bHere we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/ Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function. C lustered regularly interspaced short palindromic repeats (CRISPR) are found in roughly 50% of sequenced bacterial genomes and 90% of archaeal genomes (2, 24). CRISPR regions are composed of multiple repeated sequences ranging from 21 to 48 bp in length separated by 26-to 72-bp spacers (1, 2). The sequences of spacer regions are variable but are often identical to sequences found within phages, plasmids, or other foreign DNA (24). CRISPR loci are transcribed as one large transcript that is processed within the identical repeat sequence into mature, small CRISPR RNAs (crRNAs) by either CRISPR-associated (Cas) proteins alone or RNase III associated with Cas proteins (5, 11, 15). These mature crRNAs are then complexed with subtype-specific Cas proteins, and this complex specifically interacts with nucleotide target sequences complementary to spacer sequences (5,14,16,26). Complementarity between mature crRNAs and sequences found within phages or plasmids leads to inhibition of their replication through target nucleotide cleavage (12,14,25). The ability of CRISPR/Cas systems to also incorporate DNA sequences from newly encountered foreign DNA and subsequently resist phages or plasmids containing these sequences has led these systems to be referred to as bacterial adaptive immune systems (24).Pseudomonas aeruginosa is an opportunistic pathogen of humans and animals that is capable of becoming highly antibiotic resistant (18); thus, it has become a model for new or previously overlooked antibacterial ...

No abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting.

When activated by high NaCl, the transcription factor TonEBP/OREBP increases transcription of osmoprotective genes. High NaCl activates CDK5 kinase, which directly phosphorylates TonEBP/OREBP on threonine 135. This contributes to rapid nuclear translocation of TonEBP/OREBP, accelerating transcription of its osmoprotective target genes.