Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5-5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.bacteriophage ͉ genome evolution ͉ type III secretion system

Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1–Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1–Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities.

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.

Genomic diversity of enterohemorrhagic Escherichia coli strains

Comparing the genomes of O157 and non-O157 enterohemorrhagic Escherichia coli (EHEC) strains reveals the selective conservation of a large number of virulence determinants.