Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Detection of Escherichia coli O157:H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses.

Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.     

Comparison and genomic sizing of Escherichia coli O157:H7 isolates by pulsed-field gel electrophoresis.

Genomic DNAs of Escherichia coli O157:H7 strains isolated from patients and food samples were analyzed by pulsed-field gel electrophoresis. The rare-cutting endonucleases SfiI and XbaI generated 6 and 10 distinct genomic profiles, respectively, for the 22 strains analyzed, indicating that this technique may find application for epidemiologic studies. Summation of XbaI fragments from five E. coli O157:H7 strains estimated the genomic length at ca. 4.7 Mb.     

Variation between pathogenic serovars within Salmonella pathogenicity islands

Although four of the five Salmonella pathogenicity islands (SPIs) have been characterized in detail for Salmonella enterica serovar Typhimurium, and the fifth has been characterized for Salmonella enterica serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of S. enterica. The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions. Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs. With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs. Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing. While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions. In SPI-1 the avrA gene was replaced by a 200-base fragment in three serovars, as reported previously. In SPI-5, two serovars had acquired an insertion with similarity to the pagJ and pagK genes between pipC and pipD. In SPI-3 the genes sugR and rhuM were deleted in most serovars and in some were replaced by sequences that were very similar to either the Escherichia coli fimbrial operon, flanked by two distinct insertion sequence elements, or to the E. coli retron phage PhiR73. The distribution of these differences suggests that there have been a number of relatively recent horizontal transfers of genes into S. enterica and that in some cases the same event has occurred in multiple lineages of S. enterica. Thus, it seems that insertion sequences and retron phages are likely to be involved in continuing evolution of the pathogenicity islands of pathogenic Salmonella serovars.     

Phylogeny of Shiga toxin-producing Escherichia coli O157 isolated from cattle and clinically ill humans

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H(-) (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.     

Archaeology and evolution of transfer RNA genes in the Escherichia coli genome

Transfer RNA genes tend to be presented in multiple copies in the genomes of most organisms, from bacteria to eukaryotes. The evolution and genomic structure of tRNA genes has been a somewhat neglected area of molecular evolution. Escherichia coli, the first phylogenetic species for which more than two different strains have been sequenced, provides an invaluable framework to study the evolution of tRNA genes. In this work, a detailed analysis of the tRNA structure of the genomes of Escherichia coli strains K12, CFT073, and O157:H7, Shigella flexneri 2a 301, and Salmonella typhimurium LT2 was carried out. A phylogenetic analysis of these organisms was completed, and an archaeological map depicting the main events in the evolution of tRNA genes was drawn. It is shown that duplications, deletions, and horizontal gene transfers are the main factors driving tRNA evolution in these genomes. On average, 0.64 tRNA insertions/duplications occur every million years (Myr) per genome per lineage, while deletions occur at the slower rate of 0.30 per million years per genome per lineage. This work provides a first genomic glance at the problem of tRNA evolution as a repetitive process, and the relationship of this mechanism to genome evolution and codon usage is discussed.     

The evolving genome of Salmonella enterica serovar Pullorum

Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas. However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.     

Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Evolution of genomic content in the stepwise emergence of Escherichia coli O157:H7

Genome comparisons have demonstrated that dramatic genetic change often underlies the emergence of new bacterial pathogens. Evolutionary analysis of Escherichia coli O157:H7, a pathogen that has emerged as a worldwide public health threat in the past two decades, has posited that this toxin-producing pathogen evolved in a series of steps from O55:H7, a recent ancestor of a nontoxigenic pathogenic clone associated with infantile diarrhea. We used comparative genomic hybridization with 50-mer oligonucleotide microarrays containing probes from both pathogenic and nonpathogenic genomes to infer when genes were acquired and lost. Many ancillary virulence genes identified in the O157 genome were already present in an O55:H7-like progenitor, with 27 of 33 genomic islands of >5 kb and specific for O157:H7 (O islands) that were acquired intact before the split from this immediate ancestor. Most (85%) of variably absent or present genes are part of prophages or phage-like elements. Divergence in gene content among these closely related strains was approximately 140 times greater than divergence at the nucleotide sequence level. A >100-kb region around the O-antigen gene cluster contained highly divergent sequences and also appears to be duplicated in its entirety in one lineage, suggesting that the whole region was cotransferred in the antigenic shift from O55 to O157. The beta-glucuronidase-positive O157 variants, although phylogenetically closest to the Sakai strain, were divergent for multiple adherence factors. These observations suggest that, in addition to gains and losses of phage elements, O157:H7 genomes are rapidly diverging and radiating into new niches as the pathogen disseminates.     

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella paratyphi B.

The genomic cleavage map of Salmonella paratyphi B was determined through digestion with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 19 XbaI sites, 10 BlnI sites, and 7 CeuI sites. The fragments were arranged in order through excision of fragments from the gel, redigestion with a second enzyme, end labelling with 32P, and reelectrophoresis. Tn10 transposons inserted in 61 different genes of S. typhimurium LT2 were transduced by use of bacteriophage P22 into S. paratyphi B. The locations of Tn10 insertions on the chromosome of S. paratyphi B were determined by use of XbaI and BlnI sites in Tn10, revealing the positions of genes with Tn10 insertions in S. paratyphi B. All seven CeuI sites (in rrl genes for 23S rRNA) and most of the XbaI and BlnI sites in rrn genes for Glt-tRNA are conserved, but only about half of the XbaI and BlnI sites outside rrn genes are conserved. Gene order is identical in the 68 genes that we could compare between S. paratyphi B and S. typhimurium LT2, and the lengths of intervals between the genes are often the same, but there are several instances of differences in interval lengths, indicating that insertions or deletions of DNA have occurred during the evolutionary divergence of these bacteria.     

Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai

Prophages make up 12% of the enterohemorrhagic Escherichia coli genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of E. coli O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 × 10⁻⁴. One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing pchB and psrC, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.     

Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in northeast Ohio

To ascertain the extent to which indistinguishable strains of Escherichia coli O157:H7 are shared between farms, molecular characterization was performed on E. coli O157:H7 isolates recovered during a longitudinal study of 20 dairy farms in northeast Ohio. Of the 20 dairy farms sampled, 16 were located in a primary area and 4 were located in two other distant geographical areas. A total of 92 E. coli O157:H7 isolates obtained from bovine fecal samples, water trough sediment samples, free-stall bedding, and wild-bird excreta samples were characterized. Fifty genetic subtypes were observed among the isolates using XbaI and BlnI restriction endonucleases. Most restriction endonuclease digestion profiles (REDPs) were spatially and temporally clustered. However, four REDPs from multiple sources were found to be indistinguishable by pulsed-field gel electrophoresis between four pairs of farms. The geographical distance between farms which shared an indistinguishable E. coli O157:H7 REDP ranged from 9 to 50 km, and the on-farm sources sharing indistinguishable REDPs included cattle and wild bird feces and free-stall bedding. Within the study population, E. coli O157:H7 REDP subtypes were disseminated with considerable frequency among farms in close geographic proximity, and nonbovine sources may contribute to the transmission of this organism between farms.     

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes

Aims:  To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains.
Methods and Results:  Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes.
Conclusions:  This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes.
Significance and Impact of the Study:  ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.     

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.     

Genome signatures of Escherichia coli O157:H7 isolates from the bovine host reservoir

Cattle comprise a main reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC). The significant differences in host prevalence, transmissibility, and virulence phenotypes among strains from bovine and human sources are of major interest to the public health community and livestock industry. Genomic analysis revealed divergence into three lineages: lineage I and lineage I/II strains are commonly associated with human disease, while lineage II strains are overrepresented in the asymptomatic bovine host reservoir. Growing evidence suggests that genotypic differences between these lineages, such as polymorphisms in Shiga toxin subtypes and synergistically acting virulence factors, are correlated with phenotypic differences in virulence, host ecology, and epidemiology. To assess the genomic plasticity on a genome-wide scale, we have sequenced the whole genome of strain EC869, a bovine-associated E. coli O157:H7 isolate. Comparative phylogenomic analysis of this key isolate enabled us to place accurately bovine lineage II strains within the genetically homogenous E. coli O157:H7 clade. Identification of polymorphic loci that are anchored both in the chromosomal backbone and horizontally acquired regions allowed us to associate bovine genotypes with altered virulence phenotypes and host prevalence. This study catalogued numerous novel lineage II-specific genome signatures, some of which appear to be associated intimately with the altered pathogenic potential and niche adaptation within the bovine rumen. The presented extended list of polymorphic markers is valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies of this emerging human pathogen.     

The ETT2 gene cluster, encoding a second type III secretion system from Escherichia coli, is present in the majority of strains but has undergone widespread mutational attrition

ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.     

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago.     

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.