Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen Erwinia amylovora

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system confers acquired heritable immunity against mobile nucleic acid elements in prokaryotes, limiting phage infection and horizontal gene transfer of plasmids. In CRISPR arrays, characteristic repeats are interspersed with similarly sized nonrepetitive spacers derived from transmissible genetic elements and acquired when the cell is challenged with foreign DNA. New spacers are added sequentially and the number and type of CRISPR units can differ among strains, providing a record of phage/plasmid exposure within a species and giving a valuable typing tool. The aim of this work was to investigate CRISPR diversity in the highly homogeneous species Erwinia amylovora, the causal agent of fire blight. A total of 18 CRISPR genotypes were defined within a collection of 37 cosmopolitan strains. Strains from Spiraeoideae plants clustered in three major groups: groups II and III were composed exclusively of bacteria originating from the United States, whereas group I generally contained strains of more recent dissemination obtained in Europe, New Zealand, and the Middle East. Strains from Rosoideae and Indian hawthorn (Rhaphiolepis indica) clustered separately and displayed a higher intrinsic diversity than that of isolates from Spiraeoideae plants. Reciprocal exclusion was generally observed between plasmid content and cognate spacer sequences, supporting the role of the CRISPR/Cas system in protecting against foreign DNA elements. However, in several group III strains, retention of plasmid pEU30 is inconsistent with a functional CRISPR/Cas system.

Fig. 1.

Comparison of cas genes and associated CRRs between related Erwinia species. CRRs sharing the same repeat sequence are indicated by the same color, whereas similar colors indicate the presence of a SNP between two consensuses. In E. pyrifoliae, two independent sets of cas genes (cse and csy, corresponding to the Ecoli [red] and Ypest [blue] subtype variants, respectively) are associated with two distinct CRISPR arrays. The csy genes and one of the associated CRISPR regions show evidence of having been lost in E. amylovora, leaving only a remnant CRR4. In contrast, loss of the cse genes and both associated CRRs appears to have occurred in E. tasmaniensis. The four predicted ORFs situated between CRR1 and the cas3 gene in E. amylovora CRISPR group I and II strains (the gene arrangement of UTRJ2/pEU30 corresponds to that of CFBP1430) are remnants of the genomic island found in Rubus strains and are missing in group III strains such as OR29.

Fig. 2.

Sequences and predicted secondary structures of Erwinia amylovora CRISPRs, obtained using RNAfold. Sequence comparison shows that both CRR1 and CRR2 belong to cluster 2, while CRR4 belongs to cluster 4 of the proposed repeat-based classification of CRISPR arrays (30). The gray boxes highlight the differences in repeat consensus sequence between CRR1 and CRR2.

Fig. 3.

CRR genotypes found in a collection of 37 E. amylovora strains. CRISPR spacers are represented by boxes, with the spacer positions numbered at the top of the columns. For each single spacer, the DNA sequence and results of BLAST similarity searches are available in Table S2A to D in the supplemental material. The arrays are oriented in the 3′-to-5′ direction, meaning that the spacers next to the leader sequence are on the right side of the picture and that within the same block, the numbering generally increases chronologically from the most ancient to the more recent spacers. Identical spacers within the same block are vertically aligned and colored gray, whereas identical spacers between different blocks are represented by the same color. A white box indicates the absence of the corresponding spacer in a given genotype. A yellow “x” inside a box points to the presence of SNPs in one spacer with respect to the consensus. Duplicated spacers within the same array are characterized by the same number at the spacer position (highlighted in red). Strains or CRISPR groups encompassing one or more CRR genotypes are indicated to the right of the array.

Fig. 4.

Clustering of E. amylovora strains based on cumulative spacer patterns of CRR1, CRR2, and CRR4. The codes in brackets indicate the CRR genotype obtained in this work (shown in bold in Fig. 3), the PFGE type (27, 57), and the PCR ribotype (11, 37). An absence of data is indicated by a dash. The geographic origin of each strain is indicated in parentheses as follows: CA, Canada; CH, Switzerland; DE, Germany; ES, Spain; ET, Egypt; FR, France; IL, Israel; IT, Italy; LB, Lebanon; NL, Netherlands; NZ, New Zealand; UK, United Kingdom; and US, United States. Results of CRISPR typing were converted in a binary array according to the presence or absence of each spacer, and taxonomy was inferred using the UPGMA method (1,000 bootstrap replications; only values of >50% are shown; there were 461 positions in the final data set).

Fig. 5.

Distribution of protospacers on plasmids pEA72 (A) and pEU30 (B). Each bar indicates the position of a sequence integrated as a spacer in the CRISPR region of at least one of the E. amylovora strains considered in this work. Blue and red bars represent protospacers in forward and reverse orientations, respectively. Sections of pEU30 which were sequenced in group III strain OR29 to evaluate the presence of potential mutations in the 12 associated protospacers are represented by the shaded area.

Fig. 6.

WebLogo plot of the PAM consensus created utilizing the entire pool of 37 protospacers found on plasmid pEU30. All entries are oriented in the same way as the cognate sequences in CRR1 and CRR2 of E. amylovora and are aligned relative to the 5′ end of the protospacer (base 1). Sequences include 28 nucleotides upstream of the first base of the protospacer (containing the PAM) and the protospacer itself (positive numbers).