Molecular Characterization Of The Activity And Requirements Of A Novel And Promiscuous Bacteriophage Integrase



Mohaisen, MR
(2017) Molecular Characterization Of The Activity And Requirements Of A Novel And Promiscuous Bacteriophage Integrase. PhD thesis, University of Liverpool.

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Abstract

Stx bacteriophages are responsible for the dissemination and production of Shiga toxin genes (stx) across the Shigatoxigenic E. coli (STEC). These toxigenic bacteriophage hosts can cause severe, life-threatening illness, and Shiga toxin (Stx) is responsible for the severe nature of EHEC infection, a subset of pathogenic STEC. At the point of Stx phage infection, the injected phage DNA can direct its integration into the bacterial chromosome becoming a prophage; the host cell is then known as a lysogen. Unusually, our model Stx phage, Φ24B, can integrate into at least four distinct sites within the E. coli genome that shared no easily identifiable recognition sequence pattern. The identification of what are actually required for phage and bacterial DNAs recombination has been tested using both in vitro and in situ recombination assays. These assays enabled the simple manipulation of bacterial attachment site (attB) and phage attachment site (attP) sequences. The aim of the study is to fully characterize the requirements of this promiscuous integrase, carried by the Stx phage Φ24B (IntΦ24B), to drive integration. These assays enabled us to identify the minimal necessary flanking sequences for attB site identified (21 bp and 49 bp from the right and left the cross over region, respectively) and the attP site (200 bp each side). Furthermore, we identified that the Φ24B integrase does not need Integration Host Factor (IHF) to drive integration. Finally, as this integrase can integrate the phage genome inside at least four different bacterial attachment sites (attBs), these sites were identified, sequenced and cloned in different compatible plasmids to be transformed to one cell, and the frequency and preference of each recombination were tested by means of qPCR. The results showed, the recombination with secondary attachment sites was less frequent than that with the primary site. Furthermore, within five minutes, there was also a preference of site, as recombination occurred in both attB1 and attB2, while recombination with the attB4 and attB3 sites, took 1 hour and 2 hours, respectively.

Item Type: Thesis (PhD)
Divisions: Fac of Health & Life Sciences > Institute of Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 21 Dec 2017 13:55
Last Modified: 09 Jan 2021 06:17
DOI: 10.17638/03011837
Supervisors:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3011837