Bacterial capsules are important virulence factors for a number of different pathogens, including Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsular polysaccharides (CPSs) synthesized via the ATP-binding cassette (ABC) transporter-dependent pathway protect these bacteria from complement-mediated killing and phagocytosis, and consist of long polysaccharide chains attached to the cell surface via a phospholipid. CPSs are synthesized on the cytoplasmic face of the inner membrane before transport to the cell surface. While the enzymes that synthesize the polysaccharide have been studied in detail, very little is known about the structure and biosynthesis of the phospholipid terminus. To determine the structure of the reducing terminal glycolipid, CPS from E. coli K1, K5, and N. meningitidis group B was purified using a novel strategy and its structure was determined by mass spectrometry, nuclear magnetic resonance and chemical methods. All three polysaccharides possess terminal lyso-phosphatidylglycerol, which is connected to the CPS repeat unit by a linker consisting of multiple 3-deoxy-D-manno-octulosonic acid (Kdo) residues, forming an alternating β-2,4/β-2,7-linked structure. In addition to describing its structure, the biosynthesis of the glycolipid terminus was also investigated. KpsC and KpsS are conserved proteins encoded in the capsule loci from different bacteria with ABC transporter-dependent capsule assembly pathways but have no previously assigned function. An in vitro assay was developed to characterize KpsSC activities, leading to the finding that they are the Kdo transferases responsible for synthesis of the poly-Kdo linker. This research has contributed significantly to the understanding of the structure and biosynthesis of capsular polysaccharides.

RdoA is a bacterial protein kinase from Salmonella enterica serovar Typhimurium first noted for its regulation of dsbA expression in this organism. The crystal structure of RdoA’s homologue, YihE from Escherichia coli, revealed a basic bi-lobal kinase domain that is a hallmark of the eukaryotic Ser/Thr, Tyr protein kinase superfamily. YihE however, bears the greatest structural similarity to choline kinase and aminoglycoside 3’-phosphotransferase [APH(3’)]-IIIa which are both atypical kinases. RdoA and YihE have demonstrated the capacity for autophosphorylation in vitro and the ability to phosphorylate myelin basic protein, however, the native kinase target protein has not been identified. Based on structural alignment with APH(3’)-IIIa, predictions were made of key residues involved in ATP binding and catalysis and five YihE mutants were generated. Both the wildtype and YihE mutants were cloned for expression as N-terminal histidine-tagged proteins. In the work presented here, these proteins have been overexpressed and purified for further study. Mutational analyses revealed that four of the five mutants had decreased kinase activity in comparison to the wildtype protein, thereby establishing the mutated residues as important for enzymatic activity. Several attempts were made to elucidate the substrate of RdoA/YihE, however, it remains unknown. Further investigation is necessary to identify its substrate(s) and to pinpoint its physiological significance. RdoA is a member of the Cpx regulon and its absence stimulates Cpx activation. Since the Cpx system is involved in regulating expression of cell surface appendages and is one of three envelope stress response systems, it is hypothesized that RdoA serves to relay Cpx activation signals. This is supported by studies on the effect of pH on Cpx activity in wildtype and rdoA- cells presented here. RdoA homologues are present in at least 85 different genera. This level of conservation is indicative of an important biological role for this previously uncharacterized bacterial protein kinase. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-12-04 18:19:29.574

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An <i>Escherichia coli</i> strain carrying the <i>phs</i> mutation was isolated by other workers who were interested in determining the role of the Na /H antiport in pH homeostasis. The mutation was observed to cause impaired growth on L-glutamate amd melibiose and was also reported to cause a sensitivity to alkaline conditions due to an impairment in pH homeostasis. Since uptake of both glutamate and melibiose is energised by a Na⁺ gradient, these observations were interpreted as evidence that the <i>phs</i> mutation impairs Na⁺ /H⁺ antiport activity, and so by implication, that the Na⁺ /H⁺ antiport is involved in pH homeostasis in <i>E. coli</i>. In work for this thesis, evidence was accumulated that the <i>phs</i> mutation does not affect Na⁺ /H⁺ antiport activity. Proline uptake, which is energised by a Na⁺ gradient, was found to be normal in a mutant strain. Also, arabinose uptake via two different transport systems, neither of which is energised by a Na⁺ gradient, was found to be impaired by the mutation. K⁺ uptake by Na⁺ loaded cells, a phenomenon which is thought to give a measure of Na⁺ /H⁺ antiport activity, was not affected by the mutation. Studies on growth under alkaline conditions indicated that growth is not impaired by the mutation. Evidence was obtained however, that the <i>phs</i> mutation causes filamentation under these conditions. There is a possibility that this may be due to an effect on the penicillin-binding protein content of the cell envelope. Investigations of the effect of the mutation on pH homeostasis suggested that it affects either pH homeostasis, or the validity of the techniques used for determining the cytoplasmic pH. In view of the mapping of the mutation to the gene coding for the α-subunit of RNA polymerase (Rowland <i>et al</i>, 1985), the effects of the mutation on transcription were studied. It was found that the mutation dramatically impairs transcription of genes under the positive regulation of the <i>AraC</i> gene product. An examination of the effect of the mutation on transcription from a variety of different promotors showed that the transcription defect is highly selective. Some evidence was obtained that the mutation impairs the process of positive regulation. To test this, the effect of the mutation on transcription from AraC independent derivatives of an AraC dependent promoter was determined. In the absence of AraC, the mutation had no effect. In the presence of AraC, complex effects were observed. It was concluded that the mutation affects the process of positive regulation or CRP-cAMP mediated derepression of this promoter.