CRISPR distribution within the Escherichia coli species is not suggestive of immunity-associated diversifying selection

Abstract

In order to get further insights into the role of the clustered, regularly interspaced, short palindromic repeats (CRISPRs) in Escherichia coli, we analyzed the CRISPR diversity in a collection of 290 strains, in the phylogenetic framework of the strains represented by multilocus sequence typing (MLST). The set included 263 natural E. coli isolates exposed to various environments and isolated over a 20-year period from humans and animals, as well as 27 fully sequenced strains. Our analyses confirm that there are two largely independent pairs of CRISPR loci (CRISPR1 and -2 and CRISPR3 and -4), each associated with a different type of cas genes (Ecoli and Ypest, respectively), but that each pair of CRISPRs has similar dynamics. Strikingly, the major phylogenetic group B2 is almost devoid of CRISPRs. The majority of genomes analyzed lack Ypest cas genes and contain CRISPR3 with spacers matching Ypest cas genes. The analysis of relatedness between strains in terms of spacer repertoire and the MLST tree shows a pattern where closely related strains (MLST phylogenetic distance of <0.005 corresponding to at least hundreds of thousands of years) often exhibit identical CRISPRs while more distantly related strains (MLST distance of >0.01) exhibit completely different CRISPRs. This suggests rare but radical turnover of spacers in CRISPRs rather than CRISPR gradual change. We found no link between the presence, size, or content of CRISPRs and the lifestyle of the strains. Our data suggest that, within the E. coli species, CRISPRs do not have the expected characteristics of a classical immune system.

Fig. 1.

General features of the 4 distinct CRISPR loci in the E. coli species. In the five major phylogenetic groups of the species, namely, A, B1, B2, D, and F, we have represented the proportion of strains lacking a CRISPR in light gray, the proportion of strains containing a residual CRISPR in medium gray, and the proportion of strains having a CRISPR which we consider putatively functional in black (i.e., with a number of repeats above 5). This analysis was done for each of the 4 CRISPRs previously defined in reference . Schematic phylogenetic relationships between strains are represented on the left of the figure; E. fergusonii was used as an outgroup; the sizes of the triangles are proportional to the number of strains in each phylogenetic group. Strains of the C group are not represented.

Fig. 2.

Spacer repertoire relatedness of CRISPR1. (A) Association between spacer repertoire relatedness of CRISPR1 and phylogenetic distance between each pair of genomes. For clarity, phylogenetic distances were distributed into equidistant bins (intervals of 0.01); the average and standard error of the spacer repertoire relatedness of each bin are indicated. (B) Magnification of panel A in the distance range of 0 to 0.01. Each axis was divided into 10 equidistant bins; the point size is proportional to the number of strains present in each bin.

Fig. 3.

Spacer repertoire relatedness cladograms of CRISPR1 and CRISPR2. Spacer repertoire relatedness was used to compute a distance matrix of all pairs of genomes with a putative functional CRISPR1 (top) and CRISPR2 (bottom). Each matrix was then used to calculate a phylogenetic tree using the BIONJ algorithm (15). The cladograms were visualized using figtree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/), branch lengths are ignored, and only branching order is indicated. The Markov clustering algorithm (13) was used to cluster and organize the CRISPRs into distinct groups; the largest are represented. The phylogenetic groups of the strains are indicated with colors: A, blue; B1, green; C, grey; D, yellow; F, orange; ungrouped, black. Strains are indicated by their designation, the phylogenetic group and subgroup, the number of repeats, and the CRISPR group. esfe, E. fergusonii. Incongruent strains discussed in the text are indicated by red blots. Strains for which we have only the phylogroup determined by the Clermont et al. method (6) (see Table S1 in the supplemental material) are not represented in this figure.

Fig. 4.

Graphic representation of spacers across CRISPR1 and CRISPR2 in the clonal group C and non-group C strains belonging to the spacer repertoire relatedness group 2. Spacers are shown as boxes and are equally oriented with respect to the leader (right). Single spacers appear on a white background; identical spacers are represented using similar-colored backgrounds and identical numbers. The colors and the numbers were assigned arbitrarily and are different from those in Fig. S3 and S4 in the supplemental material. Strain phylogenetic groups, pathotypes (C, commensal; P, pathogen), and hosts (H, human; A, animal) are indicated at the left of the figure with the strain designation. (A) CRISPR1. (B) CRISPR2. S and L, small and large repeats.

Fig. 5.

Box plot of the total numbers of repeats in commensal versus pathogenic strains. (A) All strains were considered. Commensal (n = 119), mean ± standard deviation = 21.2 ± 11.7; pathogenic (n = 144), mean ± standard deviation = 16.2 ± 12; one-way analysis of variance (P < 0.0008). (B) B2 group strains were removed from the analysis. Commensal (n = 92), mean ± standard deviation = 24.8 ± 9.7; pathogenic (n = 82), mean ± standard deviation = 23.6 ± 9.8; one-way analysis of variance (P > 0.4). ***, significantly different; NS, not significant.