CRISPR-Cas immunity repressed by a biofilm-activating pathway inPseudomonas aeruginosa

Abstract

10 11 CRISPR-Cas systems are adaptive immune systems that protect bacteria from 12 bacteriophage (phage) infection. To provide immunity, RNA-guided protein 13 surveillance complexes recognize foreign nucleic acids, triggering their 14 destruction by Cas nucleases. While the essential requirements for immune 15 activity are well understood, the physiological cues that regulate CRISPR-Cas 16 expression are not. Here, a forward genetic screen identifies a two-component 17 system (KinB/AlgB), previously characterized in regulating Pseudomonas 18 aeruginosa virulence and biofilm establishment, as a regulator of the biogenesis 19 and activity of the Type I-F CRISPR-Cas system. Downstream of the KinB/AlgB 20 system, activators of biofilm production AlgU (a σ E orthologue) and AlgR, act as 21 repressors of CRISPR-Cas activity during planktonic and surface-associated 22 growth. AmrZ, another biofilm activator, functions as a surface-specific repressor 23 of CRISPR-Cas immunity. Pseudomonas phages and plasmids have taken 24 advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, 25 which are functional CRISPR-Cas repressors. This suggests that while CRISPR-26 Cas regulation may be important to limit self-toxicity, endogenous repressive 27 pathways represent a vulnerability for parasite manipulation. 29Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-30 associated (cas) genes are RNA-guided nucleases found in nearly half of all bacteria 1 . 31CRISPR-Cas systems are mechanistically diverse, with six distinct types (I-VI) identified, 32 based on signature genes, mechanisms of action, and the type of nucleic acid target 1 . 33Our strong understanding of the basic components to enable sequence specific DNA 34 and RNA cleavage have enabled functional transplantation into heterologous bacterial 2,3 35 and eukaryotic 4,5 hosts. Given that many Cas proteins encode nucleases 6 the fine-tuned 36 regulation of these systems to avoid toxicity is likely a key factor to enable safe retention 37 of a CRISPR-Cas immune system 7 . Multiple signals have been shown to activate 38 CRISPR-Cas function in diverse organisms, such as quorum sensing 8,9 , temperature 10 , 39 membrane stress 11,12 , altered host metabolite levels 13,14 , and phage infection [15][16][17][18] . 40However, relatively little is known regarding the factors and/or signals that serve to 41 temper CRISPR-Cas activity and mitigate the risk of acquiring and expressing a 42 nucleolytic immune system. 44Type I CRISPR-Cas systems are comprised of a multi-subunit RNA-guided surveillance 45 complex, a trans-acting nuclease (Cas3) [19][20][21] , and proteins dedicated to spacer 46 acquisition, Cas1 and Cas2 22,23 . Pseudomonas aeruginosa has become a powerful 47 model organism for studying Type I CRISPR-Cas mechanisms 24-29 , functions 3,23,30,31 , 48 evolution 32-34 , and interactions with phages utilizing anti-CRISPR proteins [35][36][37][38] . The P. 49aeruginosa strain PA14 possesses an active Type I-F CRISPR-Cas immune system ...