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Journal of Bacteriology, July 2003, p . 3966-3971, Vol . 185, No . 13

Genome Analysis of a Novel Shiga Toxin 1 (Stx1)-Converting Phage Which Is Closely Related to Stx2-Converting Phages but Not to Other Stx1-Converting Phages

Toshio Sato,1,2* Takeshi Shimizu,1,2 Masahisa Watarai,1,{dagger} Midori Kobayashi,1 Shigeyuki Kano,1,2 Takashi Hamabata,1,2 Yoshifumi Takeda,3,{ddagger} and Shinji Yamasaki1,2,4

Research Institute, International Medical Center of Japan, Shinjuku, Tokyo 162-8655,1 Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575,2 National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640,3 Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 591-8531, Japan4

Received 28 February 2003/ Accepted 18 April 2003


   ABSTRACT

 
Two Stx-converting phages, designated Stx1{phi} and Stx2{phi}-II, were isolated from an Escherichia coli O157:H7 strain, Morioka V526, and their entire nucleotide sequences were determined . The genomes of both phages were similar except for the stx gene-flanking regions . Comparing these phages to other known Stx-converting phages, we concluded that Stx1{phi} is a novel Stx1-converting phage closely related to Stx2-converting phages so far reported .


   TEXT

 
Infection with enterohemorrhagic Escherichia coli (EHEC) causes severe illnesses including hemorrhagic colitis, hemolytic-uremic syndrome, and encephalosis (13) . Such critical illnesses are due to Shiga toxin (Stx) produced by EHEC . EHEC produces two types of Stx, namely Stx1, which is identical to Shiga toxin produced by Shigella dysenteriae type 1 (17), and Stx2, which has immunological properties that are different from those of Stx1 but biological properties that are similar to those of Stx1 (22) . Both of these Stxs are encoded by stx genes in the genome of the lysogenic bacteriophage (Stx phage) of EHEC (12, 16) .

The fact that the expression of stx genes is linked to Stx phage induction (1, 11) is clinically quite important because DNA-damaging drugs such as quinolones, which induce an SOS response in bacteria, are supposed to enhance Stx production as well as Stx phage release from EHEC (4, 23) . In fact, several studies on the effects of antibiotics on EHEC infection have been published (2, 19, 20) . Thus, a need to analyze the nature or structure of Stx-converting phages has led to several studies on genome analysis of some Stx-converting phages (7, 9, 10, 14, 21) . We also isolated three Stx-converting phages from EHEC strains collected in Japan, i.e., Stx1{phi}, Stx2{phi}-I, and Stx2{phi}-II (18), and we determined their complete DNA sequences . In this paper, we report the genomic analysis of Stx1{phi} and Stx2{phi}-II, both derived from a single EHEC strain, Morioka V526 .

Phage isolation and DNA sequence determination. Isolation of Stx-converting phages from the EHEC Morioka V526 strain, preparation of the restriction map, and subcloning were performed as described previously (18) . DNA sequencing was done by using the Dye Terminator kit (Applied Biosystems, Norwalk, Conn.) and 377PRISM autosequencer (Applied Biosystems) with synthetic oligonucleotides as primers . It was found that the genome size of Stx1{phi} was 59,866 bp, while that of Stx2{phi}-II was 62,706 bp . As shown in Fig . 1, although these two phages carry different stx genes, their genomic structures were quite homologous . The 2.8-kb size difference was attributed mainly to the BamHI-XhoI fragment-containing stx gene (Fig . 1) . Also, insertion sequence IS1203 v (6) was found in this region in Stx2{phi}-II (Fig . 1) .


FIG . 1 . Schematic representation of Stx1{phi} and Stx2{phi}-II . (A) Comparison of DNAs of Stx2{phi}-II and Stx1{phi} . The linear sequences of XhoI fragments are shown . The open bars represent homologous portions, while the different portions of Stx1{phi} are represented by shaded bars and the corresponding regions in Stx2{phi}-II are represented by dotted bars . The DNA sizes are shown on the top of the figure in kilobases (kb) . Homology percentages are given above and below the second line of the figure . The asterisk indicates 0% homology, which is due to IS1203 v . Vertical lines on the two lower bars indicate XhoI sites (solid lines) and BamHI sites (broken lines) . (B) Comparison of ORFs of Stx2{phi}-II and Stx1{phi} . The predicted ORFs are illustrated; the boxes above and below the horizontal lines are ORFs with rightward and leftward transcription directions, respectively . Open boxes are ORFs identical to the corresponding ORFs in any Stx2-converting phage(s) described in Table 1 . Shaded boxes are ORFs characteristic of Stx1-converting phages, and dotted boxes are ORFs nearly identical to those of any Stx2-converting phage(s).

 
Comparison to other reported Stx-converting phages. The genomic structures of Stx1{phi} and Stx2{phi}-II were compared to those of other Stx-converting phages so far reported . It was found that they were quite similar to those of other known Stx2-converting phages, except for the stx-flanking regions (Fig . 1 and Table 1), but not to those of other Stx1-converting phages such as VT1-Sakai and H19B (Fig . 2) .


TABLE 1 . ORFs of Stx1{phi} and Stx2{phi}-II

 

FIG . 2 . Comparison of Stx1{phi} with other related Stx1-converting phages . The open bars represent portions homologous to Stx1{phi}, while the different portions of each Stx1-converting phage are cross-hatched . (A) Comparison between Stx1{phi} and VT1-Sakai phages; (B) comparison between Stx1{phi} and H19B phages . The DNA sequence of VT1-Sakai phage was modified from that reported by Yokoyama et al . (21) for convenience . Note that most regions of Stx1{phi} were not homologous to Stx1-converting phages, except for the stx1-flanking region.

 
ORF analysis. Open reading frames (ORFs) that showed significant homologies to the genes registered in DDBJ or that consisted of more than 80 amino acid residues were picked up . This definition enabled us to identify 167 putative ORFs in Stx1{phi} and 170 putative ORFs in Stx2{phi}-II (for detailed ORF information, please refer to DDBJ) . The ORFs that show homology to any genes in other Stx-converting phages or bacterium-associated genes were picked up and are listed in Table 1 . ORFs of Stx1{phi} and Stx2{phi}-II were also almost completely identical, reflecting the high DNA sequence homology between these two phages . The exception was the stx-flanking regions including four ORFs, B69, B73, B74, and b70 in Stx1, which are identical to or almost the same as the corresponding ORFs of VT1-Sakai (Table 1) . This region might be characteristic of Stx1-converting pages, since H19B (10) also has a homology in the corresponding region at the DNA level (data not shown) . ORFs B4, B5, and B30 of Stx1{phi} are not identical to the corresponding ORFs of Stx2{phi}-II due to frameshift mutations (data not shown) . From these data, we conclude that Stx1{phi} is closely related to other Stx2-converting phages even at the ORF level .

It is noteworthy that there are several ORFs homologous to those of Shigella sonnei phage 7888 (15) and S . dysenteriae (8) in the stx-flanking regions of Stx1{phi} and Stx2{phi}-II (Table 1) . Recently, an Stx-converting phage was isolated from Stx1-producing S . sonnei (L . Beutin, E . Strauch, and I . Fischer, Letter, Lancet 353:1498, 1999) . Treatment with mitomycin C increases Stx production and induces Stx phage from some EHEC (5) and S . sonnei (Beutin et al., letter) bacteria . It has been a focus of discussion whether Stx-converting phages in EHEC are derived from Shigella species . Our data rather support that Stx-converting phages might be derived from, or at least related to, Shigella species .

Nucleotide sequence accession numbers. The entire nucleotide sequences of Stx1{phi} and Stx2{phi}-II were submitted to DDBJ under accession numbers AP005153 and AP005154, respectively .

 


   ACKNOWLEDGMENTS

 
We thank G . Balakrish Nair for critical reading of the manuscript .

This work was supported by the Organization for Pharmaceutical Safety and Research .

This work formed a part of the Ph.D . thesis of T . Sato .


   FOOTNOTES

 
* Corresponding author . Mailing address: Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan . Phone: 81-3-3202-7181, ext . 2837 . Fax: 81-3-3202-7364 . E-mail: tsato{at}ri.imcj.go.jp .

{dagger} Present address: Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro-shi, Hokkaido 080-8555, Japan .

{ddagger} Present address: Jissen Women's University, Hino, Tokyo 191-8510, Japan .


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