Inference of the impact of insertion sequence (IS) elements on bacterial genome diversification through analysis of small-size structural polymorphisms in Escherichia coli O157 genomes

Abstract

Mobile genetic elements play important roles in the evolution and diversification of bacterial genomes. In enterohemorrhagic Escherichia coli O157, a major factor that affects genomic diversity is prophages, which generate most of the large-size structural polymorphisms (LSSPs) observed in O157 genomes. Here, we describe the results of a systematic analysis of numerous small-size structural polymorphisms (SSSPs) that were detected by comparing the genomes of eight clinical isolates with a sequenced strain, O157 Sakai. Most of the SSSPs were generated by genetic events associated with only two insertion sequence (IS) elements, IS629 and ISEc8, and a number of genes that were inactivated or deleted by these events were identified. Simple excisions of IS629 and small deletions (footprints) formed by the excision of IS629, both of which are rarely described in bacteria, were also detected. In addition, the distribution of IS elements was highly biased toward prophages, prophage-like integrative elements, and plasmids. Based on these and our previous results, we conclude that, in addition to prophages, these two IS elements are major contributors to the genomic diversification of O157 strains and that LSSPs have been generated mainly by bacteriophages and SSSPs by IS elements. We also suggest that IS elements possibly play a role in the inactivation and immobilization of incoming phages and plasmids. Taken together, our results reveal the true impact of IS elements on the diversification of bacterial genomes and highlight their novel role in genome evolution.

Figure 1.

Experimental scheme for the analysis of SSSP-containing regions. (A) The locations of the PCR primers specific to IS629 or ISEc8 (IS-insideF and -insideR and IS-outwardL and -outwardR) and to each IS-flanking genomic region (IS-outsideL and -outsideR). (B) The three strategies used to analyze the three types of SSSP-containing regions.

Figure 2.

Systematic SSSP analysis results for eight O157 strains. (Upper row) For each strain, WGPS data (Ohnishi et al. 2002) are presented; (lower row) the results of the SSSP analysis. In the WGPS data, segments that yielded PCR products with lengths identical to or different from O157 Sakai are represented by gray or yellow rectangles, respectively; (red) those that were not amplified. In this study, we analyzed all of the yellow segments, which contain SSSPs. (Blue) Segments found to contain IS629-related genomic rearrangements; (light green) those containing ISEc8-related rearrangements; (magenta) segments containing other types of structural changes; of these, segments containing strain-specific phage integrations are circled; (blue rectangles) genomic regions >500 bp in length that are not present in K-12 (Sakai-specific regions or S-loops). Loci lacking prophages (Sp1–Sp18) or prophage-like integrative elements (SpLE1–SpLE6) are indicated by a line.

Figure 3.

Distribution of IS629 and ISEc8 in the nine O157 genomes used in this study. The locations of IS629 and ISEc8 elements on each chromosome and plasmid are shown. (Black vertical lines) Insertion sites for IS629; (red lines) insertion sites for ISEc8; (blue rectangles) regions corresponding to prophages and phage-like integrative elements.

Figure 4.

Phylogenetic tree of the nine O157 strains used in this study. The phylogenetic tree was constructed based on the sequencing and analysis of 15 housekeeping genes. The locations of SNPs identified in this analysis (Table 4) are indicated.

Figure 5.

Simple excision of IS629 and the footprints generated upon excision or transposition. (Blue) IS629 sequences; (red) target site duplications (TSDs). (Slash) Deleted sequences; the size of each deletion is shown in parentheses. Homologous sequences that probably induced deletions through homologous recombination are underlined. In the four genomic loci labeled with asterisks, the phylogenetic relationships among the strains indicate that the absence of IS629 and genomic deletions in some strains were generated by a simple excision and upon the excision (or transposition) of IS629. Deletions observed at six other loci were also likely generated by a similar mechanism because they occurred precisely at IS629 insertion sites.