Title page, abstract and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / Salmonella is a genus of Gram-negative bacteria responsible for food-borne enteritis and systemic fever. Salmonellosis continues to be a serious health burden worldwide. Lipopolysaccharide (LPS) is the dominant lipid component of the outer membrane of Salmonella. LPS is composed of lipid A, an oligosaccharide core, and a polymer of O units known as O antigen. Wzz proteins control the length of O antigen by an unknown mechanism. While O antigen is essential for S. typhimurium virulence, the significance of length regulation by Wzz is not known. This study investigates the pathogenic relevance of the wzz genes in S. typhimurium. In addition to the previously recognised wzz gene (WZZ[subscript]ST), a second gene with this function (WZZ[subscript]fepE) was identified in the S. typhimurium genome. Whereas WZZ[subscript]ST specifies the production of long LPS chains with a modal length of 16-35 O antigen repeat units, WZZ[subscript]fepE conferred very long chains containing> 100 O antigen repeat units. Strains carrying mutations in one or both wzz genes were constructed to investigate the role of wzz genes in virulence. It was found that wzz-controlled regulation of O antigen length was essential for resistance to the bactericidal activity of serum complement, while studies in the mouse model of infection found that wzz genes are essential for full virulence. The wzz double mutant was complemented with heterologous wzz genes from a variety of bacterial sources. Despite variable sequence similarity of the encoded Wzz proteins each was functional in the S. typhimurium host, generating a panel of isogenic O antigen length variants. This panel of variants was used to define a minimum length requirement for complement resistance and identified relationships between O antigen length and complement consumption. Finally, the regulation of O antigen chain length was investigated. It was found that growth either in iron limiting conditions or in serum caused an increased production of LPS with very long O antigen chains (conferred by WZZ[subscript]fepE), resulting in increased complement resistance. The constitutive activation mutation of the phoPQ regulator (Phop) results in an altered O antigen distribution phenotype consistent with down-regulation of wzz genes. However PhoPQ had no effect on production of Wzz[subscript]sT as determined by immunoblotting with an antiWZZ[subscript]ST antiserum. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1152141 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005

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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. / 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. / 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