Characterization of the Late Promoter pR' from Shiga Toxin Prophages in Escherichia Coli

• Author / Creator

  Zhang, Lingxiao

• Shiga toxin producing Escherichia coli (STEC) strains are pathogens which frequently cause human intestinal diseases. The main virulence factor Shiga toxin (Stx) is encoded by the gene located on the Stx prophage and gene expression is affected by the diversity of Stx prophage genome. Nineteen candidate STEC strains were selected according to the previous research. PCR was used, and the late promoter regions, designated as pR', were amplified from STEC strains. Contig gaps were filled by sequencing, and complete sequences of target pR' fragments were obtained. To confirm the diversity of the gene structure of the target pR' region, sequences were aligned, and phylogenetic trees were built based on the sequences of target pR' and stx, respectively. According to the phylogenetic analyses, pR' fragments were highly diverse compared to the stx subtypes they harbor. To visualize Stx production, pR' fragments were fused with the rfp reporter gene DsRed constructed into pUC19. This reporter system was validated by inducing transformants E. coli O104:H4 11-3088 △stx::gfp::ampr (Prfp::chlr), E. coli O104:H4 11-3088 △stx::gfp::ampr (Pp1302::rfp::chlr) and E. coli O157:H7 CO6CE900 (Pp1302::rfp::chlr) with mitomycin C and observed by microscopy. Different combinations of pR' and target strains were made to determine the effect the late promoter pR' region conferred to Stx prophage induction. Not all prophages can be induced under the control of its parent prophage; no matter if they were controlled by the same or different prophage regulations regulatory systems, different pR' behaved diversely. In conclusion, the diversity of late promoter region sequence structure affects the behavior of the Stx prophage induction.

• Subjects / Keywords

  • Gene diversity
  • E. coli
  • Genetic analysis
  • Flow cytometry
  • Shiga toxin phage

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R39S1M12T

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.