Mechanistic insights into Xer recombination and conjugative transposition in Helicobacter pylori

Bebel, Aleksandra (2015) Mechanistic insights into Xer recombination and conjugative transposition in Helicobacter pylori. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.

Abstract

Site-specific recombinases of the Xer family are essential in most bacteria with circular chromosomes for the resolution of chromosome dimers arising after genome replication. In Helicobacter pylori, a gastric pathogen implicated in peptic ulcer disease and gastric cancer, the chromosome dimers are resolved by a single Xer recombinase, XerH. Interestingly, many H. pylori strains carry a second Xer recombinase, XerT, usually encoded on a large conjugative transposon TnPZ. Remarkably, XerT is not involved in chromosome dimer resolution, but was shown to be required for the mobilization of TnPZ in vivo. In this thesis, I investigated the molecular mechanisms of XerH- and XerT-mediated recombination by combining X-ray crystallography with protein biochemistry and microbiology. I solved the crystal structure of the XerH tetramer in a post-cleavage synaptic complex with its substrate DNA site, difH. To our knowledge, this is the first structure of an Xer recombinase bound to DNA. The structure and additional biochemical data provided key insights into the ordering and regulation of difH binding and first strand cleavage by XerH. Moreover, I investigated the regulation of XerH recombination by FtsK – a host factor usually required for Xer recombination – and found that XerH can resolve plasmids in the absence of FtsK in E. coli, but additional factors might be required for recombination of chromosome-borne difH sites. In the second part of this work, I studied the mechanism of XerT-mediated TnPZ transposition. XerT’s binding and cleavage sites on transposon ends were mapped and XerT activity was reconstituted in vitro by trapping cleavage and strand exchange products. In addition, the complete TnPZ excision has been reconstituted in vivo in E. coli, indicating that XerT is sufficient to catalyze TnPZ mobilization. Based on the results, a testable model for TnPZ excision and integration was proposed. In summary, this work provides valuable insights into the mechanisms of the two Xer recombinases of H. pylori and enhances our understanding of Xer recombination (a process essential for bacterial survival) and conjugative transposition (important in the spread of antibiotic resistance among bacteria), which in the future could help develop new therapeutic agents against deadly pathogens such as H. pylori or help control the spread of antibiotic resistance.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Xer, site specific recombination, TnPZ, conjugative transposition, X-ray crystallography
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Funder's Name: UNSPECIFIED
Supervisor's Name: Stark, Professor W. Marshall and Barabas, Dr Orsolya
Date of Award: 2015
Embargo Date: 8 January 2019
Depositing User: Ms Aleksandra Bebel
Unique ID: glathesis:2015-6975
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Jan 2016 13:22
Last Modified: 14 Jan 2016 08:50
URI: http://theses.gla.ac.uk/id/eprint/6975

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