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Journal of Clinical Microbiology, June 2003, p. 2341-2347, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2341-2347.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Distribution of the Secondary Type III Secretion System Locus Found in Enterohemorrhagic Escherichia coli O157:H7 Isolates among Shiga Toxin-Producing E. coli Strains

Sou-Ichi Makino,^1,2* Toru Tobe,³ Hiroshi Asakura,¹ Masahisa Watarai,¹ Testuya Ikeda,⁴ Koichi Takeshi,⁴ and Chihiro Sasakawa⁵

Department of Applied Veterinary Science,¹ Research Center for Animal Hygiene and Food Safety, Obihiro University of Agriculture and Veterinary Medicine,² Hokkaido Institute of Public Health, Hokkaido,⁴ Division of Applied Bacteriology, Graduate School of Medicine, Osaka University, Osaka,³ Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan⁵

Received 18 October 2002/ Returned for modification 20 January 2003/ Accepted 18 February 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enterohemorrhagic Escherichia coli (EHEC) and Shiga toxin-producing E. coli (STEC) are the most common causes of hemorrhagic colitis, bloody diarrhea, and hemolytic-uremic syndrome and also cause food-borne epidemics in humans (19). STEC is distributed widely in the environment (1, 2). It produces cytotoxin as the main virulence factor but can have a histopathologic effect on intestinal epithelial cells, causing attaching-and-effacing (A/E) lesions. Enteropathogenic E. coli (EPEC), which is a major cause of infant diarrhea in developing countries, has the same ability to cause A/E lesions. These lesions are characterized by localized destruction of brush border microvilli, intimate attachment of the organism to the host cell membrane, and formation of an actin-rich underlying structure in the host cells (5). All genes necessary for the formation of A/E lesions are located in a pathogenicity island termed the locus for enterocyte effacement (LEE) (18). This locus contains genes for structural components of a secretion apparatus that belongs to the type III secretion system, that is, genes for the adhesin intimin and its translocated receptor, Tir (7).

The type III secretion system, which is found in many gram-negative pathogens, is responsible for secretion and injection of virulence-associated factors into the cytosol of host cells. The type III apparatus comprises approximately 20 proteins, with most of them located in the inner membrane (6, 24). Most inner membrane proteins are homologous to components of the flagellar biosynthesis apparatuses of both gram-negative and gram-positive bacteria (10). In Salmonella enterica serovar Typhimurium, two independent type III secretion systems encoded by separate chromosomal loci, SPI-1 and SPI-2, have been found (8, 22). SPI-1 is required for the invasion of mammalian cells, and SPI-2 is required for replication in macrophages and for survival in mice.

The release of the complete genome sequence of EHEC O157:H7 (9) provided us with an opportunity to identify sequences specific for STEC strains. In this study, we report on a type III secretion locus in the STEC chromosome similar to the SPI-1 system. To estimate the roles of this locus in virulence expression in STEC, we show the distribution of this novel locus among various STEC strains, and we also discuss the transmission of STEC from animals to humans.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and media. Table 1 lists all STEC strains used in the study. EHEC O157:H7 strain RIMD 0509952 (referred to here as EHEC O157 Sakai) was originally isolated from a patient in an outbreak in Sakai, Japan, in 1996 (14). Four EPEC isolates from humans were used in this study: B171-8 (O111:NM, where NM indicates nonmotile) (20), E2348/69 (O127:H7), 4394-57 (O114:NM), and 1929-55(O126:NM). Some STEC strains were isolated from deer (1), sheep (2), seagulls (2), houseflies (11), and salmon roe (15). Other STEC and EPEC strains are the stock isolates in our laboratory. Bacteria were cultured in Trypticase soy broth (BBL/Becton Dickinson, Cockeysville, Md.) or on Trypticase soy agar (BBL/Becton Dickinson).

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TABLE 1. STEC strains used in this study

Southern hybridization. Chromosomal DNA was extracted from EHEC, EPEC, and E. coli K-12 strains as described previously (17) and digested with EcoRI. The DNA fragments were separated by electrophoresis in 1% agarose gels and were transferred to Hybond-N+ positively charged nylon membranes (Amersham, Little Chalfont, England). DNA fragments corresponding to three separate regions in ETT2 of EHEC O157:H7 (region A, eprH-eprK; region B, epaO-epaS; and region D, eivF-eivA) were amplified by PCR with primer sets specific for each region (Table 2) and were labeled with biotin by using the BrightStar Psoralen-Biotin nonisotopic labeling kit (Ambion, Austin, Tex.). Signals were detected by using the BrightStar BioDetect nonisotopic detection kit (Ambion).

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TABLE 2. Primers used to detect genes in five separate regions in the ETT2 locusa

PCR. Table 2 and Table 3 show the oligonucleotide primers and conditions used for PCR. All primers (20 pmol each) were mixed with TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan), and the PCR was performed in a volume of 25 µl in a model 9600 thermal cycler (Applied Biosystems Japan Ltd., Tokyo, Japan).

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TABLE 3. Primers used to detect pathogenic STEC genesa

Western blotting. Western blotting was performed as described previously (16) with rabbit polyclonal antibody raised against intimin-.

Nucleotide sequence accession number. The DNA sequence of EPEC strain B171-8 (O111:NM) has been submitted to GenBank and can be found under accession no. AB052736.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Structure of a new locus that encodes a second type III secretion system. Based on the DNA sequence of the EHEC O157 Sakai chromosome, we searched for a DNA sequence unique to EHEC O157 which was not present in the E. coli K-12 genome. One such sequence consisted of a 17-kb region inserted between the sequences homologous to those at min 64 and 65 of the E. coli K-12 chromosome (Fig. 1). The 17-kb insert showed no significant homology with the E. coli K-12 chromosome and contained 21 open reading frames (ORFs) (Table 4; Fig. 1). The G+C content of this region was 36.9%.

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FIG. 1. Primary structure of ETT2 locus and homology with SPI-1 of S. enterica serovar Typhimurium. The locations of ORFs predicted from the nucleotide sequence are shown. The structures are of the following strains, from top to bottom: ETT2 of EHEC O157 Sakai, ETT2 of EPEC O111:NM B171, chromosome of E. coli K-12 W3110 at min 64.5, and SPI-1 of S. enterica serovar Typhimurium.

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TABLE 4. ORFs in the ETT2 locus of EHEC O157 Sakai

The deduced amino acid sequences of ORF2 to ORF5 and ORF7 to ORF20 were similar to those of proteins of the type III secretion system, which are required for the export of virulence determinants in a variety of bacterial pathogens of plants and animals. The predicted proteins of ORF2 to ORF5 are similar in organization and sequences to the products of the prgH to prgK genes of Salmonella SPI-1. The predicted proteins of ORF7 to ORF20 are similar in organization and sequences to the products of the inv and spa genes of Salmonella SPI-1 but not to those of invB. They are also homologous to the mxi genes of Shigella flexneri (data not shown). For example, the predicted amino acid sequence of ORF2 is 53% identical to that of PrgK of S. enterica serovar Typhimurium and 44% identical to that of MxiJ of S. flexneri 2a. The amino acid sequences of ORF3 to ORF5 are similar to those of PrgJ (31% identical), PrgI (64%), and PrgH (31%), respectively, of S. enterica serovar Typhimurium and to those of MxiI (32%), MxiH (58%), and MxiG (25%), respectively, of S. flexneri 2a. The entire organization and amino acid sequences of the ORFs in this 17-kb region are closely related to those of SPI-1 of S. enterica serovar Typhimurium (Fig. 1). In addition to these ORFs, the predicted protein of ORF1, which is located downstream of ORF2 and extends to the homologous sequence in E. coli K-12, showed significant similarity to the N-terminal part of the protein product of orgA, downstream of prgK in SPI-1 of S. enterica serovar Typhimurium. ORF8 and ORF9 showed significant similarity to SpaR of SPI-1, but the amino acid sequence of ORF9 was separated from ORF8, similar to the sequence in the C-terminal part of SpaR, by frame shifting in the ORF9-coding sequence. ORF5 was similar to PrgH in SPI-1 of S. enterica serovar Typhimurium, but the amino acid sequence corresponding to the N-terminal part of PrgH was lost from ORF5 because of the absence of the initiation codon, even though the DNA sequence upstream of ORF5 showed a high degree of similarity to that of the sequence upstream of the prgH gene in S. enterica serovar Typhimurium. Because of the high degree of similarity of each ORF to the genes in SPI-1 of Salmonella spp., the region from ORF5 to ORF2 was designated eprH to eprK, the region from ORF12 to ORF7 was designated epaO to epaS, and the region from ORF19 to ORF13 was designated eivF to eivJ (Fig. 1).

In SPI-1 of S. enterica serovar Typhimurium, the DNA between spaS and prgH encodes secreted proteins, chaperones, and transcriptional regulatory proteins; but the region between epaS and eprH of EHEC O157 Sakai contains no ORF similar to these genes. Instead, ORF6 had a low, but significant degree of similarity to several transcriptional regulatory proteins, including NtrC of Herbaspirillum seropedicae (25% identity and 40% similarity in a 107-amino-acid overlap) and UhpA of S. enterica serovar Typhimurium (29% identity and 49% similarity in a 68-amino-acid overlap). Consequently, the 17-kb region encodes the complete set of proteins for the type III secretion system apparatus but does not include the target or chaperone proteins. This locus contains a set of genes for the type III secretion system. Therefore, to distinguish this locus from a set of genes for type III secretion in LEE, we designated this locus ETT2, for E. coli type III secretion locus 2.

Conservation of ETT2 sequences among STEC strains. To examine the degree of conservation of the ETT2 sequence among pathogenic E. coli isolates from humans, Southern hybridization was performed with probes derived from the DNA corresponding to eprH to eprK (probe A in Fig. 1; Table 2), epaO to epaS (probe B), and eivF to eivA (probe C). All seven EHEC O157 strains had DNA sequences homologous to the sequence of the ETT2 locus (Table 5). However, EHEC O26 strains from humans contained DNA sequences homologous to probes A and B, but not to probe C (Table 5). The same pattern of hybridization observed with O26 was observed with four EPEC strains, but the DNA sequence that hybridized with ETT2-specific probes was not found in nonpathogenic strain E. coli HB101. To survey the distribution of the ETT2 locus among various STEC isolates, a PCR was performed by using three primer sets specific for regions L, C, and D in the ETT2 locus (Fig. 1; Table 2). It was found that the results of the PCR coincided with those of Southern hybridization (Tables 1 and 5).

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TABLE 5. Conservation of ETT2 in EHEC and other pathogenic E. coli isolates

Thus, a PCR survey was undertaken to determine the distribution of the ETT2 locus among 56 STEC isolates of various origins (Table 1). Sixteen STEC isolates had a complete ETT2 locus of L, D, and C regions (Table 1); 15 isolates were serotype O157, and one isolate, from a deer, was Out:H25 (where Out represents all O antiserum negative). Although four EPEC isolates and two O26 isolates had only an L region, according to PCR (Table 5), eight other STEC isolates were also of this type (Table 1): four were O26 and four were O119 ONT:H- (where ONT means some O antiserum positive but not identified), O153:H-, and Out:H-. Eight STEC strains, including five O157 strains, had a complete L region and either a C region or a D region. However, among these eight strains, STEC HI-7 and HI-N had complete L and C regions and a D region with a short insertion (Table 1). Six other STEC strains had complete L and D regions but no C region (Table 1). The ETT2 locus was identified in O157 Sakai, and all O157 isolates of human origin had a complete ETT2 locus. Each of the O157 strains originating from a housefly, salmon roe, and six cattle also had a complete ETT2 locus (Table 1; Fig. 2). However, other O157 STEC strains from cattle had only the L and D regions (Table 1).

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FIG. 2. Detection of regions L, C, and D among various STEC isolates by PCR. The arrows show the expected sizes of the fragments amplified from the DNA sequence.

Distributions of other virulence factors among STEC strains. Previously, we examined the distributions of the eae and hlyA genes among STEC isolates of various origins using eae common primers and hlyA-specific primers (1, 2, 11). In the present study, the eae gene was classified by PCR and production of intimin was determined by Western blotting analysis with anti-intimin antibody. O157 STEC isolates commonly had eae- and produced intimin, regardless of their origins (Table 1; Fig. 3). In addition, all O157 STEC isolates except MB1, of cattle origin, had the hlyA gene. Among the other serotypes tested, O119 and O26 isolates commonly had the eae-ß gene and produced intimin (Table 1; Fig. 3). Among them, eight isolates had the hlyA gene; among them, five isolates had the complete L region, eae, and hlyA. Most other STEC isolates had no eae gene and did not produce intimin (Table 1). STEC strains isolated from seagulls, deer, and sheep belonged to this group (Table 1).

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FIG. 3. Western blotting analysis of intimin proteins from different STEC isolates. Western blotting was performed by using antiserum against intimin encoded by eae-. The results for representative STEC isolates are shown.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A secondary type III secretion system, which was designated ETT2 (E. coli type III secretion system 2), was found in EHEC serotype O157:H7 isolates, in addition to the first system encoded by the LEE locus. This ETT2 locus contains 19 ORFs highly homologous with the SPI-1 type III secretion system of S. enterica serovar Typhimurium; the organization of those ORFs was also similar to that of SPI-1. However, slight differences from SPI-1 were found: (i) SpaR was separated into two ORFs, epaR1 and epaR2, in the ETT2 locus, and (ii) InvB and InvH were absent from the ETT2 locus (Table 1). In the Salmonella SPI-1 system, InvH is necessary for localization of InvG onto the outer membrane, but it is not a component of the type III secretion apparatus (4), while the function of InvB remains unknown (6). Therefore, the ETT2 locus seems to encode a complete set of the type III secretion machinery. Indeed, the complete cloned sequence of ETT2 confers upon E. coli K-12 strain DH5- the ability to secrete the EspB protein (T. Tobe, unpublished data).

In SPI-1, genes coding secreted (effector) proteins such as sipBCDA, the chaperon sicP, and the regulator hilA were found in the region between the spa and prg operons. Instead of these genes, ORF6, which had 40% similarity to the NtrC transcriptional regulator (23) of H. seropedicae, was found in ETT2 (Fig. 1), consistent with the ORF6 product also being a transcriptional regulator. Genes encoding target secreted proteins of the ETT2 locus may exist elsewhere on the O157 chromosome, but we could not identify them. However, a search for other sequences unique to O157 would provide the information needed to identify those genes.

In EPEC and O26 strains, the ETT2 locus was found on the chromosome, but the chromosomes of EHEC O157 strains contained only half of the ETT2 locus. Therefore, the ETT2 DNA segment of EPEC B171-8 (O111:NM) was isolated and its DNA sequence was determined (GenBank accession no. AB052736); it was also inserted at min 64.5 of the E. coli K-12 chromosome and was highly homologous with the L region of the ETT2 sequence in EHEC O157 Sakai (Fig. 1). The sequence corresponding to the eiv genes was absent from the ETT2 loci in EPEC and EHEC O26 strains (Fig. 1). All human STEC isolates had a complete or partial ETT2 locus, and notably, all O157 isolates had a complete ETT2 locus. Interestingly, those STEC and EPEC strains commonly had the L region with the epr and epa genes, suggesting that the ETT2 locus, especially the L-region locus, might be essential for the pathogenicity of O157 STEC isolates, as might the other known virulence factors encoded by the eae and hlyA genes. Although the association of the ETT2 locus with virulence in STEC was not clear, the pathogenicity of STEC might be estimated by PCR with the primer sets described in this study. For example, although non-O157 STEC strain S-8 had the complete ETT2 locus but not stx, eae, and hlyA, it might be pathogenic for humans. As O26 STEC strains AS1 and AS15 were isolated from humans, only the L region might be essential for pathogenicity. In this sense, strains KR1, H8-55, and IK2 from cattle would be virulent strains. Also, O157 strains lacking only the C region from five cattle also would be virulent for humans. As an hlyA gene associated with pathogenicity was encoded by a large plasmid, this gene would easily be lost. Therefore, as the hlyA gene would not be a good marker for the estimation of virulence (21), O157 strains MB1, H5-26, and H8-87 might have originally been virulent. Finally, cattle should be one of the main sources of infection for the transmission of STEC to humans. In this study, O157 isolates from a fly and salmon roe were closely similar to O157 isolates of human origin. However, as the salmon roe was the food that caused the O157 outbreak in Japan (15) and the fly was caught on cow manure on a cattle farm (11), these results were not unexpected.

The G+C content of the ETT2 sequence was very low (36.9%) compared with that of the E. coli chromosome (50.8%) (3), suggesting that the ETT2 locus has been acquired laterally. Furthermore, the high degree of similarity of ETT2 to SPI-1 of S. enterica serovar Typhimurium suggests that ETT2 and SPI-1 may have originated from the same organism. However, the biological function of the ETT2 locus in the pathogenicity of STEC is not clear. The ETT2 locus might participate in the pathogenicity of these bacteria in a manner other than adherence to intestinal epithelial cells. Finally, as the ETT2 locus is common in human STEC isolates, it would be a good marker for molecular epidemiological surveys of STEC strains pathogenic for humans.

 
We thank Ben Adler for critical suggestions on the manuscript.

This work was supported in part by grants from the Ministry of Health, Labour and Welfare (Research on Emerging and Re-Emerging Infectious Diseases), by a Grant-in Aid for Scientific Research from the Japanese Society for the Promotion of Science (grant 12575029), and by a grant from The 21st Century COE Program (A-1), Ministry of Education, Culture, Sports, Science and Technology of Japan.

 
* Corresponding author. Mailing address: Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Phone and fax: 81-155-49-5386. E-mail: smakino{at}obihiro.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Asakura, H., S. Makino, T. Shirahata, T. Tsukamoto, H. Kurazono, T. Ikeda, and K. Takesi. 1998. Detection and genetical characterization of Shiga toxin-producing Escherichia coli from wild deer. Microbiol. Immunol. 42:815-822.[Medline]
2
2. Asakura, H., S. Makino, H. Kobori, M. Watarai, T. Shirahata, T. Ikeda, and K. Takeshi. 2001. Phylogenetic diversity and similarity of active sites of Shiga toxin (Stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals. Epidemiol. Infect. 127:27-36.[CrossRef][Medline]
3
3. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-1474.[Abstract/Free Full Text]
4
4. Daefler, S., and M. Russel. 1998. The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG. Mol. Microbiol. 28:1367-1380.[CrossRef][Medline]
5
5. Donnenberg, M. S., J. B. Kaper, and B. B. Finlay. 1997. Interactions between enteropathogenic Escherichia coli and host epithelial cells. Trends Microbiol. 5:109-114.[CrossRef][Medline]
6
6. Eichelberg, K., C. C. Ginocchio, and J. F. Galan. 1994. Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J. Bacteriol. 176:4501-4510.[Abstract/Free Full Text]
7
7. Elliott, S. J., L. A. Wainwright, T. K. McDaniel, K. G. Jarvis, Y. K. Deng, L. C. Lai, B. P. McNamara, M. S. Donnenberg, and J. B. Kaper. 1998. The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol. Microbiol. 28:1-4.[CrossRef][Medline]
8
8. Galan, J. E., and R. Curtiss III. 1989. Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. Proc. Natl. Acad. Sci. USA 86:6383-6387.[Abstract/Free Full Text]
9
9. Hayashi, T., K. Makino, M. Ohnishi, K. Kurokawa, K. Ishii, K. Yokoyama, C. G. Han, E. Ohtsubo, K. Nakayama, T. Murata, M. Tanaka, T. Tobe, T. Iida, H. Takami, T. Honda, C. Sasakawa, N. Ogasawara, T. Yasunaga, S. Kuhara, T. Shiba, M. Hattori, and H. Shinagawa. 2001. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 8:11-22.[Abstract]
10
10. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.[Abstract/Free Full Text]
11
11. Iwasa, M., S.-I. Makino, H. Asakura, H. Kobori, and Y. Morimoto. 1999. Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan. J. Med. Entomol. 36:108-112.[Medline]
12
12. Karch, H., H. Bohm, H. Schmidt, F. Gunzer, S. Aleksic, and J. Heesemann. 1993. Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-. J. Clin. Microbiol. 31:1200-1205.[Abstract/Free Full Text]
13
13. Levine, M. M., J. G. Xu, J. B. Kaper, H. Lior, V. Prado, R. Tall, J. Nataro, H. Karch, and I. K. Wachsmuth. 1987. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic solitis and hemolytic uremic syndrome. J. Infect. Dis. 156:176-182.
14
14. Makino, K., K. Ishii, T. Yasunaga, M. Hattori, K. Yokoyama, C. H. Yatsudo, Y. Kubota, Y. Yamaichi, T. Iida, K. Yamamoto, T. Honda, C. G. Han, E. Ohtsubo, M. Kasamatsu, T. Hayashi, S. Kuhara, and H. Shinagawa. 1998. Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak. DNA Res. 5:1-9.[Abstract]
15
15. Makino, S.-I., T. Kii, H. Asakura, T. Shirahata, T. Ikeda, K. Takeshi, and K. Itho. 2000. Does enterohemorrhagic Escherichia coli O157:H7 enter the viable but nonculturable (VNC) state in salted salmon roe? Appl. Environ. Microbiol. 66:5536-5539.[Abstract/Free Full Text]
16
16. Makino, S.-I., K. Yamamoto, S. Murakami, T. Shirahata, K. Uemera, T. Sawada, H. Wakamoto, and Y. Morita. 1998. Properties of repeat domain found in a novel protective antigen, SpaA, of Erysipelothrix rhusiopathiae. Microb. Pathog. 723:101-109.
17
17. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
18
18. McDaniel, T. K., and J. B. Kaper. 1997. A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol. Microbiol. 23:399-407.[CrossRef][Medline]
19
19. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.[Abstract/Free Full Text]
20
20. Puente, J. L., D. Bieber, S. W. Ramer, W. Murray, and G. K. Schoolnik. 1996. The bundle-forming pili of enteropathogenic Escherichia coli: transcriptional regulation by environmental signals. Mol. Microbiol. 20:87-100.[CrossRef][Medline]
21
21. Schmidt, H., H. Russmann, and H. Karch. 1993. Virulence determinants in nontoxinogenic Escherichia coli O157 strains that cause infantile diarrhea. Infect. Immun. 61:4894-4898.[Abstract/Free Full Text]
22
22. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 93:2593-2597.[Abstract/Free Full Text]
23
23. Souza, E. M., F. O. Pedrosa, L. U. Rigo, H. B. Machado, and M. G. Yates. 2000. Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element. Microbiology 146:1407-1418.[Abstract/Free Full Text]
24
24. Woestyn, S., A. Allaoui, P. Wattiau, and G. R. Cornelis. 1994. YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J. Bacteriol. 176:1561-1569.[Abstract/Free Full Text]
25
25. Yamasaki, S., Z. Lin, and H. Shirai. 1996. Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction. Microbiol. Immunol. 40:345-352.[Medline]

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Journal of Clinical Microbiology, June 2003, p. 2341-2347, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2341-2347.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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• Lefebvre, B., Gattuso, M., Moisan, H., Malouin, F., Diarra, M. S. (2009). Genotype comparison of sorbitol-negative Escherichia coli isolates from healthy broiler chickens from different commercial farms. Poult. Sci. 88: 1474-1484 [Abstract] [Full Text]  
• Rasko, D. A., Rosovitz, M. J., Myers, G. S. A., Mongodin, E. F., Fricke, W. F., Gajer, P., Crabtree, J., Sebaihia, M., Thomson, N. R., Chaudhuri, R., Henderson, I. R., Sperandio, V., Ravel, J. (2008). The Pangenome Structure of Escherichia coli: Comparative Genomic Analysis of E. coli Commensal and Pathogenic Isolates. J. Bacteriol. 190: 6881-6893 [Abstract] [Full Text]  
• Wu, G., Carter, B., Mafura, M., Liebana, E., Woodward, M. J., Anjum, M. F. (2008). Genetic Diversity among Escherichia coli O157:H7 Isolates and Identification of Genes Linked to Human Infections. Infect. Immun. 76: 845-856 [Abstract] [Full Text]  
• Liverman, A. D. B., Cheng, H.-C., Trosky, J. E., Leung, D. W., Yarbrough, M. L., Burdette, D. L., Rosen, M. K., Orth, K. (2007). Arp2/3-independent assembly of actin by Vibrio type III effector VopL. Proc. Natl. Acad. Sci. USA 104: 17117-17122 [Abstract] [Full Text]  
• Troisfontaines, P., Cornelis, G. R. (2005). Type III Secretion: More Systems Than You Think. Physiology 20: 326-339 [Abstract] [Full Text]  
• van Diemen, P. M., Dziva, F., Stevens, M. P., Wallis, T. S. (2005). Identification of Enterohemorrhagic Escherichia coli O26:H- Genes Required for Intestinal Colonization in Calves. Infect. Immun. 73: 1735-1743 [Abstract] [Full Text]  
• Zhang, L., Chaudhuri, R. R., Constantinidou, C., Hobman, J. L., Patel, M. D., Jones, A. C., Sarti, D., Roe, A. J., Vlisidou, I., Shaw, R. K., Falciani, F., Stevens, M. P., Gally, D. L., Knutton, S., Frankel, G., Penn, C. W., Pallen, M. J. (2004). Regulators Encoded in the Escherichia coli Type III Secretion System 2 Gene Cluster Influence Expression of Genes within the Locus for Enterocyte Effacement in Enterohemorrhagic E. coli O157:H7. Infect. Immun. 72: 7282-7293 [Abstract] [Full Text]  
• Nakano, M., Iida, T., Honda, T. (2004). Urease activity of enterohaemorrhagic Escherichia coli depends on a specific one-base substitution in ureD. Microbiology 150: 3483-3489 [Abstract] [Full Text]  
• Ren, C.-P., Chaudhuri, R. R., Fivian, A., Bailey, C. M., Antonio, M., Barnes, W. M., Pallen, M. J. (2004). 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. J. Bacteriol. 186: 3547-3560 [Abstract] [Full Text]  

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