1887

Abstract

The key virulence factor in Shiga-toxigenic is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to phage. However, contrary to the superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Φ24. Here, the Φ24 integrase gene is identified, and the preferred site of integration defined. Although an gene was identified close to the Φ24 integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to and are therefore unlikely to support phage integration. A Φ24 gene, possessing similarity to the well-characterized P22 gene, was identified. RT-PCR was used to demonstrate that is transcribed in a Φ24 lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Φ24 to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the gene may lead to an increase in toxin production and consequently increased virulence.

Keyword(s): Stx, Shiga toxin
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2007-12-01
2019-11-12
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References

  1. Allison, H. E. ( 2007; ). Stx-phages: drivers and mediators of the evolution of STEC and STEC-like pathogens. Future Microbiol 2, 165–174.[CrossRef]
    [Google Scholar]
  2. Allison, H. E., Sergeant, M. J., James, C. E., Saunders, J. R., Smith, D. L., Sharp, R. J., Marks, T. S. & McCarthy, A. J. ( 2003; ). Immunity profiles of wild-type and recombinant Shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens. Infect Immun 71, 3409–3418.[CrossRef]
    [Google Scholar]
  3. Bagdasarian, M., Lurz, R., Ruckert, B., Franklin, F. C., Bagdasarian, M. M., Frey, J. & Timmis, K. N. ( 1981; ). Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16, 237–247.[CrossRef]
    [Google Scholar]
  4. Balding, C., Bromley, S. A., Pickup, R. W. & Saunders, J. R. ( 2005; ). Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages. Environ Microbiol 7, 1558–1567.[CrossRef]
    [Google Scholar]
  5. Barreiro, V. & Haggard-Ljungquist, E. ( 1992; ). Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E. coli K-12 derivatives. J Bacteriol 174, 4086–4093.
    [Google Scholar]
  6. Bielaszewska, M., Prager, R., Zhang, W., Friedrich, A. W., Mellmann, A., Tschape, H. & Karch, H. ( 2006; ). Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157 : NM. Appl Environ Microbiol 72, 1900–1909.[CrossRef]
    [Google Scholar]
  7. Bielaszewska, M., Prager, R., Kock, R., Mellmann, A., Zhang, W., Tschape, H., Tarr, P. I. & Karch, H. ( 2007; ). Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans. Appl Environ Microbiol 73, 3144–3150.[CrossRef]
    [Google Scholar]
  8. Biswas, T., Aihara, H., Radman-Livaja, M., Filman, D., Landy, A. & Ellenberger, T. ( 2005; ). A structural basis for allosteric control of DNA recombination by λ integrase. Nature 435, 1059 [CrossRef]
    [Google Scholar]
  9. Blaisdell, B. E., Campbell, A. M. & Karlin, S. ( 1996; ). Similarities and dissimilarities of phage genomes. Proc Natl Acad Sci U S A 93, 5854–5859.[CrossRef]
    [Google Scholar]
  10. Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K. & other authors ( 1997; ). The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1462.[CrossRef]
    [Google Scholar]
  11. Botstein, D. ( 1980; ). A theory of modular evolution for bacteriophages. Ann N Y Acad Sci 354, 484–490.[CrossRef]
    [Google Scholar]
  12. Botstein, K., Lew, K. K., Jarvik, V. & Swanson, C. A. ( 1975; ). Role of antirepressor in the bipartite control of repression and immunity by bacteriophage P22. J Mol Biol 91, 439–462.[CrossRef]
    [Google Scholar]
  13. Boyd, E. F. & Brussow, H. ( 2002; ). Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol 10, 521 [CrossRef]
    [Google Scholar]
  14. Brussow, H., Canchaya, C. & Hardt, W.-D. ( 2004; ). Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 68, 560–602.[CrossRef]
    [Google Scholar]
  15. Calef, E. ( 1967; ). Mapping of integration and excision crossovers in superinfection double lysogens for phage lambda in Escherichia coli. Genetics 55, 547–556.
    [Google Scholar]
  16. Campbell, A. ( 1994; ). Comparative molecular biology of lambdoid phages. Annu Rev Microbiol 48, 193–222.[CrossRef]
    [Google Scholar]
  17. Campbell, A. ( 2003; ). Prophage insertion sites. Res Microbiol 154, 277–282.[CrossRef]
    [Google Scholar]
  18. Casjens, S., Winn-Stapley, D. A., Gilcrease, E. B., Morona, R., Kuhlewein, C., Chua, J. E. H., Manning, P. A., Inwood, W. & Clark, A. J. ( 2004; ). The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging. J Mol Biol 339, 379–394.[CrossRef]
    [Google Scholar]
  19. Chen, Y., Narendra, U., Iype, L. E., Cox, M. M. & Rice, P. A. ( 2000; ). Crystal structure of a Flp recombinase–Holliday junction complex: assembly of an active oligomer by helix swapping. Mol Cell 6, 885–897.
    [Google Scholar]
  20. Clark, A. J., Inwood, W., Cloutier, T. & Dhillon, T. S. ( 2001; ). Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages. J Mol Biol 311, 657–679.[CrossRef]
    [Google Scholar]
  21. Craig, N. L. ( 2001; ). Mobile DNA II. Washington, DC: American Society for Microbiology.
  22. Creuzburg, K., Kohler, B., Hempel, H., Schreier, P., Jacobs, E. & Schmidt, H. ( 2005; ). Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1. Microbiology 151, 941–950.[CrossRef]
    [Google Scholar]
  23. De Greve, H., Qizhi, C., Deboeck, F. & Hernalsteens, J. P. ( 2002; ). The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome. Biochim Biophys Acta 1579, 196–202.[CrossRef]
    [Google Scholar]
  24. Esposito, D. & Scocca, J. J. ( 1997; ). The integrase family of tyrosine recombinases: evolution of a conserved active site domain. Nucleic Acids Res 25, 3605–3614.[CrossRef]
    [Google Scholar]
  25. Fattah, K. R., Mizutani, S., Fattah, F. J., Matsushiro, A. & Sugino, Y. ( 2000; ). A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity. Genes Genet Syst 75, 223–232.[CrossRef]
    [Google Scholar]
  26. Freifelder, D. & Kirschner, I. ( 1971; ). The formation of homoimmune double lysogens of phage lambda and the segregation of single lysogens from them. Virology 44, 633–637.[CrossRef]
    [Google Scholar]
  27. Gerdes, S. Y., Scholle, M. D., Campbell, J. W., Balázsi, G., Ravasz, E., Daugherty, M. D., Somera, A. L., Kyrpides, N. C., Anderson, I. & other authors ( 2003; ). Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J Bacteriol 185, 5673–5684.[CrossRef]
    [Google Scholar]
  28. Hall, T. A. ( 1999; ). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
    [Google Scholar]
  29. Hatfull, G. F., Pedulla, M. L., Jacobs-Sera, D., Balázsi, G., Ravasz, E., Daugherty, M. D., Somera, A. L., Kyrpides, N. C., Anderson, I. & other authors ( 2006; ). Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet 2, e92 [CrossRef]
    [Google Scholar]
  30. Herold, S., Karch, H. & Schmidt, H. ( 2004; ). Shiga toxin-encoding bacteriophages – genomes in motion. Int J Med Microbiol 294, 115–121.[CrossRef]
    [Google Scholar]
  31. James, C. E., Stanley, K. N., Allison, H. E., Flint, H. J., Stewart, C. S., Sharp, R. J., Saunders, J. R. & McCarthy, A. J. ( 2001; ). Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage. Appl Environ Microbiol 67, 4335–4337.[CrossRef]
    [Google Scholar]
  32. Johansen, B. K., Wasteson, Y., Granum, P. E. & Brynestad, S. ( 2001; ). Mosaic structure of Shiga-toxin-2-encoding phages isolated from Escherichia coli O157 : H7 indicates frequent gene exchange between lambdoid phage genomes. Microbiology 147, 1929–1936.
    [Google Scholar]
  33. Kaniga, K., Delor, I. & Cornelis, G. R. ( 1991; ). A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109, 137–141.[CrossRef]
    [Google Scholar]
  34. Koch, C., Hertwig, S. & Appel, B. ( 2003; ). Nucleotide sequence of the integration site of the temperate bacteriophage 6220, which carries the Shiga toxin gene stx(1ox3). J Bacteriol 185, 6463–6466.[CrossRef]
    [Google Scholar]
  35. Koudelka, A. P., Hufnagel, L. A. & Koudelka, G. B. ( 2004; ). Purification and characterization of the repressor of the Shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage. J Bacteriol 186, 7659–7669.[CrossRef]
    [Google Scholar]
  36. Livny, J. & Friedman, D. I. ( 2004; ). Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system. Mol Microbiol 51, 1691–1704.[CrossRef]
    [Google Scholar]
  37. Makino, K., Yokoyama, K., Kubota, Y., Yutsudo, C. H., Kimura, S., Kurokawa, K., Ishii, K., Hattori, M., Tatsuno, I. & other authors ( 1999; ). Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157 : H7 derived from the Sakai outbreak. Genes Genet Syst 74, 227–239.[CrossRef]
    [Google Scholar]
  38. Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II ( 1997; ). GeneDoc: analysis and visualization of genetic variation. EMBnet News 4 (2). http://www.embnet.org/files/shared/EMBnetNews/embnet_news_4_2.pdf.
  39. Nunes-Duby, S. E., Kwon, H. J., Tirumalai, R. S., Ellenberger, T. & Landy, A. ( 1998; ). Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26, 391–406.[CrossRef]
    [Google Scholar]
  40. O'Brien, A. D., Newland, J. W., Miller, S. F., Holmes, R. K., Smith, H. W. & Formal, S. B. ( 1984; ). Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 226, 694–696.[CrossRef]
    [Google Scholar]
  41. Ohnishi, M. & Hayashi, T. ( 2002; ). Genetic diversity of enterohemorrhagic Escherichia coli. Nippon Rinsho 60, 1077–1082 (in Japanese).
    [Google Scholar]
  42. Penfold, R. J. & Pemberton, J. M. ( 1992; ). An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118, 145–146.[CrossRef]
    [Google Scholar]
  43. Plunkett, G., III, Rose, D. J., Durfee, T. J. & Blattner, F. R. ( 1999; ). Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157 : H7: Shiga toxin as a phage late-gene product. J Bacteriol 181, 1767–1778.
    [Google Scholar]
  44. Ptashne, M. ( 2004; ). A Genetic Switch: Phage Lambda Revisited, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  45. Recktenwald, J. & Schmidt, H. ( 2002; ). The nucleotide sequence of Shiga toxin (Stx) 2e-encoding phage φP27 is not related to other Stx phage genomes, but the modular genetic structure is conserved. Infect Immun 70, 1896–1908.[CrossRef]
    [Google Scholar]
  46. Rice, P. A. ( 2005; ). Resolving integral questions in site-specific recombination. Nat Struct Mol Biol 12, 641 [CrossRef]
    [Google Scholar]
  47. Riley, M., Abe, T., Arnaud, M. B., Berlyn, M. K., Blattner, F. R., Chaudhuri, R. R., Glasner, J. D., Horiuchi, T., Keseler, I. M. & other authors ( 2006; ). Escherichia coli K-12: a cooperatively developed annotation snapshot – 2005. Nucleic Acids Res 34, 1–9.[CrossRef]
    [Google Scholar]
  48. Rutkai, E., Dorgai, L., Sirot, R., Yagil, E. & Weisberg, R. A. ( 2003; ). Analysis of insertion into secondary attachment sites by phage λ and by int mutants with altered recombination specificity. J Mol Biol 329, 983–996.[CrossRef]
    [Google Scholar]
  49. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  50. Saunders, J. R., Allison, H. E., James, C. E., McCarthy, A. J. & Sharp, R. ( 2001; ). Phage-mediated transfer of virulence genes. J Chem Tech Biotech 76, 662–666.[CrossRef]
    [Google Scholar]
  51. Schaefer, K. L. & McClure, W. R. ( 1997; ). Antisense RNA control of gene expression in bacteriophage P22. I. Structures of sar RNA and its target, ant mRNA. RNA 3, 141–156.
    [Google Scholar]
  52. Sherratt, D. J., Soballe, B., Barre, F. X., Filipe, S., Lau, I., Massey, T. & Yates, J. ( 2004; ). Recombination and chromosome segregation. Philos Trans R Soc Lond B Biol Sci 359, 61–69.[CrossRef]
    [Google Scholar]
  53. Siegler, R. L., Pysher, T. J., Tesh, V. L. & Taylor, F. B., Jr ( 2001; ). Response to single and divided doses of Shiga toxin-1 in a primate model of hemolytic uremic syndrome. J Am Soc Nephrol 12, 1458–1467.
    [Google Scholar]
  54. Susskind, M. M. & Botstein, D. ( 1975; ). Mechanism of action of Salmonella phage P22 antirepressor. J Mol Biol 98, 413–424.[CrossRef]
    [Google Scholar]
  55. Susskind, M. M. & Botstein, D. ( 1978; ). Molecular genetics of bacteriophage P22. Microbiol Rev 42, 385–413.
    [Google Scholar]
  56. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ( 1994; ). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef]
    [Google Scholar]
  57. Tyler, J. S., Mills, M. J. & Friedman, D. I. ( 2004; ). The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression. J Bacteriol 186, 7670–7679.[CrossRef]
    [Google Scholar]
  58. Van Duyne, G. D. ( 2001; ). A structural view of Cre-loxP site-specific recombination. Annu Rev Biophys Biomol Struct 30, 87–104.[CrossRef]
    [Google Scholar]
  59. Willshaw, G. A., Smith, H. R., Scotland, S. M., Field, A. M. & Rowe, B. ( 1987; ). Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol 133, 1309–1317.
    [Google Scholar]
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