CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPRassociated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) comprise a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans, including the analysis of its interaction with crRNA and also AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa. The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5′ handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5′-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5′-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 Å, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity.

Genome-based population structure analysis of the strawberry plant pathogen Xanthomonas fragariae reveals two distinct groups that evolved independently before its species description

“…Evolutionary classification of this CRISPR/ cas system indicated that it belongs to class 1, type I-F, initially identified in Yersinia pseudotuberculosis [ 57 ]. The same type of CRISPR/cas system has been reported very recently in Xanthomonas albilineans [ 58 ].…”

Structural and mechanistic insights into the CRISPR inhibition of AcrIF7

“…Using individually purified recombinant proteins, we asked whether AcrIF7 interacts with the X. albilineans Cas8f–Cas5f heterodimer. We previously showed that X. albilineans Cas8f and Cas5f proteins stably form a heterodimeric complex that is capable of binding the 5′ crRNA handle ( 24 ). AcrIF2, a previously characterized type I-F Acr protein, associates tightly with the X. albilineans Cas8f–Cas5f heterodimer with an equilibrium dissociation constant ( K D ) of 7.2 nM ( 24 ).…”

“…We previously showed that X. albilineans Cas8f and Cas5f proteins stably form a heterodimeric complex that is capable of binding the 5′ crRNA handle ( 24 ). AcrIF2, a previously characterized type I-F Acr protein, associates tightly with the X. albilineans Cas8f–Cas5f heterodimer with an equilibrium dissociation constant ( K D ) of 7.2 nM ( 24 ). AcrIF2 also shows similar affinity for the complete P. aeruginosa Csy complex ( 10 , 11 ).…”

Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity

“…All Acrs characterized to date function by inhibiting either the CRISPR surveillance complexes or the effector nucleases ( Bondy-Denomy et al., 2015 , Pawluk et al., 2016b , Pawluk et al., 2017 , Wang et al., 2016a , Wang et al., 2016b , Chowdhury et al., 2017 , Dong et al., 2017 , Guo et al., 2017 , Harrington et al., 2017 , Hynes et al., 2017 , Peng et al., 2017 , Rauch et al., 2017 , Shin et al., 2017 , Yang and Patel, 2017 , Hong et al., 2018 ). These acr genes were first identified in temperate Pseudomonas phages ( Bondy-Denomy et al., 2013 ) and can rescue phage from CRISPR-mediated extinction ( van Houte et al., 2016b ).…”