In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism.

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

“…1C). These results are consistent with pull-down experiments performed in the type I-F CRISPRCas system of Pectobacterium atrosepticum (20) and suggest that Cas1 interacts with the amino-terminal Cas2-like domain of the Cas2/3 fusion protein (22)(23)(24).…”

“…In fact, early evidence supporting this functional overlap came from bioinformatic studies that identified a unique fusion in type I-F systems that links a Cas2-like adaptation protein to the amino terminus of the Cas3 interference protein (i.e., Cas2/3) (54). Indeed, Cas1 and Cas2/3 have been shown to interact in P. atrosepticum, but the structural and functional implications of the Cas2/3 fusion have not been determined (20). Here we show that Cas2/3 from P. aeruginosa stably associates with the Cas1 adaptation protein, forming a 375-kDa propeller-shaped complex consisting of two Cas2/3 proteins and four Cas1 proteins (Cas1 4 :Cas2/3 2 ) (Fig.…”

“…Like the majority of Cas3 proteins, the cas3 gene in type I-F systems encodes both the HD-nuclease and SF2-helicase domains, but unlike all other Cas3 proteins, the type I-F Cas3 is fused to a Cas2-like domain (Fig. 1A) (7,(18)(19)(20). Cas2 proteins play a critical role in CRISPR adaptation (i.e., the integration of foreign DNA into CRISPR loci) (21)(22)(23)(24)(25), and fusion of the Cas2 adaptation protein to the Cas3 interference protein suggests a functional connection between the two stages of CRISPR immunity.…”

Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria

“…18 Furthermore, we have shown that a Csy interference complex is formed, composed of Csy1, Csy2, Csy3 and Cas6f. 19 The Csy complex also interacts with Cas2-Cas3, 19 which is likely to be recruited for target DNA unwinding and degradation.…”

“…The recent evidence that Cas1 and Cas2 from the related type I-E system are involved in acquisition 9 led to the proposal that the Cas1:Cas2-Cas3 complex is important for acquisition of new spacers. 19 Therefore, Cas2-Cas3 is hypothesized to play crucial roles in both acquisition and interference steps.…”