Requirement for Dam DNA-methyltransferase for the host-range of the Shiga-toxin-encoding bacteriophage 933W.

Main area:Molecular biology
Target group:Biology, Molecular Biomedicine, Biochemistry
Educational level:Masters, Bachelor
Project description:
Project Description Enterohemorrhagic Escherichia coli O157:H7 (EHEC) produce Shiga toxin that, during infection, can produce potentially fatal damage to the host kidneys. The genes encoding Shiga toxin are located on a resident bacteriophage (prophage), 933W, in the bacterial genome (1, 2, 3). Almost all of these prophages also contain a dam gene encoding a DNA methyltransferase that has identical specificity to the dam gene on the host chromosome. Deletion of the prophage dam gene has no effect on bacteriophage growth in wildtype EHEC but growth is inhibited in EHEC strains in which the host dam gene has been deleted (4). This result indicates that the host Dam methyltransferase is required for a vital function in the bacteriophage growth cycle. To further understand what this role might be, we have identified second-site mutations that allow growth of dam-deleted phage on a dam-deleted host (Anders Løbner-Olesen unpublished data). These second-site mutations in the bacteriophage map to a gene contiguous with the dam gene and downstream of it. The predicted protein produced by this gene has no known function but is present in the genomes of many prophages of both Gram-positive and –negative bacteria. In this project we will study the requirement for DNA methylation for propagation of the Shiga toxin encoding phages using phage 933W as our model, and determine how the loss of the downstream gene may overcome the dam requirement. These studies are particularly important as they may shed light on the horizontal spread and host range of the 933W phage. A recent example of this horizontal spread occurred in 2011 when the phage moved from the O157:H7 strain into an enteroaggregative (EAEC) E. coli O104:H4 strain which caused infection, through consumption of contaminated bean sprouts, of more than 3.500 people primarily in Germany, with 53 fatalities. It should be emphasized that the use of an attenuated version of phage 933W (and all relevant mutants), where the genes encoding the Shiga toxins have been deleted, will be employed in this project. Contact Professor Anders Løbner-Olesen ( or Associate Professor Sine Lo Svenningsen ( if you are interested in undertaking this project. References 1. Donnenberg, M. S., and T. S. Whittam. 2001. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J. Clin. Investig. 107:539–548. 2. Kaper, J. B., J. P. Nataro, and H. L. Mobley. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2:123–140. 3. Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529–533. 4. Kenan C. Murphy, Jennifer M. Ritchie, Matthew K. Waldor, Anders Løbner-Olesen and M. G. Marinus. 2008. Dam Methyltransferase Is Required for Stable Lysogeny of the Shiga Toxin (Stx2)-Encoding Bacteriophage 933W of Enterohemorrhagic Escherichia coli O157:H7. J. Bacteriol 190(1):438. 5. Lobner-Olesen,A., Skovgaard,O. and Marinus,M.G. (2005) Dam methylation: coordinating cellular processes. Curr. Opin. Microbiol., 8, 154-160.
Methods used:cloning, site-directed mutagenesis, pcr, plaque assays, general techniques for molecular microbiology
Keywords:molecular microbiology, shiga toxin, bacteriophage, genetics
Supervisor(s): Anders Løbner-Olesen, Sine Lo Svenningsen /