Non-Identity-Mediated CRISPR-Bacteriophage Interaction Mediated via the Csy and Cas3 Proteins

Abstract

Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs. Further, we present evidence that the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens. We also determined that only spacer 1, which is not identical to any region of the DMS3 genome, mediates the CRISPR-dependent loss of biofilm formation. Our evidence suggests that gene 42 of phage DMS3 (DMS3-42) is targeted by CRISPR2 spacer 1 and that this targeting tolerates multiple point mutations between the spacer and DMS3-42 target sequence. This work demonstrates how the interaction between P. aeruginosa strain UCBPP-PA14 and bacteriophage DMS3 can be used to further our understanding of the diverse roles of CRISPR system function in bacteria.

Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

“…By comparing the amino acid sequences of these three Cas3 domains in Escherichia coli UT189, PA2192, P. mendocina and P. stutzeri, we noticed conserved residues in each domain [15] (also seen in Supplementary information, Figure S4). We generated strains carrying point mutations at these conserved residues to identify the necessary domains required for repression of lasR ( Figure 2H).…”

“…We set out to explore whether additional CRISPR-Cas systems are employed by bacteria to target their endogenous genes in order to alter virulence and impact host immunity. To this end, we first measured transcript levels of algC, which controls P. aeruginosa LPS synthesis [14], in PA14 WT and total Type I CRISPR-Cas knockout (KO) mutant strain (∆TCR, including two repeat-rich CRISPR regions and six cas genes, Figure 1A and Supplementary information, Figure S1A) [15]. However, transcript levels of algC were similar between these two PA14 strains (Supplementary information, Figure S2A).…”

“…The P. aeruginosa strain PA14 and its CRISPR-Cas component mutants, complementary strains were kindly provided by Dr George A O'Toole (Dartmouth Medical School) [15]. Other PA14 mutant strains were constructed in this study (Supplementary information, Table S3).…”

“…Cas3 contains three major conserved domains: an N-terminal HD-type phosphohydrolase (HD) domain, a central DExD/H helicase (DExD/H) domain with multiple conserved motifs and a C-terminal helicase (HelC) domain [15]. By comparing the amino acid sequences of these three Cas3 domains in Escherichia coli UT189, PA2192, P. mendocina and P. stutzeri, we noticed conserved residues in each domain [15] (also seen in Supplementary information, Figure S4).…”

CRISPR control of virulence in Pseudomonas aeruginosa

“…While some of the spacers are perfectly complementary, others contain several mismatches. In 2011, Cady et al [7] showed that one of the spacers with five mismatches to an integrated viral genome (i.e., a prophage called DMS3) was involved in prophage-dependent inhibition of biofilm formation. At the time it was thought that phage-dependent inhibition of biofilm formation might be due to crRNA-guided targeting of the RNA, but it was later shown that imperfect base pairing between the crRNA and the integrated prophage DNA caused DNA damage (i.e., low level autoimmunity) and cell death during biofilm formation [8].…”