gb-2007-8-7-r138 GBJ Research <p>Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic <it>Escherichia coli </it>strains of O157 and non-O157 serotypes</p> Ogura Yoshitoshi y-ogura@med.miyazaki-u.ac.jp Ooka Tadasuke taohoka@med.miyazaki-u.ac.jp Asadulghani asadu@med.miyazaki-u.ac.jp Terajima Jun terajima@nih.go.jp Nougayrède Jean-Philippe jp.nougayrede@envt.fr Kurokawa Ken ken@is.naist.jp Tashiro Kousuke ktashiro@grt.kyushu-u.ac.jp Tobe Toru torutobe@bact.med.osaka-u.ac.jp Nakayama Keisuke knakaya@bg.mbn.or.jp Kuhara Satoru kuhara@grt.kyushu-u.ac.jp Oswald Eric e.oswald@envt.fr Watanabe Haruo haruwata@nih.go.jp Hayashi Tetsuya thayash@med.miyazaki-u.ac.jp

Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki,5200 Kihara, Kiyotake, Miyazaki, 889-1692, Japan

Division of Microbiology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki,5200 Kihara, Kiyotake, Miyazaki, 889-1692, Japan

Department of Bacteriology, National Institute for Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640, Japan

UMR1225, INRA-ENVT, 23 chemin des Capelles, 31076 Toulouse, France

Laboratory of Comparative Genomics, Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

Laboratory of Molecular Gene Technics, Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakosaki, Fukuoka, 812-8581, Japan

Division of Applied Bacteriology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan

 Genome Biology 1465-6906 2007 8 7 R138 http://genomebiology.com/2007/8/7/R138 1771159610.1186/gb-2007-8-7-r138 7 3 2007 6 6 2007 10 7 2007 10 7 2007 2007 Ogura et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Abstract

Background

Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the 'parallel' evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed.

Results

Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157.

Conclusion

Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs.

 Evolution Genome studies Microbiology and parasitology Bioinformatics

Background

Escherichia coli is a commensal intestinal inhabitant of vertebrates and rarely cause diseases except in compromised hosts. Several types of strains, however, cause diverse intestinal and extra-intestinal diseases in healthy humans and animals by means of individually acquired virulence factors 1. Enterohemorragic E. coli (EHEC) is one of the most devastating pathogenic E. coli, which can cause diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome (HUS) 2. Shiga toxin (Stx) is the key virulence factor responsible for the induction of hemorrhagic colitis with such complications 3. In addition, typical EHEC strains possess a pathogenicity island called 'the locus of enterocyte effacement (LEE)', which encodes a set of proteins constituting type III secretion system (T3SS) machinery. The LEE also encodes several effector proteins secreted by the T3SS, and an adhesin called intimin (encoded by the eaeA gene). The system confers on the bacteria the ability to induce attaching and effacing (A/E) lesions on the host colonic epithelial cells, enabling it to colonize tightly at the lesions 4. The LEE has also been found in enteropathogenic E. coli (EPEC), which cause severe diarrhea in infants, and in several other animal pathogens, including Citrobacter rodentium and rabbit EPEC 56. It is also known that EHEC strains harbor a large plasmid encoding several virulence factors, such as enterohemolysin 2.

Our previous genome sequence comparison of O157:H7 strain RIMD 0509952 (referred to as O157 Sakai) with the benign laboratory strain K-12 MG1655 revealed that the O157 Sakai chromosome is composed of 4.1 Mb sequences conserved in K-12, and 1.4 Mb sequences absent from K-12 (referred to as the backbone and S-loops, respectively) 78. Importantly, most of the large S-loops are prophages and prophage-like elements, and O157 Sakai contains 18 prophages (Sp1-Sp18) and 6 prophage-like elements (SpLE1-SpLE6; these elements contain phage integrase-like genes but no other phage-related genes). These Sps and SpLEs carry most of the virulence-related genes of O157, including the stx genes (stx1AB on Sp15 and stx2AB on Sp5). The LEE pathogenicity island corresponds to SpLE4. Of particular importance is that, in addition to 7 LEE-encoded effectors, 32 proteins encoded in non-LEE loci have been identified as effectors secreted by LEE-encoded T3SS (non-LEE effectors) 9101112131415. Among these, TccP has already been shown to play a pivotal role for the induction of A/E lesions in EHEC 1617. Others are also suspected to be involved in EHEC pathogenesis. Nearly all of these non-LEE effectors are encoded on the Sps and SpLEs 15.

We have recently performed a whole genome comparison of eight O157 strains by whole genome PCR scanning (WGPScanning) and comparative genomic hybridization (CGH) using O157 oligoDNA microarray analysis 1819. These analyses revealed that O157 strains are significantly divergent in the genomic structure and gene repertoire. In particular, Sp and SpLE regions exhibit remarkable diversity. We identified about 400 genes that are variably present in the O157 strains. They include several virulence-related genes, suggesting that some level of strain-to-strain variations in the potential virulence exist among O157 strains.

Although numerous EHEC outbreaks have been attributed to strains of the O157 serotype (O157 EHEC), it has increasingly been more frequently recognized that EHEC strains belonging to a wide range of other serotypes also cause similar gastrointestinal diseases in humans. Among these non-O157 EHECs, O26, O111, and O103 are the serotypes most frequently associated with human illness in many countries 20. By multilocus sequencing typing (MLST) of housekeeping genes, Reid et al. 21 have shown that these non-O157 EHEC strains belong to clonal groups distinct from O157 EHEC. Based on this finding, they proposed a 'parallel' evolution model of EHEC; each EHEC lineage has independently acquired the same major virulence factors, stx, LEE, and plasmid-encoded enterohemolysin 21. However, our knowledge on the prevalence of virulence factors among non-O157 EHEC strains is very limited. Many other virulence factors found on the O157 genome, such as fimbrial and non-fimbrial adhesins, iron uptake systems, and non-LEE effectors, are also thought to be required for the full virulence of EHEC, but their prevalence among non-O157 EHEC strains has not yet been systematically analyzed. Differences (or conservation) in the genomic structure between O157 and non-O157 EHEC strains are also yet to be determined.

In this study, we selected 20 non-O157 EHEC strains, 8 of which belong to O26, six to O111, and six to O103 serotypes, and performed a whole genome comparison with O157 EHEC strains by O157 oligoDNA microarray and WGPScanning. Our data indicate that the backbone regions are highly conserved also in non-O157 EHEC strains, while most S-loops are very poorly conserved. Among the genes on S-loops, only 8.5% were detected in all the EHEC strains examined, and around 20% were fully conserved in each non-O157 serotype. Besides, we found that the genome sizes of non-O157 EHEC strains are similar or rather larger than those of O157 strains, indicating that non-O157 EHEC strains have a huge amount of serotype- or strain-specific genes. Interestingly, virulence-related genes, particularly those for non-LEE effectors and non-fimbrial adhesions, were relatively well conserved in the non-O157 EHEC strains.

Results

Phylogeny and other features of non-O157 EHEC strains

EHEC strains used in this study were isolated from patients in Japan, Italy, or France (Table 1). The XbaI digestion patterns examined by pulsed field gel electrophoresis (PFGE) showed that the genomic DNA of EHEC strains is significantly divergent (Additional data file 1), while all possess stx1 and/or stx2 genes, and the eaeA gene encoding intimin (see 'Detection and subtyping of stx and eaeA genes' in Materials and methods). The results of the fluorescent actin staining (FAS) assay 22 indicated that all strains are potentially capable of inducing A/E lesions except for O111 strain 1. The efficiency, however, somewhat varied from strain-to-strain (data not shown).

<p>Table 1</p>

EHEC strains tested in this study

No.

Strain

Serotype

Source

Country

Symptoms

Shiga toxin

Intimin type

Sakai

RIMD 0509952

O157:H7

Human

Japan

(Sequenced strain)

stx1, stx2

γ1

O157 #2

980938

O157:H7

Human

Japan

Abdominal pain, fever

stx1, stx2vh-b

γ1

O157 #3

980706

O157:H7

Human

Japan

Diarrhea, bloody stool, abdominal pain

stx1, stx2, stx2vh-a

γ1

O157 #4

990281

O157:H7

Human

Japan

Asymptomatic carrier

stx2vh-a

γ1

O157 #5

980551

O157:H7

Human

Japan

Diarrhea, bloody stool

stx1, stx2

γ1

O157 #6

990570

O157:H7

Human

Japan

Diarrhea, bloody stool, fever

stx2vh-a

γ1

O157 #7

981456

O157:H7

Human

Japan

Diarrhea

stx1, stx2vh-a

γ1

O157 #8

982243

O157:H-

Human

Japan

Diarrhea, fever

stx1, stx2vh-a

γ1

O157 #9

981795

O157:H7

Human

Japan

Diarrhea, bloody stool, abdominal pain

stx1, stx2

γ1

O26 #1

11044

O26:H11

Human

Japan

Diarrhea, bloody stool

stx1

β1

O26 #2

11368

O26:H11

Human

Japan

Diarrhea

stx1

β1

O26 #3

11656

O26:H-

Human

Japan

Diarrhea, fever

stx1

β1

O26 #4

12719

O26:H-

Human

Japan

Diarrhea

stx1

β1

O26 #5

12929

O26:H-

Human

Japan

Diarrhea

stx1

β1

O26 #6

13065

O26:H11

Human

Japan

Diarrhea, abdominal pain

stx1

β1

O26 #7

13247

O26:H11

Human

Japan

Diarrhea, abdominal pain

stx1

β1

O26 #8

ED411

O26:H11

Human

Italy

stx2

β1

O111 #1

11109

O111:H-

Human

Japan

Diarrhea, abdominal pain

stx1

γy

O111 #2

11128

O111:H-

Human

Japan

Diarrhea, bloody stool

stx1, stx2

γy

O111 #3

11619

O111:H-

Human

Japan

Asymptomatic carrier

stx1, stx2

γy

O111 #4

11788

O111:H-

Human

Japan

Diarrhea

stx1

γy

O111 #5

13369

O111:H-

Human

Japan

Diarrhea, abdominal pain, bloody stool

stx1

γy

O111 #6

ED71

O111:H-

Human

Italy

stx1

γy

O103 #1

10828

O103:H2

Human

Japan

Diarrhea, abdominal pain

stx1

ε

O103 #2

11117

O103:H2

Human

Japan

Diarrhea, fever

stx1

ε

O103 #3

11711

O103:H2

Human

Japan

Diarrhea, fever

stx1

ε

O103 #4

11845

O103:H2

Human

Japan

Diarrhea, abdominal pain

stx1

ε

O103 #5

12009

O103:H2

Human

Japan

Diarrhea, bloody stool

stx1, stx2

ε

O103 #6

PMK5

O103:H2

Human

France

HUS

stx1

ε

The MLST analysis using seven housekeeping genes (aspC, clpX, fadD, icdA, lysP, mdh, and uidA) indicated that strains belonging to the O157, O26, O111, and O103 serotypes were clustered into three different phylogenic groups (O26 and O111 strains were clustered together; Additional data file 2). This result is basically consistent with those from previous MLST analyses using different genetic loci 2123. The type of intimin was classified as γ1, β1, γ2, and ε for O157, O26, O111, and O103, respectively.

Chromosome sizes and plasmid profiles

The I-CeuI digestion of chromosomal DNA yielded seven fragments in 26 out of 29 EHEC strains (data not shown). Because I-CeuI specifically cleaves a 19 base-pair sequence in the 23S ribosomal RNA gene, it demonstrated that these strains have seven copies of the ribosomal operon (rrn), as in K-12 and O157. Estimated chromosome sizes of these strains were all much larger than that of K-12, with diverged sizes ranging from 5,102 to 5,945 kb (Table 2). O111 and O103 strains contained slightly smaller chromosomes than O157 strains. In contrast, most O26 strains contained relatively larger chromosomes. We could not estimate the chromosome sizes in two O157 strains (2 and 9) and one O103 strain (4), because all or the largest fragments repeatedly exhibited smear patterns.

<p>Table 2</p>

Estimated genome sizes of EHEC strains

Estimated sizes (kb)

K-12*

Sakai*

O157

O26

O111

O103

In silico

Exp

In silico

Exp

#2

#3

#4

#5

#6

#7

#8

#9

#1

#2

#3

#4

#5

#6

#7

#8

#1

#2

#3

#4

#5

#6

#1

#2

#3

#4

#5

#6

I-ceuI-fragmant no.

1

2,498

2,686

3,216

3,191

ND

3,342

3,325

3,277

3,226

3,358

3,325

ND

3,185

3,386

3,345

3,414

3,571

3,513

3,630

3,374

2,941

3,044

2,912

2,898

2,884

2,814

2,911

2,959

3,291

ND

2,923

2,961

2

698

687

712

720

722

722

713

713

693

718

708

ND

777

777

782

823

751

787

782

734

824

803

808

808

803

808

889

923

941

872

883

761

3

657

649

709

707

698

679

679

657

670

679

674

ND

746

751

751

741

720

720

720

720

698

698

698

693

693

698

709

720

797

714

756

712

4

521

525

579

591

574

574

574

574

574

582

574

ND

382

382

458

382

385

385

385

537

519

519

519

519

519

519

517

517

346

521

362

514

5

131

127

144

142

144

142

179

142

142

144

144

ND

295

295

301

295

298

298

298

143

140

137

137

135

135

135

137

136

317

133

320

136

6

94

83

96

89

89

88

88

88

91

88

89

ND

97

97

96

97

97

97

97

99

92

92

92

91

86

88

98

101

97

98

97

93

7

41

41

41

41

43

42

42

42

42

42

42

ND

41

41

41

41

41

41

33

41

41

41

41

41

41

41

41

43

43

43

43

43

Chromosome total

4,640

4,797

5,498

5,480

ND

5,589

5,600

5,492

5,437

5,610

5,556

ND

5,524

5,731

5,773

5,794

5,864

5,842

5,945

5,647

5,256

5,334

5,207

5,185

5,160

5,102

5,303

5,398

5,833

ND

5,384

5,220

Plasmid no.

1

93

93

93

93

101

93

93

93

93

ND

7

85

91

98

98

98

98

137

77

205

125

81

87

155

74

ND

89

89

72

52

2

3

3

6

7

3

ND

63

65

73

49

91

107

98

77

51

47

7

ND

72

63

3

3

ND

6

4

7

68

25

78

7

7

7

5

ND

4

ND

4

7

3

8

5

5

ND

5

ND

7

ND

Plasmid total

-

-

96

96

93

93

101

93

102

99

95

ND

7

158

156

175

154

98

263

273

77

395

208

144

145

166

74

ND

160

152

72

52

Genome total

4,640

4,797

5,594

5,576

NE

5,682

5,701

5,585

5,539

5,709

5,651

ND

5,530

5,889

5,929

5,969

6,018

5,940

6,208

5,920

5,333

5,729

5,415

5,328

5,305

5,268

5,377

ND

5,993

ND

5,456

5,273

*Lengths of each band estimated from experimental data and in silico analyses are shown. ND, not detected.

Plasmid profiles indicated that all but one O157 strain contain one large plasmid of a similar size (Table 2; Additional data file 3). All of the non-O157 EHEC strains also contained at least one large plasmid except for O26 strain 1 (one small plasmid was present) and O103 strain 2 (no plasmid was detected). Several O26 and O111 strains possessed two or three large plasmids. The estimated total genome sizes of EHEC strains ranged from 5.27 Mb to 6.21 Mb.

Overview of the CGH analysis of non-O157 EHEC

We analyzed the gene contents of non-O157 EHEC strains by using the O157 oligoDNA microarray, and compared the results with those of O157 strains in our previous report 18 (Figures 1 and 2). More Sakai genes were absent from the non-O157 EHEC strains. In O157 strains, the absent genes were found mostly in Sp and SpLE regions, but in non-O157 EHEC strains, they were found not only in Sp and SpLE regions but also in various S-loops. The conservation tended to exhibit a serotype-specific pattern, but remarkable strain-to-strain diversity was also observed in each serotype.

<p>Figure 1</p>

Summary of the CGH and WGPScanning analyses of O157 and non-O157 EHEC strains

Summary of the CGH and WGPScanning analyses of O157 and non-O157 EHEC strains. Results from the CGH analysis of 29 EHEC strains using an O157 oligoDNA microarray are shown in the upper half of each segment, and those from the genome structural analysis by the WGPScaning method in the lower half. Above the CGH data, genes on prophages (Sps), prophage-like elements (SpLEs), and plasmids are indicated in red (the first row), repeated genes in black (the second row), and genes conserved or partially conserved in K-12 in green or pink, respectively (the third row). Genes judged as present in the CGH analysis are indicated in blue and those absent in yellow. Singleton and repeated genes classified as 'uncertain' are indicated in pink and gray, respectively. Results from the WGPScanning analysis are presented as follows. Segments of the same sizes as those from O157 Sakai are indicated in gray, and those with large (≥5 kb) and small (<5 kb) size reductions in blue and light blue, respectively. The segments with large (≥5 kb) and small (<5 kb) size increments are indicated in orange and yellow, respectively, and those not amplified in red. When Sps, SpLEs, or their corresponding elements were not integrated in relevant loci, such regions are depicted as blank areas. The segments containing potential integration sites for large genomic elements are indicated by arrowheads. Positions of known and newly identified integration sites for Stx phages and LEE elements are indicated between the panels for the CGH and WGPScanning data. In this figure, each segment is not drawn to scale but to the gene position in the data presentation of the CGH analyses. The data from the first half of EHEC chromosomes are shown in this figure, and those from the second half and plasmids in Figure 2.

 <p>Figure 2</p>

Summary of the CGH and WGPScanning analyses of O157 and non-O157 EHEC strains

Summary of the CGH and WGPScanning analyses of O157 and non-O157 EHEC strains. The data from CGH and WGPScanning analyses of 29 EHEC strains are shown. The data from the second half of EHEC chromosomes and plasmids are shown in this figure. See the legend of Figure 1 for details.

To more precisely analyze the CGH data, we categorized the Sakai genes into three groups 18. Since most Sakai genes were represented by two oligonucleotide probes in our microarray, we first classified the probes into two groups by their homologies to the K-12 genome sequence; those with ≥90% identity into 'conserved in K-12' probes and others into 'Sakai-specific' probes. Each gene was then classified into 'conserved in K-12' genes, 'partly conserved in K-12' genes (genes represented by one 'conserved in K-12' probe and one 'Sakai-specific' probe), or 'Sakai-specific' genes. Repeated gene families that occurred in O157 Sakai more than once were analyzed separately from singleton genes (see Materials and methods for details on the classification and the presence or absence determination).

'Conserved in K-12' singleton genes were highly conserved in all serotypes: 3,596 (98.5%), 3,450 (94.5%), 3,331 (91.2%), and 3,542 (97.0%) out of 3,651 genes were fully conserved in O157, O26, O111 and O103, respectively, and 3,240 (88.7%) in all the test strains (Figure 3; Additional data file 4). 'Sakai-specific' singleton genes were relatively well conserved in O157 strains, but very poorly in non-O157 EHEC strains: 741 (64.3%), 221 (19.2%), 300 (26.0%), and 231 (20.0%) out of 1,153 genes were fully conserved in O157, O26, O111, and O103, respectively. Only 98 (8.5%) were conserved in all the test strains.

<p>Figure 3</p>

Conservation of O157 Sakai genes in O157 and non-O157 EHEC strains

Conservation of O157 Sakai genes in O157 and non-O157 EHEC strains. The data from CGH analyses of O157 and non-O157 EHEC strains using an O157 Sakai oligoDNA microarray are summarized. Among the 4,905 singleton genes on the O157 Sakai genome, 3,651 were categorized as 'conserved in K-12', 101 as 'partly conserved in K-12', and 1,153 as 'Sakai-specific'. Among the 151 repeated gene families, 23 were categorized as 'conserved in K-12' and 128 as 'Sakai-specific'. Genes that were judged as 'present' in all the tested strains were categorized as 'Fully conserved' genes, those judged as 'absent' in all the strains as 'Fully absent' genes, and others as 'Variably absent or present' genes. In the CGH analysis, because repeated gene families with reduced copy numbers were often judged as 'absent', all the repeated gene families judged as 'absent' were categorized as 'uncertain'. See Additional data file 4 for further details.

Among the 4,905 singleton genes, 101 were categorized as 'partly conserved in K-12' genes. They included 81 genes that are encoded on the backbone and 20 genes on S-loops or backbone/S-loop junctions. In O157, all but 5 (95.0%) of the 'partly conserved in K-12' genes were fully conserved. In non-O157 EHECs, however, many 'partly conserved in K-12' genes were categorized as 'uncertain' (7 to 42 genes in each non-O157 EHEC strain, 28 genes on average), because only one of the two probes yielded positive results. Therefore, only 44 (43.6%), 40 (39.6%), and 58 (57.4%) were fully conserved in O26, O111, and O103, respectively (Figure 3; Additional data file 4). This result suggests that most of the 'partly conserved in K-12' genes are present in the non-O157 EHEC strains but many have significantly divergent sequences from those of O157 Sakai.

O157 Sakai contains many repeated genes (542 out of 5,447 genes), such as transposase- and phage-related genes. They can be grouped into 151 families. Compared with the singleton genes, the repeated gene families were relatively well conserved in non-O157 EHECs. About half of the 'conserved in K-12' repeated gene families (11 out of the 23 families (47.8%)) were fully conserved in all the test strains, and 81 (63.3%), 74 (57.8%), 60 (46.9%), and 77 (60.2%) out of the 128 'Sakai-specific' repeated gene families were fully conserved in O157, O26, O111, and O103, respectively (Figure 3; Additional data file 4). Because most of the repeated genes were from lambda-like prophages and IS elements 818, this result indicates that non-O157 EHEC strains also contain multiple lambda-like prophages and IS elements very similar to those found in O157 Sakai.

Absent 'conserved in K-12' genes in EHEC strains

Among the 3,651 'conserved in K-12' singleton genes, 224 (6.1%) were absent in at least one test strain. These genes were found to be absent more frequently in non-O157 EHEC strains than in O157 strains: 75 genes (2.1%) in O26 strains, 184 (5.0%) in O111, and 61 (1.7%) in O103, while only 37 (1.0%) in O157 (here we counted only the genes that were judged as 'absent' in at least one strain; therefore, these results do not include the genes that were 'uncertain' in some strains but 'absent' in no strain). These genes were dispersed on the chromosome and belonged to various functional categories (Additional data file 5); but as expected, none of them was listed as essential, either in the 'profiling of E. coli chromosome' (PEC) database 24 or in a systematic single-gene deletion study of E. coli K-12 25. We also identified 46, 83, and 30 'conserved in K-12' singleton genes that are fully absent in O26, O111, and O103, respectively. Among these, 22 genes, which are located in 12 different chromosomal loci, were absent in all non-O157 EHEC strains, and 10, 44, and 3 genes were specifically missing in O26, O111, and O103, respectively.

Conservation of 'Sakai-specific' genes in non-O157 EHEC strains

We categorized 'Sakai-specific' singleton genes according to the COG (clusters of orthologous groups of proteins) classification 26, and analyzed the gene conservation of each functional category (Figure 4). In O157, most genes were well conserved in all categories. Many genes for 'replication, recombination and repair' and for 'transcription' were variably present among O157 strains, but most of them were on Sps and SpLEs. In the non-O157 serotypes, however, the 'Sakai-specific' singleton genes belonging to almost every COG functional category exhibited poor conservation (many were classified as 'Fully absent'). The level of conservation was similar to that observed for the four sequenced pathogenic E. coli strains of different pathotypes 27282930 (Additional data file 4).

<p>Figure 4</p>

Conservation of 'Sakai-specific' singleton genes in each functional group

Conservation of 'Sakai-specific' singleton genes in each functional group. 'Sakai-specific' singleton genes were categorized according to the COG classification. In each functional category, the numbers of genes fully conserved, variably absent or present, and fully absent are shown for each serotype.

A relatively large number of genes for 'carbohydrate transport and metabolism' were fully conserved in non-O157 EHECs. Among these, genes for the sugar ABC transporter system (ECs0374-0378), and the N-acetylgalactosamine-specific PTS system (ECs4013-4014), and two genes for sugar utilization (ECs3242: fructokinase and ECs3243: sucrose-6 phosphate hydrolase) were conserved in all the tested strains. A relatively large number of genes for the 'cell wall/membrane biogenesis' category were also fully conserved. Most of them were the genes for lipopolysaccharide core biosynthesis (ECs2831 and ECs2836-2845). This is consistent with the fact that four serotypes examined here share the same core type (R3) 3132.

SpLE1 carries gene clusters for urease (ECs1321-1327) and tellurite resistance (ECs1343, 1351-1358). In an earlier report, the urease genes were found specifically associated with EHEC strains irrespective of their serotypes 33. Our present data, however, demonstrate that five EHEC strains (one O157, one O26, and three O103 strains) lack the urease genes. The tellurite resistance genes were also well conserved in non-O157 EHECs but absent in one O26 and two O103 strains.

Distribution of O157 Sakai virulence-related genes in non-O157 EHECs

In the COG classification, many of the virulence-related genes were classified into the 'not in COGs' category. We thus picked up all the known or suspected O157 virulence-related genes, and analyzed their conservation in non-O157 EHECs. Fimbria are important for virulence as an initial attachment factors to the host intestine. The O157 Sakai genome contained 14 fimbrial biosynthesis gene clusters (loci 1 to 14), all of which were completely conserved in every O157 strain except for strain 8, in which locus 11 was partially conserved (Table 3). Among the 14 clusters, four (loci 3, 5, 7, and 14) were completely conserved in K-12 and three (loci 1, 8, and 11) partially conserved. These seven loci were also completely or partially conserved in the non-O157 EHEC strains, suggesting that these gene clusters are widely conserved in various E. coli strains irrespective of their pathotypes. Genes on the remaining seven loci were almost completely absent in all non-O157 serotypes. Only loci 9 and 10 were partially conserved in several non-O157 EHEC strains. Thus, we may regard them as O157-specifc fimbrial gene clusters.

<p>Table 3</p>

Conservation of fimbrial loci in each EHEC strain

K-12

O157

O26

O111

O103

Locus no.

ECs number

#2

#3

#4

#5

#6

#7

#8

#9

#1

#2

#3

#4

#5

#6

#7

#8

#1

#2

#3

#4

#5

#6

#1

#2

#3

#4

#5

#6

1

ECs0019-0024

p

+

+

+

+

+

+

+

+

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

p

2

ECs0139-0145

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

ECs0592-0597

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

4

ECs0741-0744

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5

ECs1021-1028

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

6

ECs1276-1281

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

7

ECs0267&1414-1421

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

8

ECs2107-2114

p

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

p

p

+

p

p

+

9

ECs2914-2918

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

p

-

-

p

10

ECs3216-3222

-

+

+

+

+

+

+

+

+

p

-

-

p

-

-

p

p

p

p

-

p

-

p

p

p

p

p

p

p

11

ECs4020-4023&4026

p

+

+

+

+

+

+

p

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

12

ECs4426-4431

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

13

ECs4665-4670

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

14

ECs5271-5279

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

p

+

+

+

+

+

+

+

+

+

+

Symbols: '+' indicates a locus where all genes were conserved; '-' a locus where all genes were absent; and 'P' a locus where one or more genes, but not all genes, were absent. Genes judged as 'uncertain' were not considered.

In addition to the fimbrial genes, 14 Sakai genes have been demonstrated or suspected to encode non-fimbrial adhesins (Table 4). They were relatively well conserved in the non-O157 EHEC strains. 'Regulators' and 'Toxins and their activators' showed similar levels of conservation as the genes related to adhesion (Table 4).

<p>Table 4</p>

Conservation of Sakai virulence-related genes in EHECs and other sequenced pathogenic E. coli strains

In silico*

No. of strains conserved†

Gene

Location

Conservation in K-12

Common name/description

CFT073

UTI89

536

APEC

O157 (8)

O26 (8)

O111 (6)

O103 (6)

Genes related to adhsion (not fimbrial genes)

ECs0336

S-loop20

Partly conserved

Putative invasin

+

+

+

+

[8]

[8]

[6]

[6]

ECs0350

S-loop23

Absent

HmwA-like protein

-

-

+

-

(7)

0

0

0

ECs0362

S-loop24

Absent

AidA-I adhesin-like protein

-

-

-

-

[8]

[8]

[6]

[6]

ECs0548

S-loop43

Absent

Saa-like protein

-

+

-

+

[8]

0

0

0

ECs1360

SpLE1

Absent

Iha adhesin

+

-

-

-

[8]

[8]

[6]

2

ECs1396

SpLE1

Conserved

AidA-I adhesin-like protein

-

-

-

-

(7)

[8]

0

[6]

ECs1772

Sp9

Absent

Paa

-

-

-

-

[8]

(7)

[6]

4

ECs2006

Backbone

Absent

BigA-like protein

-

-

-

-

[8]

0

0

0

ECs2007

Backbone

Absent

BigB-like protein

-

-

-

-

[8]

0

0

0

ECs2567

Backbone

Conserved

Putative adhesin

+

+

+

+

[8]

[8]

[6]

[6]

ECs3860

SpLE3

Absent

Efa1 (interrupted)

-

-

-

-

(7)

[8]

[6]

[6]

ECs3861

SpLE3

Absent

Efa1 (partial)

-

-

-

-

(7)

[8]

[6]

[6]

ECs4559

SpLE4 (LEE)

Absent

Gamma intimin

-

-

-

-

[8]

0

0

0

ECs5290

S-loop288

Absent

Putative invasin

-

-

-

-

(7)

0

0

0

Genes confer resistance to host immune response

ECs0218

S-loop14

Absent

IcmF-like protein

-

-

-

-

[8]

[8]

[6]

[6]

ECs1236

Sp5

Absent

Lom

-

-

-

-

5

1

2

0

ECs1312

SpLE1

Absent

TraT

-

-

-

-

(7)

(7)

0

3

ECs1956

Sp10

Absent

IrsA-like protein

-

-

-

+

5

1

(5)

1

ECs3850

SpLE3

Absent

PagC-like protein

-

-

-

-

[8]

0

[6]

2

RF001

Sp5, 8

Conserved

Bor

+

+

+

+

6

[8]

1

5

RF098

Sp4, 10

Absent

Copper/zinc-superoxide dismutase

-

-

-

-

4

1

(5)

[6]

RF115

Sp3, 4, 8 - 12, 14, 15

Absent

Lom

-

-

-

+

[8]

[8]

[6]

[6]

Toxins and activators

ECs0541

S-loop42

Absent

RTX-like protein

-

-

-

-

[8]

0

0

0

ECs0542

S-loop42

Absent

RTX-like protein

-

-

-

-

[8]

0

0

0

ECs0814

Sp3

Absent

SfpA (systemic factor protein A)-like protein

-

-

-

-

[8]

[8]

[6]

[6]

ECs1205

Sp5

Absent

Shiga toxin 2 subunit A

-

-

-

-

4

1

2

1

ECs1206

Sp5

Absent

Shiga toxin 2 subunit B

-

-

-

-

3

1

2

1

ECs1282

S-loop71

Absent

Hemagglutinin/hemolysin - related protein

-

-

-

-

[8]

0

0

0

ECs1283

S-loop71

Absent

Hemolysin activator - related protein

-

-

-

-

[8]

0

0

0

ECs1382

SpLE1

Absent

HecB-like protein

-

-

-

-

5

(7)

[6]

2

ECs1652

Sp8

Absent

Putative catalase

-

-

-

-

[8]

0

0

0

ECs1677

Backbone

Conserved

Hemolysin E

-

-

-

-

[8]

[8]

[6]

[6]

ECs2973

Sp15

Absent

Stx1B

-

-

-

-

6

(7)

[6]

[6]

ECs2974

Sp15

Absent

Stx1A

-

-

-

-

6

(7)

[6]

[6]

Regulators

ECs1274

s-loop71

Absent

GrvA

-

-

-

-

[8]

0

0

0

ECs1388

SpLE1

Absent

PchD

+

-

-

+

4

(7)

2

2

ECs1588

Sp7

Absent

PchE

-

-

-

-

(7)

[8]

0

4

ECs3105

Backbone

Conserved

RcsD

+

+

+

+

[8]

[8]

[6]

[6]

ECs3106

Backbone

Conserved

RcsB

+

+

+

+

[8]

[8]

[6]

[6]

ECs3107

Backbone

Conserved

RcsC

+

+

+

+

[8]

[8]

[6]

[6]

ECs3720

ETT2

Absent

EtrA

-

-

-

-

[8]

[8]

4

4

ECs3734

ETT2

Absent

EivF

-

-

-

-

[8]

0

0

0

ECs4577

SpLE4 (LEE)

Absent

GrlA

-

-

-

-

[8]

[8]

4

(5)

ECs4578

SpLE4 (LEE)

Absent

GrlR

-

-

-

-

[8]

[8]

4

4

ECs4588

SpLE4 (LEE)

Absent

Ler

-

-

-

-

[8]

[8]

(5)

[6]

RF132

Sp4, 11, 14

Absent

PchA, B, C

-

-

-

-

[8]

(7)

[6]

[6]

Secretion machineries

ECs0540

S-loop42

Absent

CyaE-like protein

-

-

-

-

[8]

0

0

0

ECs0543

S-loop43

Absent

Putative RTX toxin secretion ATP-binding protein

-

-

-

-

[8]

0

0

0

ECs0544

S-loop44

Absent

Putative RTX toxin secretion membrane fusion protein

-

-

-

-

[8]

0

0

0

ECs3716

ETT2

Absent

EprK

-

-

-

-

[8]

[8]

(5)

(5)

ECs3717

ETT2

Absent

EprJ

-

-

-

-

[8]

[8]

4

(5)

ECs3718

ETT2

Absent

EprI

-

-

-

-

[8]

[8]

(5)

4

ECs3719

ETT2

Absent

EprH

-

-

-

-

[8]

[8]

4

(5)

ECs3721

ETT2

Absent

EpaS

-

-

-

-

[8]

[8]

(5)

(5)

ECs3722

ETT2

Absent

EpaR2

-

-

-

-

[8]

[8]

4

4

ECs3723

ETT2

Absent

EpaR1

-

-

-

-

[8]

[8]

(5)

4

ECs3724

ETT2

Absent

EpaQ

-

-

-

-

[8]

[8]

(5)

(5)

ECs3725

ETT2

Absent

EpaP

-

-

-

-

[8]

[8]

4

4

ECs3726

ETT2

Absent

EpaO

-

-

-

-

[8]

1

0

2

ECs3727

ETT2

Absent

EivJ

-

-

-

-

[8]

0

0

0

ECs3729

ETT2

Absent

EivI

-

-

-

-

[8]

0

0

0

ECs3730

ETT2

Absent

EivC

-

-

-

-

[8]

0

0

0

ECs3731

ETT2

Absent

EivA

-

-

-

-

[8]

0

0

0

ECs3732

ETT2

Absent

EivE

-

-

-

-

[8]

0

0

0

ECs3733

ETT2

Absent

EivG

-

-

-

-

[8]

0

0

0

ECs4551

SpLE4 (LEE)

Absent

Orf29

-

-

-

-

[8]

3

[6]

(5)

ECs4552

SpLE4 (LEE)

Absent

EscF

-

-

-

-

[8]

[8]

[6]

[6]

ECs4553

SpLE4 (LEE)

Absent

CesD2

-

-

-

-

[8]

[8]

[6]

[6]

ECs4555

SpLE4 (LEE)

Absent

EspD

-

-

-

-

[8]

0

0

1

ECs4556

SpLE4 (LEE)

Absent

EspA

-

-

-

-

[8]

1

0

1

ECs4557

SpLE4 (LEE)

Absent

SepL

-

-

-

-

[8]

[8]

[6]

(5)

ECs4558

SpLE4 (LEE)

Absent

EscD

-

-

-

-

[8]

0

3

2

ECs4560

SpLE4 (LEE)

Absent

CesT

-

-

-

-

[8]

6

[6]

[6]

ECs4563

SpLE4 (LEE)

Absent

CesF

-

-

-

-

[8]

0

0

0

ECs4565

SpLE4 (LEE)

Absent

SepQ

-

-

-

-

[8]

0

0

1

ECs4566

SpLE4 (LEE)

Absent

Orf16

-

-

-

-

[8]

0

(5)

0

ECs4567

SpLE4 (LEE)

Absent

Orf15

-

-

-

-

[8]

6

(5)

4

ECs4568

SpLE4 (LEE)

Absent

EscN

-

-

-

-

[8]

4

(5)

3

ECs4569

SpLE4 (LEE)

Absent

EscV

-

-

-

-

[8]

6

(5)

2

ECs4570

SpLE4 (LEE)

Absent

Orf12

-

-

-

-

[8]

4

4

2

ECs4572

SpLE4 (LEE)

Absent

Rorf8

-

-

-

-

[8]

0

0

0

ECs4573

SpLE4 (LEE)

Absent

EscJ

-

-

-

-

[8]

3

4

2

ECs4574

SpLE4 (LEE)

Absent

SepD

-

-

-

-

[8]

0

4

2

ECs4575

SpLE4 (LEE)

Absent

EscC

-

-

-

-

[8]

[8]

[6]

4

ECs4576

SpLE4 (LEE)

Absent

CesD

-

-

-

-

[8]

5

4

3

ECs4579

SpLE4 (LEE)

Absent

Rorf3

-

-

-

-

[8]

1

4

2

ECs4580

SpLE4 (LEE)

Absent

EscU

-

-

-

-

[8]

(7)

(5)

4

ECs4581

SpLE4 (LEE)

Absent

EscT

-

-

-

-

[8]

(7)

4

3

ECs4582

SpLE4 (LEE)

Absent

EscS

-

-

-

-

[8]

[8]

(5)

(5)

ECs4583

SpLE4 (LEE)

Absent

EscR

-

-

-

-

[8]

[8]

4

(5)

ECs4584

SpLE4 (LEE)

Absent

Orf5

-

-

-

-

[8]

[8]

[6]

(5)

ECs4585

SpLE4 (LEE)

Absent

Orf4

-

-

-

-

[8]

[8]

4

(5)

ECs4586

SpLE4 (LEE)

Absent

Orf3

-

-

-

-

[8]

6

[6]

(5)

ECs4587

SpLE4 (LEE)

Absent

Orf2

-

-

-

-

[8]

3

1

2

T3SS effectors (LEE encoded)

ECs4550

SpLE4 (LEE)

Absent

EspF1

-

-

-

-

[8]

0

0

0

ECs4554

SpLE4 (LEE)

Absent

EspB

-

-

-

-

[8]

0

0

0

ECs4561

SpLE4 (LEE)

Absent

Tir

-

-

-

-

[8]

0

0

0

ECs4562

SpLE4 (LEE)

Absent

Map

-

-

-

-

[8]

0

0

2

ECs4564

SpLE4 (LEE)

Absent

EspH

-

-

-

-

[8]

[8]

0

[6]

ECs4571

SpLE4 (LEE)

Absent

SepZ

-

-

-

-

[8]

0

0

0

ECs4590

SpLE4 (LEE)

Absent

EspG

-

-

-

-

[8]

1

3

2

T3SS effectors (non-LEE encoded)

ECs0061

Backbone

Conserved

EspY1

-

-

-

-

[8]

0

0

0

ECs0847

Sp3

Absent

NleC

-

-

-

-

[8]

(7)

1

1

ECs0848

Sp3

Absent

NleH1-1

-

-

-

-

[8]

1

0

3

ECs0850

Sp3

Absent

NleD

-

-

-

-

[8]

0

0

4

ECs0876

S-loop57

Absent

EspX2

-

-

-

-

[8]

0

0

0

ECs1127

Sp4

Absent

EspK

-

-

-

-

[8]

[8]

[6]

[6]

ECs1560

Sp6

Absent

EspX7

-

-

-

-

[8]

(7)

[6]

[6]

ECs1561

Sp6

Absent

EspN

-

-

-

-

[8]

(7)

(5)

[6]

ECs1567

Sp6

Absent

EspO1-1

-

-

-

-

[8]

0

[6]

1

ECs1568

Sp6

Absent

EspR1

-

-

-

-

[8]

[8]

[6]

[6]

ECs1810

Sp9

Absent

NleG2-1

-

-

-

-

(7)

(7)

[6]

1

ECs1811

Sp9

Absent

NleG2-1

-

-

-

-

(7)

(7)

0

2

ECs1812

Sp9

Absent

NleA/EspI

-

-

-

-

(7)

(7)

0

1

ECs1814

Sp9

Absent

NleH1-2

-

-

-

-

4

6

[6]

[6]

ECs1815

Sp9

Absent

NleF

-

-

-

-

4

6

[6]

[6]

ECs1824

Sp9

Absent

NleG

-

-

-

-

[8]

(7)

[6]

0

ECs1825

Sp9

Absent

EspM1

-

-

-

-

[8]

(7)

[6]

0

ECs2226

Sp12

Absent

NleG7

-

-

-

-

[8]

[8]

[6]

[6]

ECs2714

Sp14

Absent

EspJ

-

-

-

-

[8]

0

0

1

ECs3485

Sp17

Absent

EspM2

-

-

-

-

[8]

(7)

[6]

2

ECs3486

Sp17

Absent

NleG8-2

-

-

-

-

[8]

(7)

[6]

3

ECs3487

Sp17

Absent

EspW

-

-

-

-

[8]

(7)

[6]

3

ECs3855

SpLE3

Absent

EspL2

-

-

-

-

[8]

[8]

[6]

[6]

ECs3857

SpLE3

Absent

NleB1

-

-

-

-

[8]

[8]

[6]

[6]

ECs3858

SpLE3

Absent

NleE

-

-

-

-

[8]

[8]

[6]

[6]

ECs4653

S-loop252

Absent

EspY4

-

-

-

-

[8]

0

0

0

RF004

Sp4, 14

Absent

Tccp, TccP2

-

-

-

-

[8]

1

2

4

RF067

Sp10, 11

Absent

NleG2-2, NleG2-3

-

-

-

-

[8]

[8]

[6]

4

RF069

Sp10, 11, 17

Absent

NleG6-1, NleG6-2, NleG6-3

-

-

-

-

[8]

(7)

[6]

3

RF070

Sp10, 11

Absent

NleG5-1, NleG5-2

-

-

-

-

[8]

(7)

(5)

1

Plasmid encoded

pO157_01

pO157

Absent

Metalloprotease StcE

-

-

-

-

[8]

0

0

0

pO157_02

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_03

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_04

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_05

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

0

0

2

pO157_06

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

2

0

3

pO157_07

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_08

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

0

0

3

pO157_09

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_10

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

0

0

1

pO157_11

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_12

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

1

0

3

pO157_13

pO157

Absent

Type II secretion pathway related protein

-

-

-

-

[8]

0

0

1

pO157_14

pO157

Absent

Type II secretion pathway related protein

-

-

+

+

[8]

0

0

1

pO157_17

pO157

Absent

Hemolysin C

-

-

-

-

[8]

[8]

(5)

3

pO157_18

pO157

Absent

Hemolysin A

-

-

-

-

[8]

(7)

(5)

4

pO157_19

pO157

Absent

Hemolysin B

-

-

-

-

[8]

(7)

(5)

4

pO157_20

pO157

Absent

Hemolysin D

-

-

-

-

[8]

(7)

(5)

4

pO157_39

pO157

Absent

Hemagglutinin-associated protein

-

+

-

-

[8]

5

0

1

pO157_59

pO157

Absent

Putative adherence factor, Efa1 homolog

-

-

-

-

[8]

3

0

(5)

pO157_76

pO157

Absent

KatP

-

-

-

-

6

6

(5)

2

pO157_79

pO157

Absent

EspP

-

-

-

-

[8]

(7)

[6]

4

pO157_80

pO157

Absent

Putative polysaccharide deacetylase (ecf1)

-

-

-

-

[8]

(7)

(5)

4

pO157_81

pO157

Absent

Putative LPS-1,7-N-acetylglucosamine transferase (ecf2)

-

-

-

-

[8]

(7)

(5)

4

pO157_82

pO157

Absent

Putative membrane protein (ecf3)

-

-

-

-

[8]

(7)

(5)

4

pO157_83

pO157

Absent

Putative lipid A myristoyl transferase, MsbB2 (ecf4)

-

-

-

-

[8]

(7)

(5)

4

*Conservation of O157 Sakai virulence genes in four sequenced pathogenic E. coli strains was determined according to the results of homology search using the BLASTP program. The threshold for presence (+) or absence (-) determination was ≥90% sequence identity and ≥50% aligned length coverage of a query sequence. † Genes conserved in all strains are indicated by brackets in bold, and those absent only in one strain by parentheses in bold.

Iron uptake systems are also important for bacterial survival in host environments. O157 Sakai contains seven gene clusters for iron uptake. All were conserved in every O157 strain except for strains 4 and 7, where locus 4 was missing (Table 5). In non-O157 EHECs, although three clusters common with K-12 were present in all strains, another four clusters were completely missing.

<p>Table 5</p>

Conservation of the loci for iron uptake systems in each EHEC strain

K-12

O157

O26

O111

O103

Locus no.

ECs number

#2

#3

#4

#5

#6

#7

#8

#9

#1

#2

#3

#4

#5

#6

#7

#8

#1

#2

#3

#4

#5

#6

#1

#2

#3

#4

#5

#6

1

ECs0154-0157, 1752, 3889, 3890

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

2

ECs0413-0415

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

ECs0622-0635

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

4

ECs1693-1699

-

+

+

-

+

+

-

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5

ECs3913-3917

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

6

ECs4250&4251

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

7

ECs4380-4387

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Symbols: '+' indicates a locus where all genes were conserved; '-' a locus where all genes were absent. Genes judged as 'uncertain' were not considered.

LEE is a T3SS-encoding pathogenicity island (SpLE4 in O157 Sakai) acquired by lateral gene transfer (LGT). Although LEE has been found in various EHEC and EPEC strains, they are genetically divergent. Based on the sequence polymorphism of the eaeA gene encoding intimin, 28 alleles have been identified so far 34. Although the core regions of each type of LEE encode nearly the same set of genes, their DNA sequences are known to be significantly divergent. For example, the sequence identity of the LEE core region between O157 Sakai (intimin γ1) and the O26:NM strain 413/89-1 (intimin β1) (accession number: AJ277443) is around 93% on average, and that between O157 Sakai and the O103:H2 strain RW1374 (intimin ε) 35 (accession number: AJ303141) is also 93%. In our CGH analysis, many probes for LEE core genes exhibited reduced signal intensities, just below borderline for presence/absence calls in all the non-O157 EHEC strains, and thus many LEE core genes were judged as 'absent' (Table 4). This indicates that the core genes of the non-O157 EHEC strains, which include seven LEE-encoded effector genes, also have significantly diverged nucleotide sequences.

Of the 32 non-LEE effectors, all but three are encoded on Sps and SpLEs 15. These non-LEE effectors on Sps and SpLEs, which are composed of 22 singleton genes and 4 repeated gene families, exhibited an unexpectedly high level of conservation in non-O157 EHECs. Six were conserved in all strains, eighteen in more than half of the strains, and all in at least one strain (Table 4). In contrast, three non-LEE effectors on non-prophage regions were fully absent in all non-O157 EHEC strains.

Plasmid-encoded virulence-related genes

O157 Sakai contains a 93 kb virulence plasmid (pO157) and a small cryptic plasmid (pOSAK1) 36. As previously reported 18, genes on pO157 were almost completely conserved in O157 strains excepted for strain 2, where 18 genes were missing. In contrast, these plasmid genes exhibited poor and highly variable conservation patterns in the non-O157 EHEC strains (Figures 1 and 2). Consistent with the plasmid profiles, all the pO157 genes except for an IS-related gene were absent in O26 strain 1 and O103 strain 2, in which no large plasmid was detected (Table 2; Additional data file 3). In other non-O157 EHEC strains that contained one or more large plasmids, pO157 genes were variably conserved: percentages of genes judged as 'present' in each strain ranged from 18% to 59%.

Importantly, genes for enterohemolysin, KatP catalase, and EspP protease, all of which are suspected to be involved in O157 virulence, were also well conserved in non-O157 EHECs (Table 4). The ecf operon (ecf1 to ecf4), encoding a lipid A modification system that has recently been found to be related to colonization of bovine intestine 37, was also well conserved in the non-O157 EHEC strains.

Comparative analysis of genomic structures in EHEC strains by WGPScanning

Although the gene composition of each strain can be easily analyzed by CGH, it does not provide positional information, such as strain-specific translocations and strain-specific insertions. To obtain more details on the genomic differences between O157 and non-O157 EHECs, we analyzed the non-O157 EHEC strains by WGPScanning, and compared the results with earlier information on O157 strains 19 (Figures 1 and 2). Remarkable structural variations had been found mainly in Sp and SpLE regions in the O157 strains. In the non-O157 EHEC strains, Sp and SpLE regions exhibited much higher levels of structural change, and various other chromosomal loci containing S-loops also showed remarkable structural alterations. Because the PCR products obtained from most of these loci were reduced in size, we consider that S-loops have been deleted. This supposition is in good agreement with the CGH data.

We were able to obtain PCR products rarely from most Sp and SpLE regions in the non-O157 EHEC strains. Only SpLE1 and Sp10 regions of a few non-O157 EHEC strains yielded PCR products from their entire regions, indicating that only these strains contained genetic elements closely related to SpLE1 and Sp10 at the same loci as in Sakai. At other Sp- and SpLE-integration sites, it is likely that no insertion exists or different types of genomic elements have been inserted. We performed further PCR analyses to confirm this, using primer pairs targeting the flanking regions of each Sp and SpLE. We obtained PCR products from many Sp and SpLE loci through this analysis, and the results suggest that no large insertions exist at these loci (indicated by blank areas in Figures 1 and 2). At the remaining sites, it appears that large inserts different from those of O157 Sakai have been integrated. Of interest was the finding that no PCR product was obtained for many genes detected by the CGH analysis on the Sp and SpLE loci (see the Sp4 region of Figure 1 as an example). These results indicate that non-O157 EHEC strains also contain Sp- and SpLE-like elements, which are structurally and/or positionally highly divergent from those in O157 Sakai.

In non-prophage regions, a number of segments (49 in total) were again not amplified by PCR, suggesting that these loci contain large insertions or some other types of genomic rearrangements (indicated by arrowheads in Figures 1 and 2). In these regions, we identified several alternative integration sites for LEEs and Stx phages, as described in the next section.

Although a significant number of pO157 genes were detected in the CGH analysis, pO157-targeted primer pairs yielded no PCR product in all the non-O157 EHEC strains with a single exception (a small segment in one O26 strain; Figures 1 and 2). This indicates that plasmids harbored by non-O157 EHEC strains are highly divergent from pO157 in structure.

Integration sites of Stx phages and LEE islands

All the non-O157 EHEC strains examined in this study carried Stx phage(s) and the LEE. The results of WGPScanning analyses, however, implied that their integration sites are different from those in O157 Sakai (Figures 1 and 2). We thus searched for integration sites of these elements in the non-O157 EHEC strains. We first searched for LEE integration sites by long PCR using primer pairs, one targeting eaeA and the other the flanking regions of known LEE integration sites. This analysis revealed that LEEs are located at the pheU locus in all O26 strains and the pheV locus in all O111 and O103 strains (Figure 5a).

<p>Figure 5</p>

Variation in the integration sites for Stx phages and LEE islands in O157 and non-O157 EHEC strains

Variation in the integration sites for Stx phages and LEE islands in O157 and non-O157 EHEC strains. (a) Locations of the Stx phages and the LEE islands on each chromosome are shown. Integration sites of Stx2 phages in O157 strain 3, O26 strain 8, and O111 strain 3, and those of Stx1 phages in O111 strains 1, 4, 5, and 6, and O103 strain 3 are unknown. (b-e) Schematic presentation of newly identified integration sites for Stx phages. DNA sequences of left or right junctions for each integration site are also shown. (b) The torS-tort intergenic region in O103 strains 1, 2, 4, and 6. The torS and torT genes encode a sensor for a two-component regulatory system and a periplasmic protein of unknown function, respectively. The right junction was not identified. (c) The argW region in O157 strain 8 and O103 strain 5. The argW gene encodes an arginine tRNA. (d) The ssrA region in O111 strains 2 and 3. The ssrA gene encodes the tmRNA. The right junction was not identified. (e) The prfC gene in O103 strain 5. The prfC gene encodes the peptide chain release factor (RF-3). Integration of Stx1 phage into the prfC gene changes the amino acid sequence of a short amino-terminal region of RF-3, and removes the authentic ribosome binding site and promoter sequences. It is not known whether the prfC gene is transcribed and/or translated in the strain. The prfC gene, however, is not listed as an essential gene of E. coli [24].

Although Stx1 and Stx2 phages are integrated into the wrbA and yehV genes, respectively, in the two sequenced O157 strains (Sakai and EDL933), several alternative integration sites of Stx phages have been described in other O157 strains; one site for Stx1 phage (sbcB) and two for Stx2 phages (sbcB and yehV) 19. The yecE locus has also been identified as an integration site of the Stx2 phage in an ONT:H - strain 38. We consecutively analyzed these sites of the non-O157 EHEC strains by long PCR using primer pairs specific to stx1A (or stx2A) and each of these integration sites. We could find Stx1 phages at the wrbA locus in only seven O26 strains (1 to 7) and a Stx2 phage at the yecE locus in only one O111 strain (strain 2) (Figure 5a). We then constructed fosmid libraries of six EHEC strains (O157 strain 8, O26 strain 2, O111 strains 2 and 3, and O103 strains 1 and 5), and screened for stx1- or stx2-containing clones. By this systematic screening, we identified four new integration sites (torS-torT intergenic region, argW, ssrA, and prfC) for Stx phages. Based on this finding, the long PCR strategy also enabled us to find the Stx1 phages integrated at the torS-torT intergenic region in three O103 strains (2, 4, and 6) (Figure 5a). These results indicate that Stx phages are extremely divergent not only in genomic structure but also in integration site among EHEC strains. DNA sequences of these newly identified integration sites are shown in Figure 5b-e).

Discussion

A previous whole genome comparison of two E. coli strains, K-12 and O157 Sakai, revealed that their chromosomes are large mosaics of conserved core sequences and strain-specific sequences 8. Our present CGH analysis of O157 and non-O157 EHEC strains demonstrates that the core sequences are also well conserved in the non-O157 EHEC strains. Although the EHEC strains analyzed here were derived from three different clonal lineages, 3,240 out of 3,651 'conserved in K-12' singleton genes and 11 out of 23 'conserved in K-12' repeated gene families were perfectly conserved in all EHEC strains. The number of 'E. coli core genes' proposed by several array-based genome comparisons ranges from 2,800 to 3,782 genes 3940414243. The difference would come from the number and types of tested strains and types of microarrays used in each study.

More than 1,600 O157 Sakai genes that are absent from K-12 encode a great variety of proteins, including various virulence factors. Most of these 'Sakai-specific' genes are also absent from four sequenced extra-intestinal pathogenic E. coli strains (Table 4; Additional data file 4), which belong to a different phylogenetic group (Additional data file 2). The highly biased GC content of 'Sakai-specific' genes implies that most of them have been acquired by LGT 8. In fact, two-thirds of the S-loops are prophages and prophage-like elements. Our CGH analysis demonstrated a very poor conservation of 'Sakai-specific' genes in non-O157 EHECs: only 98 'Sakai-specific' singleton genes were conserved in all tested EHEC strains (Figure 3; Additional data file 4). Among these, 16 genes were also present in all the sequenced extra-intestinal pathogenic E. coli strains, indicating that they have been specifically deleted in the K-12 lineage.

Interestingly, however, a significant number of virulence-related genes, particularly those for non-LEE effectors and non-fimbrial adhesins, were well conserved in the non-O157 EHEC strains (Table 4). All four sequenced extra-intestinal pathogenic strains do not contain homologues of 31 non-LEE effectors and 11 non-fimbrial adhesins that are absent in K-12, except for three non-fimbrial adhesin genes (ECs0350, Ecs0362, and ECs1360). It is thus assumed that these virulence-related genes were selectively acquired and retained in multiple EHEC lineages like the genes for Stx, LEE, and enterohemolysin. Most of these virulence genes are on prophages, prophage-like elements, or the plasmid 81544. 'Sakai-specific' repeated gene families derived from prophages were also well conserved in the non-O157 EHEC strains, suggesting that they contain multiple prophages similar to those of O157 Sakai. These results indicate that infection of similar bacteriophages is deeply involved in the evolution of O157 and non-O157 EHEC lineages.

Prophages in non-O157 EHEC strains are remarkably divergent in their structure and integration site from those in O157 Sakai. With respect to this, another important achievement of this study is the identification of a set of alternative integration sites for prophages and other large genomic elements in the non-O157 EHEC genomes (Figures 1 and 2). They include those for the Stx phages and LEE islands (Figure 5).

Selective conservation of pO157-associated virulence-related genes in non-O157 EHECs is also intriguing. Most non-O157 EHEC strains contained one or two large plasmids, but their structures were very different from pO157, and pO157 genes other than virulence-related genes were very poorly conserved in non-O157 EHEC strains. (Figures 1 and 2; Table 4). Although more information on the large plasmids of non-O157 EHECs is necessary, similar but significantly diverged plasmids may have independently carried these virulence-determinants into each EHEC lineage.

The genome sizes of the non-O157 EHEC strains were similar or rather larger than those of O157 strains (Table 2). The CGH data, on the other hand, indicates that 83% of 4,905 singleton genes and 78% of 151 repeated gene families (composed of 542 repeated genes) are conserved in non-O157 EHEC strains on average. Taken together, we can expect that each non-O157 EHEC strain contains around 950 serotype- or strain-specific genes that do not exist in O157 Sakai. The presence of such a huge amount of serotype- or strain-specific genes may explain why non-O157 EHECs exhibit several phenotypes distinct from O157. For example, O26, O111, and O103 EHEC can cause diseases in cattle, goats, pigs, and rabbits, while O157 rarely does 6. In this regard, the absence of several gene clusters for fimbrial biosynthesis and iron utilization systems in non-O157 EHECs may suggest that non-O157 EHEC lineages have acquired alternative gene clusters for virulence towards these animals, which may confer different types of host tropisms to these lineages. To address these issues, more detailed analyses of non-O157 EHEC strains, including whole genome sequence determination, will be required.

Conclusion

We describe the first systematic whole genome comparison between O157 and non-O157 EHEC strains based on the O157 Sakai sequence. Chromosomal backbone regions were highly conserved both in O157 and non-O157 EHEC strains of O26, O111, and O103 serotypes. In contrast, O157 Sakai-specific regions were very poorly conserved in the non-O157 EHEC strains, even though their total genome sizes were the same or rather larger than that of O157. It is assumed, therefore, that O157 and non-O157 EHEC strains have independently acquired a huge amount of lineage- or strain-specific genes by LGT. On the other hand, an unexpectedly large number of virulence genes, especially those for non-LEE effectors and non-fimbrial adhesions, were well conserved in non-O157 EHEC strains in addition to the stx genes and LEE island. In O157, most of them were encoded on prophages and the plasmid. Although non-O157 EHEC strains contained multiple prophages similar to those of O157, these prophages exhibited remarkable structural and positional diversity. These data suggest that infections of similar but distinct bacteriophages are deeply involved in the evolution of EHEC strains belonging to different E. coli lineages.

Materials and methods

Bacterial strains, growth conditions, and DNA preparation

Bacterial strains used in this study are listed in Table 1. O157 Sakai and K-12 MG1655 were used as references in CGH and WGPScanning analyses. Eight EHEC O157:H7 (or H-) strains were previously described 19. Of the tested non-O157 EHEC strains, ED71, ED80, and ED411 were isolated in Italy (kindly provided by S Morabito, Istituto Superiore di Sanità, Rome), PMK5 in France 45, and the others in Japan in 2001. Growth conditions and the protocol for genomic DNA preparation were described previously 18.

Detection and subtyping of stx and eae genes

Detection of stx1 genes was done by PCR amplification with primers stx1-F (5'-caggggataatttgtttgcagttg-3') and stx1-R (5'-gacacatagaaggaaactcatcag-3'), using 10 ng of genomic DNA as template with a EX taq PCR kit (Takara Bio, Kyoto, Japan) by 30 amplification cycles of denaturation for 20 s at 98°C, annealing for 30 s at 60°C, and primer extension for 45 s at 72°C. The amplified DNA was analyzed by electrophoresis on 2% agarose gel. Detection and subtyping of stx2 and eae were done by restriction fragment length polymorphism (RFLP) analysis of PCR products as described previously 4647.

Multi-locus sequence typing

Internal regions of each of seven housekeeping genes, aspC, clpX, fadD, icdA, lysP, mdh, and uidA, were amplified and sequenced for each test strain. Primer designs and PCR conditions were determined according to the 'multil-locus sequence typing database for pathogenic E. coli 48. The sequences of seven loci were concatenated and aligned with those of other pathogenic E. coli strains in the EcMLST database by using the ClustalW program 49 in the MEGA3 software 50, and then a neighbor-joining (NJ) tree was generated by using the Tamura-Nei evolutionary model.

Pulsed-field gel electrophoresis

PFGE analyses were performed according to the method described by Terajima et al. 51 with some modification. In brief, bacterial cells were embedded in 0.9% Certified Low Melt Agarose (Bio-Rad Laboratories, Inc., Tokyo, Japan), lysed with a buffer containing 0.2% sodium deoxycholate, 0.5% N-lauroylsarcosine, and 0.5% Brij-58, and treated with 100 μg/ml proteinase K. XbaI-digested genomic DNA was separated by using CHEF MAPPER (Bio-Rad Laboratories, Inc.) with 1% Pulsed Field Certified Agarose (Bio-Rad Laboratories, Inc.,) at 6.0 V/cm for 22 h and 18 minutes with pulsed times ranging from 47 to 44.69 s. I-CeuI-digested DNA was with 1% Pulsed Field Certified Agarose at 6.0 V/cm for 23 h and 52 minutes with pulsed times ranging from 1.19 to 83.55 s or with 0.8% Agarose at 3.0 V/cm for 24 h with pulsed times ranging from 600 to 800 s. Sizes of each DNA band were estimated by Lane Analyzer (ATTO Corp., Tokyo, Japan).

Plasmid profile

Plasmid DNA was purified from overnight culture of each EHEC strain using a plasmid midi kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions, and was separated by the CHEF MAPPER with 1% Pulsed Field Certified Agarose at 6.0 V/cm for 12 h with pulsed times ranging from 0.14 to 21.79 s. Band sizes were estimated by Lane Analyzer.

Microarray analysis

The protocol of CGH analysis using an O157 oligoDNA microarray has been described previously 18. In brief, oligonucleotide probes were prepared for all the protein-coding genes in the Sakai genome (5,447 genes in total). The probes were principally 60-mer in length and two probes were prepared for each gene. Repeated genes sharing various lengths of almost identical sequences (542 genes in total) were grouped into 151 repeated gene families, and each family was represented by a single probe. Genomic DNA (3 μg) from the reference strain (O157 Sakai) and each test strain was used to generate Cy3- and Cy5-labeled samples, respectively, and cohybridized on a single array. For each test strain, DNA labeling and hybridization were performed twice independently. Fluorescence intensities of the spots were collected using the ArrayVision 8.0 software (Imaging Research Inc., Ontario, Canada). After filtrating the spots with slide abnormalities or low signal intensities in the reference channels, the fluorescence intensity in each cannel was log2-transformed. Presence or absence of each probe was then determined by using the array-based genotyping software GACK 52. The presence or absence of each gene was finally determined according to each probe result obtained from two independent hybridizations as described previously 18. Processed datasets were displayed in genomic order using the TREEVIEW program 53.

In our previous test experiment using K-12 strain MG1655, 96.9% of the genes that were predicted as 'present' by an in silico analysis (threshold value ≥90% identity in each 60-mer probe sequence) were judged as 'present' in the microarray analysis, and 97.8% of the genes predicted as 'absent' were judged as 'absent' 18.

The microarray data have been submitted to the Gene Expression Omnibus (series record number GSE7931). Final processed data are presented in Additional data file 6.

WGPScanning analysis

The WGPScanning method was described previously 19. In brief, we used a total of 1,120 primers that can amplify the entire O157 Sakai genome by 560 long PCRs (549 for the chromosome and 11 for the plasmids). All the primer sequences are available at our web site 54. Long PCR was performed using the LA taq PCR kit (Takara Shuzo, Kyoto, Japan) and 1 ng of genomic DNA as template with 30 cycles of a two-step amplification program: 20 s at 98°C and 16 minutes at 69°C. PCR products were separated by field inversion gel electrophoresis (FIGE), and product sizes were estimated by Lane Analyzer.

Construction of fosmid libraries

To identify alternative integration sites of Stx phages, we constructed fosmid libraries for O157 strain 8, O26 strain 2, O111 strains 2 and 3, and O103 strains 1 and 5 by using a Copycontrol Fosmid Library Production Kit (Epicentre, Medison, WI, USA) according to the manufacturer's instructions. Insert sizes and redundancies of each library were, on average, 40 kb and 20 times, respectively. The stx1- and stx2-containing clones were screened by PCR using the following primer pairs; stx1-F, 5'-gacacatagaaggaaactcatcag-3', and stx1-R, 5'-caggggataatttgtttgcagttg-3' for stx1; and stx2-F, 5'-ggcgcgttttgaccatcttcgt-3', and stx2-R, 5'-tacctttagcacaatccgccgc-3' for stx2. End sequences of each insert were determined by direct sequencing, and were used to roughly map the integration sites on the E. coli chromosome. Precise integration sites were determined by the primer-walking method.

Additional data files

The following additional data files are available with the online version of this paper. Additional file 1 is a figure showing the gel of a PFGE analysis of XbaI-digested genomic DNA in O157 and non-O157 EHEC strains. Additional file 2 shows the phylogeny of O157 and non-O157 EHEC strains determined by MLST. Additional file 3 is a figure showing the gel of a PFGE analysis of plasmids isolated from O157 and non-O157 EHEC strains. Additional file 4 is a summary of the CGH analyses. Additional file 5 presents the data on conservation of the 'conserved in K-12' singleton genes belonging to each COG category in the EHEC strains. Additional file 6 is a table listing all the result of CGH analyses in non-O157 EHEC strains (processed data only).

<p>Additional data file 1</p>

XbaI-digestion patterns of EHEC genomic DNA

XbaI-digestion patterns of EHEC genomic DNA are shown.

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 <p>Additional data file 2</p>

Phylogeny of O157 and non-O157 EHEC strains determined by MLST

The MLST analysis of EHEC strains of the present study with other pathogenic E. coli strains in the EcMLST database was conducted by using concatenated DNA sequences of seven loci (aspC, clpX, fadD, icdA, lysP, mdh and uidA). The sequences of reference strains were obtained from EcMLST. Multiple sequence alignments were made by using the ClustalW program in the MEGA3 software. The NJ tree was generated by using the Tamura-Nei evolutionary model. Bootstrap values greater than 50% are indicated. The scale bar represents the number of substitutions per site. Accession numbers in EcMLST, strain names, serotypes, and classes in EcMLST of each strain are indicated. MLST20 is an undefined clonal group.

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 <p>Additional data file 3</p>

Plasmid profiles of O157 and non-O157 EHEC strains

Plasmid profiles of O157 and non-O157 EHEC strains are shown.

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 <p>Additional data file 4</p>

Summary of the CGH analyses

The results of CGH analyses of O157 and non-O157 EHEC strains are summarized.

Click here for file

 <p>Additional data file 5</p>

Conservation of the 'conserved in K-12' singleton genes belonging to each COG category in the EHEC strains

Conservation of the 'conserved in K-12' singleton genes belonging to each COG category was analyzed in each EHEC serotype.

Click here for file

 <p>Additional data file 6</p>

CGH analyses in non-O157 EHEC strains (processed data only)

The processed data of CGH analyses of non-O157 EHEC strains are shown.

Click here for file

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas "Applied Genomics", the 21st Century COE Program (Life Science) from the Ministry of Education, Science, and Technology of Japan, by a Grant-in-Aid of Ministry of Health, Labor and Welfare of Japan (H17-Sinkou-ippan-019), and a grant from the Yakult Foundation. We thank Dr Stefano Morabito for providing EHEC strains, Dr Taku Ohshima for valuable advice, Yoshiyuki Maki for assistances in primer design, Akemi Yoshida and Yumiko Takeshita for technical assistance, and Yumiko Hayashi for language assistance.

 <p>(Patho-)Genomics of <it>Escherichia coli</it>.</p> Dobrindt U Int J Med Microbiol 2005 295 357 371 10.1016/j.ijmm.2005.07.009 16238013 <p>Enterohaemorrhagic <it>Escherichia coli</it>: emerging issues on virulence and modes of transmission.</p> Caprioli A Morabito S Brugere H Oswald E Vet Res 2005 36 289 311 10.1051/vetres:2005002 15845227 <p>Diarrheagenic <it>Escherichia coli</it>.</p> Nataro JP Kaper JB Clin Microbiol Rev 1998 11 142 201 121379 9457432 <p>Pathogenic <it>Escherichia coli</it>.</p> Kaper JB Nataro JP Mobley HL Nat Rev Microbiol 2004 2 123 140 10.1038/nrmicro818 15040260 <p>Impact of the locus of enterocyte effacement pathogenicity island on the evolution of pathogenic <it>Escherichia coli</it>.</p> Jores J Rumer L Wieler LH Int J Med Microbiol 2004 294 103 113 10.1016/j.ijmm.2004.06.024 15493820 <p>Attaching-effacing bacteria in animals.</p> Wales AD Woodward MJ Pearson GR J Comp Pathol 2005 132 1 26 10.1016/j.jcpa.2004.09.005 15629476 <p>The complete genome sequence of <it>Escherichia coli </it>K-12.</p> Blattner FR Plunkett G 3rd Bloch CA Perna NT Burland V Riley M Collado-Vides J Glasner JD Rode CK Mayhew GF Science 1997 277 1453 1474 10.1126/science.277.5331.1453 9278503 <p>Complete genome sequence of enterohemorrhagic <it>Escherichia coli </it>O157:H7 and genomic comparison with a laboratory strain K-12.</p> Hayashi T Makino K Ohnishi M Kurokawa K Ishii K Yokoyama K Han CG Ohtsubo E Nakayama K Murata T DNA Res 2001 8 11 22 10.1093/dnares/8.1.11 11258796 <p>Dissecting virulence: systematic and functional analyses of a pathogenicity island.</p> Deng W Puente JL Gruenheid S Li Y Vallance BA Vázquez A Barba J Ibarra JA O'Donnell P Metalnikov P Proc Natl Acad Sci USA 2004 101 3597 3602 373508 14988506 10.1073/pnas.0400326101 <p>TccP is an enterohaemorrhagic <it>Escherichia coli </it>O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.</p> Garmendia J Phillips AD Carlier MF Chong Y Schüller S Marches O Dahan S Oswald E Shaw RK Knutton S Cell Microbiol 2004 6 1167 1183 10.1111/j.1462-5822.2004.00459.x 15527496 <p>Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic <it>Escherichia coli </it>O157:H7.</p> Gruenheid S Sekirov I Thomas NA Deng W O'Donnell P Goode D Li Y Frey EA Brown NF Metalnikov P Mol Microbiol 2004 51 1233 1249 10.1046/j.1365-2958.2003.03911.x 14982621 <p>Identification of a novel <it>Citrobacter rodentium </it>type III secreted protein, EspI, and roles of this and other secreted proteins in infection.</p> Mundy R Petrovska L Smollett K Simpson N Wilson RK Yu J Tu X Rosenshine I Clare S Dougan G Infect Immun 2004 72 2288 2302 375195 15039354 10.1128/IAI.72.4.2288-2302.2004 <p>EspJ is a prophage-carried type III effector protein of attaching and effacing pathogens that modulates infection dynamics.</p> Dahan S Wiles S La Ragione RM Best A Woodward MJ Stevens MP Shaw RK Chong Y Knutton S Phillips A Infect Immun 2005 73 679 686 547035 15664905 10.1128/IAI.73.2.679-686.2005 <p>Comparative analysis of EspF from enteropathogenic and enterohemorrhagic <it>Escherichia coli </it>in alteration of epithelial barrier function.</p> Viswanathan VK Koutsouris A Lukic S Pilkinton M Simonovic I Simonovic M Hecht G Infect Immun 2004 72 3218 3227 415647 15155623 10.1128/IAI.72.6.3218-3227.2004 <p>An extensive repertoire of type III secretion effectors in <it>Escherichia coli </it>O157 and the role of lambdoid phages in their dissemination.</p> Tobe T Beatson SA Taniguchi H Abe H Bailey CM Fivian A Younis R Matthews S Marches O Frankel G Proc Natl Acad Sci USA 2006 103 14941 14946 1595455 16990433 10.1073/pnas.0604891103 <p>TccP2 of O157:H7 and non-O157 enterohemorrhagic <it>Escherichia coli </it>(EHEC): challenging the dogma of EHEC-induced actin polymerization.</p> Ogura Y Ooka T Whale A Garmendia J Beutin L Tennant S Krause G Morabito S Chinen I Tobe T Infect Immun 2007 75 604 612 1828498 17101643 10.1128/IAI.01491-06 <p>Subversion of actin dynamics by EPEC and EHEC.</p> Caron E Crepin VF Simpson N Knutton S Garmendia J Frankel G Curr Opin Microbiol 2006 9 40 45 10.1016/j.mib.2005.12.008 16406772 <p>Complexity of the genomic diversity in enterohemorrhagic <it>Escherichia coli </it>O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.</p> Ogura Y Kurokawa K Ooka T Tashiro K Tobe T Ohnishi M Nakayama K Morimoto T Terajima J Watanabe H DNA Res 2006 13 3 14 10.1093/dnares/dsi026 16766508 <p>Genomic diversity of enterohemorrhagic <it>Escherichia coli </it>O157 revealed by whole genome PCR scanning.</p> Ohnishi M Terajima J Kurokawa K Nakayama K Murata T Tamura K Ogura Y Watanabe H Hayashi T Proc Natl Acad Sci USA 2002 99 17043 17048 139266 12481030 10.1073/pnas.262441699 <p>Zoonotic non-O157 Shiga toxin-producing <it>Escherichia Coli </it>(STEC).</p> World Health Organization Report of a WHO Scientific Working Group Meeting, Berlin, Germany, 23-26 June 1998 http://whqlibdoc.who.int/hq/1998/WHO_CSR_APH_98.8.pdf <p>Parallel evolution of virulence in pathogenic <it>Escherichia coli</it>.</p> Reid SD Herbelin CJ Bumbaugh AC Selander RK Whittam TS Nature 2000 406 64 67 10.1038/35017546 10894541 <p>Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic <it>Escherichia coli</it>.</p> Knutton S Baldwin T Williams PH McNeish AS Infect Immun 1989 57 1290 1298 313264 2647635 <p>Molecular characterization of a serotype O121:H19 clone, a distinct Shiga toxin-producing clone of pathogenic <it>Escherichia coli</it>.</p> Tarr CL Large TM Moeller CL Lacher DW Tarr PI Acheson DW Whittam TS Infect Immun 2002 70 6853 6859 133070 12438362 10.1128/IAI.70.12.6853-6859.2002 <p>Cell size and nucleoid organization of engineered <it>Escherichia coli </it>cells with a reduced genome.</p> Hashimoto M Ichimura T Mizoguchi H Tanaka K Fujimitsu K Keyamura K Ote T Yamakawa T Yamazaki Y Mori H Mol Microbiol 2005 55 137 149 10.1111/j.1365-2958.2004.04386.x 15612923 <p>Construction of <it>Escherichia coli </it>K-12 in-frame, single-gene knockout mutants: the Keio collection.</p> Baba T Ara T Hasegawa M Takai Y Okumura Y Baba M Datsenko KA Tomita M Wanner BL Mori H Mol Syst Biol 2006 2 2006.0008 <p>The COG database: an updated version includes eukaryotes.</p> Tatusov RL Fedorova ND Jackson JD Jacobs AR Kiryutin B Koonin EV Krylov DM Mazumder R Mekhedov SL Nikolskaya AN BMC Bioinformatics 2003 4 41 222959 12969510 10.1186/1471-2105-4-41 <p>Identification of genes subject to positive selection in uropathogenic strains of <it>Escherichia coli</it>: a comparative genomics approach.</p> Chen SL Hung CS Xu J Reigstad CS Magrini V Sabo A Blasiar D Bieri T Meyer RR Ozersky P Proc Natl Acad Sci USA 2006 103 5977 5982 1424661 16585510 10.1073/pnas.0600938103 <p>How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic <it>Escherichia coli </it>strains.</p> Brzuszkiewicz E Bruggemann H Liesegang H Emmerth M Olschlager T Nagy G Albermann K Wagner C Buchrieser C Emody L Proc Natl Acad Sci USA 2006 103 12879 12884 1568941 16912116 10.1073/pnas.0603038103 <p>Extensive mosaic structure revealed by the complete genome sequence of uropathogenic <it>Escherichia coli</it>.</p> Welch RA Burland V Plunkett G 3rd Redford P Roesch P Rasko D Buckles EL Liou SR Boutin A Hackett J Proc Natl Acad Sci USA 2002 99 17020 17024 139262 12471157 10.1073/pnas.252529799 <p>The genome sequence of avian pathogenic <it>Escherichia coli </it>strain O1:K1:H7 shares strong similarities with human extraintestinal pathogenic <it>E. coli </it>genomes.</p> Johnson TJ Kariyawasam S Wannemuehler Y Mangiamele P Johnson SJ Doetkott C Skyberg JA Lynne AM Johnson JR Nolan LK J Bacteriol 2007 189 3228 3236 10.1128/JB.01726-06 17293413 <p>The lipopolysaccharide core type of <it>Escherichia coli </it>O157:H7 and other non-O157 verotoxin-producing <it>E. coli</it>.</p> Currie CG Poxton IR FEMS Immunol Med Microbiol 1999 24 57 62 10.1111/j.1574-695X.1999.tb01265.x 10340713 <p>Distribution of core oligosaccharide types in lipopolysaccharides from <it>Escherichia coli</it>.</p> Amor K Heinrichs DE Frirdich E Ziebell K Johnson RP Whitfield C Infect Immun 2000 68 1116 1124 97256 10678915 10.1128/IAI.68.3.1116-1124.2000 <p>Association of the urease gene with enterohemorrhagic <it>Escherichia coli </it>strains irrespective of their serogroups.</p> Nakano M Iida T Ohnishi M Kurokawa K Takahashi A Tsukamoto T Yasunaga T Hayashi T Honda T J Clin Microbiol 2001 39 4541 4543 88583 11724879 10.1128/JCM.39.12.4541-4543.2001 <p>Molecular evolution of typical enteropathogenic <it>Escherichia coli</it>: clonal analysis by multilocus sequence typing and virulence gene allelic profiling.</p> Lacher DW Steinsland H Blank TE Donnenberg MS Whittam TS J Bacteriol 2007 189 342 350 1797380 17098897 10.1128/JB.01472-06 <p>Description of a 111-kb pathogenicity island (PAI) encoding various virulence features in the enterohemorrhagic <it>E. coli </it>(EHEC) strain RW1374 (O103:H2) and detection of a similar PAI in other EHEC strains of serotype 0103:H2.</p> Jores J Wagner S Rumer L Eichberg J Laturnus C Kirsch P Schierack P Tschape H Wieler LH Int J Med Microbiol 2005 294 417 425 10.1016/j.ijmm.2004.09.009 15715170 <p>Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic <it>Escherichia coli </it>O157:H7 derived from Sakai outbreak.</p> Makino K Ishii K Yasunaga T Hattori M Yokoyama K Yutsudo CH Kubota Y Yamaichi Y Iida T Yamamoto K DNA Res 1998 5 1 9 10.1093/dnares/5.1.1 9628576 <p>Involvement of the <it>Escherichia coli </it>O157:H7(pO157) ecf operon and lipid A myristoyl transferase activity in bacterial survival in the bovine gastrointestinal tract and bacterial persistence in farm water troughs.</p> Yoon JW Lim JY Park YH Hovde CJ Infect Immun 2005 73 2367 2378 1087426 15784583 10.1128/IAI.73.4.2367-2378.2005 <p>The nucleotide sequence of Shiga toxin (Stx) 2e-encoding phage phiP27 is not related to other Stx phage genomes, but the modular genetic structure is conserved.</p> Recktenwald J Schmidt H Infect Immun 2002 70 1896 1908 127862 11895953 10.1128/IAI.70.4.1896-1908.2002 <p>Evolutionary dynamics of full genome content in <it>Escherichia coli</it>.</p> Ochman H Jones IB EMBO J 2000 19 6637 6643 305890 11118198 10.1093/emboj/19.24.6637 <p>Analysis of genome plasticity in pathogenic and commensal <it>Escherichia coli </it>isolates by use of DNA arrays.</p> Dobrindt U Agerer F Michaelis K Janka A Buchrieser C Samuelson M Svanborg C Gottschalk G Karch H Hacker J J Bacteriol 2003 185 1831 1840 150128 12618447 10.1128/JB.185.6.1831-1840.2003 <p>Comparative genomic indexing reveals the phylogenomics of <it>Escherichia coli </it>pathogens.</p> Anjum MF Lucchini S Thompson A Hinton JC Woodward MJ Infect Immun 2003 71 4674 4683 165989 12874348 10.1128/IAI.71.8.4674-4683.2003 <p>Extensive genomic diversity in pathogenic <it>Escherichia coli </it>and <it>Shigella </it>strains revealed by comparative genomic hybridization microarray.</p> Fukiya S Mizoguchi H Tobe T Mori H J Bacteriol 2004 186 3911 3921 419953 15175305 10.1128/JB.186.12.3911-3921.2004 <p>Design of a seven-genome <it>Escherichia coli </it>microarray for comparative genomic profiling.</p> Willenbrock H Petersen A Sekse C Kiil K Wasteson Y Ussery DW J Bacteriol 2006 188 7713 7721 1636325 16963574 10.1128/JB.01043-06 <p>Diversification of <it>Escherichia coli </it>genomes: are bacteriophages the major contributors?</p> Ohnishi M Kurokawa K Hayashi T Trends Microbiol 2001 9 481 485 10.1016/S0966-842X(01)02173-4 11597449 <p>Identification of a clone of <it>Escherichia coli </it>O103:H2 as a potential agent of hemolytic-uremic syndrome in France.</p> Mariani-Kurkdjian P Denamur E Milon A Picard B Cave H Lambert-Zechovsky N Loirat C Goullet P Sansonetti PJ Elion J J Clin Microbiol 1993 31 296 301 262753 8432815 <p>Identification of new verocytotoxin type 2 variant B-subunit genes in human and animal <it>Escherichia coli </it>isolates.</p> Piérard D Muyldermans G Moriau L Stevens D Lauwers S J Clin Microbiol 1998 36 3317 3322 105321 9774585 <p>Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic <it>Escherichia coli</it>: characterization of a new intimin variant.</p> Oswald E Schmidt H Morabito S Karch H Marchès O Caprioli A Infect Immun 2000 68 64 71 97102 10603369 <p>EcMLST Database</p> http://www.shigatox.net/cgi-bin/mlst7/index <p>The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.</p> Thompson JD Gibson TJ Plewniak F Jeanmougin F Higgins DG Nucleic Acids Res 1997 25 4876 4882 147148 9396791 10.1093/nar/25.24.4876 <p>MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment.</p> Kumar S Tamura K Nei M Brief Bioinform 2004 5 150 163 10.1093/bib/5.2.150 15260895 <p>High genomic diversity of enterohemorrhagic <it>Escherichia coli </it>isolates in Japan and its applicability for the detection of diffuse outbreak.</p> Terajima J Izumiya H Iyoda S Tamura K Watanabe H Jpn J Infect Dis 2002 55 19 22 11971157 <p>Improved analytical methods for microarray-based genome-composition analysis.</p> Kim CC Joyce EA Chan K Falkow S Genome Biol 2002 3 RESEARCH0065 133449 12429064 10.1186/gb-2002-3-11-research0065 <p>Cluster analysis and display of genome-wide expression patterns.</p> Eisen MB Spellman PT Brown PO Botstein D Proc Natl Acad Sci USA 1998 95 14863 14868 24541 9843981 10.1073/pnas.95.25.14863 <p>E. coli O157: H7 Sakai Genome Project</p> http://genome.naist.jp/bacteria/o157/pcrscan.html