Diversity and abundance of ring nucleases in type III CRISPR-Cas loci

Abstract

Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Owing to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38 742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focusing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.

Keywords: CRISPR; cyclic nucleotide; phosphodiesterase; ring nuclease.

Figure 1.

Structural modelling and RN activity of Csx15, Csx16 and Csx20. (A) Dimeric protein AF3 models are shown with 2 AMP molecules (yellow sticks) modelled to mimic the cOA binding site, subunits are coloured differently for ease of interpretation. (B) RN activity of Csx15, Csx16 and Csx20 against cA₃, cA₄ and cA₆, monitored by HPLC. Csx16 and Csx20 degrade cA₄ into linear products. Standards and characterized reaction products are labelled. (>p represents a 2′,3′-cyclic phosphate).

Figure 2.

Phylogenetic tree of Cas10 with ring nuclease distribution. The signalling molecule used by each system is predicted based on effector content of the locus, as defined in [6], and coloured according to the key. Instances of candidate ring nucleases Crn1, Crn2, Crn3, Csx15, Csx16 and Csx20 are indicated in concentric circles. Cas10s lacking a clear active cyclase domain are indicated by a red dot in the subtype ring.

Figure 3.

Co-occurrences of ring nucleases and effectors in type III CRISPR loci. (A) Gephi plot showing co-occurrence patterns between effectors and ring nucleases. Ring nucleases are in yellow, cA₃, cA₄ and cA₆-activated effectors in purple, blue and green, respectively and SAM-AMP-activated effectors in orange. (B) The proportion of effector instances that are associated with a RN. The data only includes loci with one effector and zero or one RNs.