Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

Abstract

CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR-Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

Figure 1.

A proposed classification of type IV CRISPR–Cas systems based on their genome loci architectures and evolutionary relationships. Phylogenetic tree depicting the typical operon organization of the identified subtype IV loci. A selected representative locus is shown for each clade wherein genes are colour-coded and labelled according to the protein families they encode, using both the cas (upper) and csf (lower) nomenclatures. Genes or CRISPR arrays that are not invariably present are represented with dashed lines on the gene maps. The number of loci identified for each clade is given on the right. Hypothesized gene gain/loss events over the course of evolution are shown on the left.

Figure 2.

Distribution of type IV loci across prokaryotic taxa and MGE types. Phylogenetic tree based on 16S rRNA gene sequences of all bacteria and archaea that carry type IV CRISPR–Cas systems. Concentric rings denote the presence or absence of type IV and other co-occurring non-type IV CRISPR–Cas loci in the same genomes, colour-coded according to the subtype/variant to which they belong. All non-type IV systems, except I-E (light green) and I-F (pink), were merged into one lane (orange) for visualization purposes. Type IV effector cas operons for which an associated CRISPR array was detected are shown (black). Based on genomic context analyses (Methods), CRISPR–Cas systems predicted to be encoded by plasmid-like elements (grey) or (pro)phages/viruses (black) are shown (Supplementary Data S2), for both type IV and non-type IV loci (two outermost ring lanes). 758 (of 883) identified type IV loci are displayed on the tree; for the remainder no 16S rRNA gene sequence was found in the genome.

Figure 3.

Spacers from type IV systems preferentially target plasmid-borne protospacers. (A). Comparison of spacer–protospacer matches detected for type IV systems (left) and the co-occurring non-type IV systems (right). A more detailed breakdown, by CRISPR–Cas subtype/variant, is presented in Supplementary Table S4. (B). Distribution of type IV spacer hits on plasmids as a function of predicted plasmid mobility. (C). Size distribution of the targeted plasmids. The mobility prediction and size for the collection of PLSDB plasmids are displayed as a reference in both ‘B’ and ‘C’ plots.

Figure 4.

Interactions between type IV CRISPR–Cas systems and other co-encoded CRISPR–Cas systems in a host. (A) Co-occurrence analysis between type IV and non-type IV systems. Estimates are from phylogenetic logistic regressions, with P-values fdr-adjusted. Only estimates with standard errors <10 are shown. (B) Unrooted phylogenetic tree for Cas6/Csf3 built with representatives covering the diversity of type IV and type I subtypes/variants detected in this study. Each cluster is coloured according to the cas6-like family it corresponds to, and the coloured dot at the end of branches indicates the specific RISPR–Cas subtype/variant encoding such a Cas6-like protein. (C) Heat map depicting CRISPR repeat similarity of co-occurring CRISPR–Cas subtypes/variants clustered by average linkage hierarchical clustering. (D) PAM, consensus CRISPR repeat and leader sequence logos for the positively correlated subtypes IV-A3 and I-E. The short semi-palindromic repeats at the centre of the consensus repeat that are used as anchor sequences by the Cas1–Cas2e complex are highlighted in grey, as well as the conserved leader sequences comprising the binding sites for the Cas1–Cas2e complex (left) and the IHF (right).