CRISPR regulation of intraspecies diversification by limiting IS transposition and intercellular recombination

Abstract

Mobile genetic elements (MGEs) and genetic rearrangement are considered as major driving forces of bacterial diversification. Previous comparative genome analysis of Porphyromonas gingivalis, a pathogen related to periodontitis, implied such an important relationship. As a counterpart system to MGEs, clustered regularly interspaced short palindromic repeats (CRISPRs) in bacteria may be useful for genetic typing. We found that CRISPR typing could be a reasonable alternative to conventional methods for characterizing phylogenetic relationships among 60 highly diverse P. gingivalis isolates. Examination of genetic recombination along with multilocus sequence typing suggests the importance of such events between different isolates. MGEs appear to be strategically located at the breakpoint gaps of complicated genome rearrangements. Of these MGEs, insertion sequences (ISs) were found most frequently. CRISPR analysis identified 2,150 spacers that were clustered into 1,187 unique ones. Most of these spacers exhibited no significant nucleotide similarity to known sequences (97.6%: 1,158/1,187). Surprisingly, CRISPR spacers exhibiting high nucleotide similarity to regions of P. gingivalis genomes including ISs were predominant. The proportion of such spacers to all the unique spacers (1.6%: 19/1,187) was the highest among previous studies, suggesting novel functions for these CRISPRs. These results indicate that P. gingivalis is a bacterium with high intraspecies diversity caused by frequent insertion sequence (IS) transposition, whereas both the introduction of foreign DNA, primarily from other P. gingivalis cells, and IS transposition are limited by CRISPR interference. It is suggested that P. gingivalis CRISPRs could be an important source for understanding the role of CRISPRs in the development of bacterial diversity.

Keywords: Porphyromonas gingivalis; clustered regularly interspaced short palindromic repeat (CRISPR); diversification; genome rearrangement; intercellular recombination; mobile genetic element (MGE).

Fig. 1.—

Clustering by spacer content in CRISPR type 36.2 of Porphyromonas gingivalis. In type 36.2, the presence of each unique spacer is shown using a heatmap. The dendrogram was constructed from Euclidian distances. In the heatmap, the boxes indicate unique spacers and are arrayed horizontally. In the heatmap, 2 colors were used according to the bit score; red: ≥50, yellow: <50. To the right of the isolate’s name, the following information is indicated: geographic origin (black: Japan; outlined: overseas or unspecified), patient source (seven patients) and fimA type. Eight colors are used to emphasize the clusters.

Fig. 2.—

Spacer contents of Porphyromonas gingivalis isolates from seven patients in four CRISPR loci. Spacer arrays of 26 isolates from 7 patients are shown at each CRISPR locus. Each box indicates one spacer. The spacers in the arrays exhibit high nucleotide similarity to each other among the isolates if they are aligned vertically and have the same color. Blank boxes indicate absent spacers in the particular isolates. In patient no. 2, two colors are used because the D5 isolate has a type 30 spacer array that is distinct from those of D8 and D9. The spacers in type 36.2, shared among seven isolates of three patients, are indicated by deep yellow boxes and emphasized by dark gray belts.

Fig. 3.—

Split network of 60 Porphyromonas gingivalis isolates obtained from concatenated seven loci sequences. A split network tree based upon the MLST data is shown. Circles indicate external nodes (each isolate) and are colored according to geographic origin (black: Japan; outlined: overseas or unspecified). fimA types are shown by light gray shadows. The numbers outside the isolate’s name indicate the patient source. Eleven colors are used to emphasize the clusters.

Fig. 4.—

Characteristics of recombination breakpoints among three Porphyromonas gingivalis genomes. (A) Fragments are shown in the alignment of two genome sequences (TDC60, ATCC 33277). The positions of MGEs or rRNA operons in the breakpoint gaps are indicated by colored broken lines, connecting the gaps and the bars (indicating the positions of the features on the genome), which are arrayed along the outside of the plot area. The red boxes on the plot area are the regions shown in (B) in detail. (B) Breakpoint gaps of TDC60 are enlarged in light gray areas surrounded by broken lines. The regions of ATCC 33277, which correspond to the enlarged gap of TDC60, are enlarged similarly. The fragments in TDC60 and ATCC 33277 are colored by red and deep blue, respectively. The regions exhibiting high nucleotide similarity to each other are shown by a yellow belt between two fragments. The 3-kb regions of the breakpoints are indicated by dark gray rectangles on the upper or lower side of the fragments. (i) rRNA operons in the breakpoint gap. The black arrows indicate rRNA genes. The light blue-filled boxes with arrows inside indicate ISs. (ii) ISs in the breakpoint gap. (C) The number of each feature in the breakpoint gap is plotted. The regions without any characteristic features are included under “Others.” The mean and standard deviations are provided by the horizontal and vertical lines, respectively. Statistical significance is indicated by an asterisk (P < 0.05, two-tailed paired t test).

Fig. 5.—

Regions exhibiting high nucleotide similarity to P. gingivalis CRISPR spacers. Two examples of the 19 spacers exhibiting high nucleotide similarity to the P. gingivalis genome are shown. The white and black arrows indicate CDSs and rRNA genes, respectively. The arrows within the light blue-filled boxes indicate ISs. The orange regions indicate the sequences exhibiting high nucleotide similarity to CRISPR spacers. (i) Region exhibiting high nucleotide similarity to spacer 37_259: the transposase gene in ISPg2, in the TDC60 genome. (ii) Region exhibiting high nucleotide similarity to spacer 37_90: close to the IS both 2-kb upstream and 2-kb downstream in the 3 genomes.