetic elements and into the arms race between phages and their bacterial hosts.Ph.D.2019-11-19 00:00:0