Skip to main content

Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system

Abstract

In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work and reporters for transcriptional analyses. In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157. The deletion and replacement of the LEE4 operon of E. coli O157 is detailed to show the advantages and limitations of the technology.

References

  1. Falkow S. Molecular Koch’s postulates applied to bacterial pathogenicity — a personal recollection 15 years later. Nat Rev Microbiol 1994; 2:67–72.

    Article  Google Scholar 

  2. Miller JH. Experiments in Molecular Genetics. New York: Cold Spring Harbour Laboratory Press; 1972.

    Google Scholar 

  3. Parker B, Marinus MG. A simple and rapid method to obtain substitution mutations in Escherichia coli: isolation of a dam deletion/insertion mutation. Gene 1988; 73:531–535.

    Article  PubMed  CAS  Google Scholar 

  4. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000; 97:6640–6645.

    Article  PubMed  CAS  Google Scholar 

  5. Perna NT et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 2001; 409:529–533.

    Article  PubMed  CAS  Google Scholar 

  6. Spears KJ, Roe AJ, Gally DL. A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis. FEMS Microbiol Letts 2005; 255:187–202.

    Article  Google Scholar 

  7. Campellone KG, Robbins D, Leong JM. EspF(U) is a translocated EHEC effector that interacts with Tir and N-WASP and promotes nck-independent actin assembly. Dev Cell 2004; 7:217–228.

    Article  PubMed  CAS  Google Scholar 

  8. Roe AJ et al. Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level. Infect Immun 2003; 71:5900–5909.

    Article  PubMed  CAS  Google Scholar 

  9. Naylor SW et al. Escherichia coli O157:H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon. Microbiol 2005; 151:2773–2781.

    Article  CAS  Google Scholar 

  10. Cohen SN, Chang ACY. Revised Interpretation of the Origin of the pSC101 Plasmid. J Bacteriol 1997; 132:734–737.

    Google Scholar 

  11. Hashimoto-Gotoh TSM, Sekiguchi M. Mutations to Temperature Sensitivity in R Plasmid pSC101. J Bacteriol 1977; 131:405–412.

    PubMed  CAS  Google Scholar 

  12. Hamilton CM et al. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 1989; 171:4617–4622.

    PubMed  CAS  Google Scholar 

  13. Blomfield IC et al. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol 1991; 5:1447–1457.

    Article  PubMed  CAS  Google Scholar 

  14. Merlin C et al. Tools for characterization of Escherichia coli genes of unknown function. J Bacteriol 2002; 184:4573–4581.

    Article  PubMed  CAS  Google Scholar 

  15. Low AS et al. Analysis of fimbrial gene clusters and their expression in enterohaemorrhagic Escherichia coli O157:H7. Env Microbiol 2006; 8:1033–1047.

    Article  CAS  Google Scholar 

  16. Roe AJ et al. Co-ordinate single-cell expression of LEE4 — and LEE5-encoded proteins of Escherichia coli O157:H7. Mol Microbiol 2004; 54:337–352.

    Article  PubMed  CAS  Google Scholar 

  17. Porter ME et al. The LEE1 promoters from both enteropathogenic and enterohemorrhagic Escherichia coli can be activated by PerC-like proteins from either organism. J Bacteriol 2005; 187:458–472.

    Article  PubMed  CAS  Google Scholar 

  18. Tsunekawa H et al. Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry. Appl Environ Microbiol 1992; 58:2081–2088.

    PubMed  CAS  Google Scholar 

  19. Johnson JR et al. High-Frequency Secondary Mutations after Suicide-Driven Allelic Exchange Mutagenesis in Extraintestinal Pathogenic Escherichia coli. J Bacteriol 2003; 185:5301–5305.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Andrew J. Roe.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

Emmerson, J.R., Gally, D.L. & Roe, A.J. Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Biol. Proced. Online 8, 153–162 (2006). https://doi.org/10.1251/bpo123

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo123

Indexing terms

  • Gene Deletion
  • Escherichia coli O157:H7