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Structural and Functional Characterization of O-Antigen Translocation and Polymerization in Pseudomonas aeruginosa PAO1

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dc.contributor.advisor Lam, Joseph Islam, Salim Timo 2013-06-07T18:23:48Z 2013-06-07T18:23:48Z 2013-06 2013-05-30 2013-06-07
dc.description Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. en_US
dc.description.abstract Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. en_US
dc.description.sponsorship 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC en_US
dc.language.iso en en_US
dc.publisher University of Guelph en_US
dc.subject lipopolysaccharide en_US
dc.subject flippase en_US
dc.subject polymerase en_US
dc.subject chain-length regulator en_US
dc.subject polysaccharide co-polymerase en_US
dc.subject O antigen en_US
dc.subject Wzx/Wzy-dependent en_US
dc.subject membrane protein en_US
dc.subject topology mapping en_US
dc.subject C-terminal reporter en_US
dc.subject iodide efflux en_US
dc.subject iodide flux en_US
dc.subject anion flux en_US
dc.subject proteoliposome en_US
dc.subject Wzx en_US
dc.subject Wzy en_US
dc.subject Wzz en_US
dc.subject Wzz1 en_US
dc.subject Wzz2 en_US
dc.subject site-directed mutagenesis en_US
dc.subject protein purification en_US
dc.subject detergent solubilization en_US
dc.subject vesicle reconstitution en_US
dc.subject alkaline phosphatase en_US
dc.subject beta-galactosidase en_US
dc.subject normalized activity ratio en_US
dc.subject antiport en_US
dc.subject GFP en_US
dc.subject topology prediction en_US
dc.subject homology model en_US
dc.subject Western immunoblot en_US
dc.subject silver stain en_US
dc.subject structure prediction en_US
dc.subject proton-dependent gating en_US
dc.subject coupling ion en_US
dc.subject proton uptake en_US
dc.subject catch-and-release mechanism en_US
dc.subject arginine en_US
dc.subject remote homologue detection en_US
dc.subject biochemistry en_US
dc.subject biophysics en_US
dc.subject microbiology en_US
dc.subject structural biology en_US
dc.subject genetics en_US
dc.subject bioinformatics en_US
dc.title Structural and Functional Characterization of O-Antigen Translocation and Polymerization in Pseudomonas aeruginosa PAO1 en_US
dc.type Thesis en_US Molecular and Cellular Biology en_US Doctor of Philosophy en_US Department of Molecular and Cellular Biology en_US
dc.rights.license All items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated. University of Guelph en_US

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