C. M. Fogg, Paul
(2007)
Stx-Phage Integration and Multiple Lysogeny in Escherichia coli.
PhD thesis, University of Liverpool.
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Abstract
The characteristic virulence factor of shigatoxigenic E. coli strains (STEC) is the expression of Shiga-toxin encoded in the late gene region of temperate lambdoid phages. The use of differentially labelled isogenic recombinant Stx phage (cD24B) had demonstrated the production of true double lysogens in E. coli, contrary to the lambda immunity model, however, the insertion sites and underlying mechanisms had not been determined. Here, the bacteriophage <1>24B integrase gene and the preferred site of insertion in the E. coli genome have been identified. The integrase encoded by <1>24B differs significantly from the integrases of all other previously characterised phages, and the insertion site shares 24 bp of complimentary sequence with the phage aUP site. Furthermore, an additional 3 integration sites in the E. coli genome were elucidated, each with a decreased level of ide'ntity with the aUP site. Triple lysogens could be produced by means of isogenic phage in which the stx gene was interrupted with kanamycin, chloramphenicol and spectinomycin resistance genes as reporters. There were seven possible sites of integration identified, two of which lay within predicted essential genes in the E. coli genome, and thus could not be occupied, and integration at another site was never observed. The preferred integration site described above was close to an integrase gene on the E. coli chromosome but it was demonstrated unequivocally that this host integrase was not responsible for the multiple integration events. The complete partially annotated genome sequence of <1>24B revealed the presence of a gene homologous to the putative antirepressor encoding genes ofVT2-Sa and Lahul and similarity to the well-characterised phage P22 ant gene. Antirepressors bind to the lambdoid phage cI repressor responsible for superinfection immunity and it is therefore possible that the presence of an ant gene on <D24Bis involved in the absence or the classical lambda immunity response in <D24B. Application of reverse transcriptase PCR showed that the gene is transcribed in a lysogenised E. coli host as well as E. coli cells undergoing infection, with some evidence for expression levels correlating with multiplicity of infection. An existing bank of 11 integrase primer sets was supplemented with specific <D24B integrase primers, and applied in the identification of integrase types from 113 wildtype Stx phage isolates with the aim of examining integrase diversity within these populations of phage induced from a collection of STEC isolates. Despite the inherent mosaic nature of lambdoid phage, the diversity of integrases was limited and dominated by 2 groups, 933\V-like and <1>24B-like. Demonstration of the ability of <D24B to form multiple lysogens, as well as the presence of different integrases within a population of Stx phages, has consequences for the evolution of pathogens, as novel Stx phages can emerge due to intracellular recombination events. Multiple lysogens may also produce more Stx-toxin and thus have increased virulence. Supplied by The British Library - 'The world's knowledge'
| Item Type: | Thesis (PhD) |
|---|---|
| Depositing User: | Symplectic Admin |
| Date Deposited: | 20 Oct 2023 09:25 |
| Last Modified: | 20 Oct 2023 09:30 |
| DOI: | 10.17638/03174569 |
| Copyright Statement: | Copyright © and Moral Rights for this thesis and any accompanying data (where applicable) are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3174569 |
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