The impact of anti-CRISPRs on horizontal gene transfer (HGT) and their regulation in mobile genetic elements

Abstract

CRISPR-Cas systems are common in bacterial genomes, providing adaptive immunity against bacterial viruses (bacteriophages). CRISPR-Cas systems are diverse, with two classes, six types and thirty-three subtypes. Bacteriophages circumvent this host defence by using anti-CRISPRs. Anti-CRISPR proteins may have a massive impact on microbial evolution and bacteriophage biology. This thesis explores the potential of anti-CRISPRs for the horizontal transmission of acquired traits (e.g. antibiotic resistance) across microbiomes. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally-acquired antibiotic resistance genes. Our genome-wide correlation analysis of over 100,000 bacterial genomes, with a focus on pathogens, found a significant positive association between the presence of anti-CRISPRs and acquired antibiotic resistance in Pseudomonas aeruginosa. Furthermore, genome mining identified novel type I anti-CRISPRs from human and plant pathogens. We, along with our collaborators uncovered 11 type I-F and/or I-E anti-CRISPR genes encoded on chromosomal and extrachromosomal mobile genetic elements (MGEs) within Enterobacteriaceae and Pseudomonas. These were cloned and tested experimentally in Pectobacterium and Serratia systems, validating the anti-CRISPR activity of some. The new acr genes cluster with genes encoding inhibitors of other distinct bacterial defence systems. In most cases, anti-CRISPR genes are located before of DNA binding helix-turn-helix (HTH) transcription factors, named anti-CRISPR associated proteins (Aca). Recent studies had shown three of these Acas act as repressors of anti-CRISPR expression. Here, we computationally analysed bacterial, phage, and plasmid genomes to determine the possible mode of actions of Aca families. The investigation of the promoter regions of acr genes suggests a widespread DNA repression mechanism of regulation by the Aca proteins in the control of anti-CRISPR expression. Overall, the study has broadened our knowledge of anti-CRISPR mediated HGT and its regulation in MGEs.