No description available.

No description available.

Seventy eight serologically distinct strains of Klebsiella bacteria are known to exist. The capsular polysaccharide surrounding the bacterial cell of these pathogenic Enterobacteria is of immunological significance. Structures of the capsular polysaccharides of nearly sixty seven strains of Klebsiella have been established, and each one found to be unique. The structures of the K antigens from Klebsiella, serotypes K67 and K80 are presented as a contribution to the continuing program of elucidation of the chemical structures of these antigens in an attempt to explain their immunological responses. Chemical methods of structural elucidation were employed and the following two structures were obtained. [formula omitted] The polysaccharide from K67 was unique among the Klebsiella K antigens in having a four-plus-two-plus-one repeating-unit (indicating a branch on a side chain), while K80 was unique as it was the first instance that a pyruvic acetal was found on a terminal rhamnose unit. The importance of bacteriophage-borne enzymes in the generation of single repeating-units containing labile substituents is demonstrated. Klebsiella K44 polysaccharide was degraded using a crude solution of Φ44 bacteriophage. The oligosaccharides obtained were crucial in the determination of the position of the 0-acetate group. In the case of the polysaccharide from Klebsiella K26, the degradation performed using a crude solution of Φ26 bacteriophage resulted in the isolation of a single repeating-unit containing a pyruvic acetal together with an oligosaccharide corresponding to a single repeating-unit devoid of its terminal pyruvate containing sugar. The structures of these compounds which are as follows, were useful in further confirmation of the structures of the original polysaccharides. [formula omitted] Escherichia coli. another pathogenic Enterobacteria possessing immunologically significant K antigens, has been found to contain capsular polysaccharides bearing a strong resemblance to those of Klebsiella. Recently it was discovered that some strains of E. coli contained K antigens comprising amino sugars. A preliminary study on the chemical behaviour of amino sugars, and chemical methods of structure elucidation of such polysaccharides have been included in an appendix as this has been a new area of research in this laboratory. An appendix containing compilations of the cross-reactions, known structures, and chemotypes of the Klebsiella K antigens has also been included. / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

The structural elucidation of bacterial capsular polysaccharides is traditionally performed by using "wet chemical procedures" but instrumental methods such as nuclear magnetic resonance spectroscopy and novel mass spectrometric techniques are coming into prominence. In this thesis three different mass spectrometric techniques were investigated to establish their applicability for the sequencing of bacterial capsular polysaccharides. These techniques included fast atom bombardment (FAB), desorption chemical ionisation (DCI) and laser desorption ionisation Fourier transform ion cyclotron resonance spectroscopy (LDI-FTICR). The soft ionisation produced by these methods allows sequential loss of individual sugar residues without excess thermal decomposition of the ring. Thus sequencing of oligosaccharides could be achieved. The most common of all these three techniques is FAB which is already considered to be a well established form of soft ionisation, although the exact mechanism of ionisation is unknown. The utilisation of DCI has not been thoroughly exploited in carbohydrate research, due to the non-volatility of oligosaccharides and the possible thermal decomposition of the sample in the source. LDI-FTICR due to the general unavailability of the instrument has only been used for model studies of "shelf carbohydrates". In the course of this work it was found that FAB MS and DCIMS complement each other. The sequence of linear oligosaccharides of up to five sugar units can be deduced from either the native or permethylated sample. If the oligosaccharides investigated were generated by phage-borne enzyme, the total sequence of the native polysaccharide can be established. This was illustrated by the use of FABMS on Klebsiella K44 de-O-acetylated oligosaccharide and the reduced oligosaccharide. The sequence of the polysaccharide was shown to be: [Formula Omitted] The structural elucidation of bacterial capsular polysaccharides is traditionally performed by using "wet chemical procedures" but instrumental methods such as nuclear magnetic resonance spectroscopy and novel mass spectrometric techniques are coming into prominence. In this thesis three different mass spectrometric techniques were investigated to establish their applicability for the sequencing of bacterial capsular polysaccharides. These techniques included fast atom bombardment (FAB), desorption chemical ionisation (DCI) and laser desorption ionisation Fourier transform ion cyclotron resonance spectroscopy (LDI-FTICR). The soft ionisation produced by these methods allows sequential loss of individual sugar residues without excess thermal decomposition of the ring. Thus sequencing of oligosaccharides could be achieved. The most common of all these three techniques is FAB which is already considered to be a well established form of soft ionisation, although the exact mechanism of ionisation is unknown. The utilisation of DCI has not been thoroughly exploited in carbohydrate research, due to the non-volatility of oligosaccharides and the possible thermal decomposition of the sample in the source. LDI-FTICR due to the general unavailability of the instrument has only been used for model studies of "shelf carbohydrates". In the course of this work it was found that FAB MS and DCIMS complement each other. The sequence of linear oligosaccharides of up to five sugar units can be deduced from either the native or permethylated sample. If the oligosaccharides investigated were generated by phage-borne enzyme, the total sequence of the native polysaccharide can be established. This was illustrated by the use of FABMS on Klebsiella K44 de-O-acetylated oligosaccharide and the reduced oligosaccharide. The sequence of the polysaccharide was shown to be: [Formula Omitted] The location of acid-labile pyruvic acid acetal group, like base-labile acetate group, is also difficult to establish chemically. The fragment ions arising from permethylated oligosaccharides were mostly non-reducing end residues. This is due to the stability of oxonium ion formation. However, when an amino sugar was investigated, oxonium ions were not observed. Instead the cleavage took place between the glycosidic oxygen and the reducing end residue. This fragmentation route is in sharp contrast to previously reported spectral data. This may be due to the fact that amino sugars strengthen the glycosidic bond between the oxygen and the carbon-1. LDI-FTICR was investigated for its applicability to a "real" sample. The sequence of the linear Klebsiella K44 de-O-acetylated, phage degraded oligosaccharide was determined from the spectrum. Furthermore, a few positions of linkage were also deduced from ring cleavage fragments. Although linkage positions can be obtained from methylation analysis data, some sugar residues such as deoxyhexoses are more labile than others, thus positions of linkage obtained from LDI-FTICR can be used for confirmation. / Science, Faculty of / Chemistry, Department of / Graduate

Many Gram-positive and Gram-negative bacteria produce an additional thick layer of carbohydrate polymers on the cell wall surface. These capsules (capsular polysaccharides; CPS) play critical roles in interactions between bacteria and their environments (Whitfield, 2006). This is especially important in infection processes since for both Gram-negative and Gram-positive pathogens CPS is the point of first contact with the host immune system (Whitfield, 2006). However, the details of CPS biosynthesis and assembly mechanisms are still unclear. Therefore, we embarked on structural and kinetic studies of the proteins Wzc, Wza and Wzb/ Cps4B from the Wzy-dependent pathway, as well as the protein WbdD from the ATP-binding cassette (ABC) transporter dependent system. Full-length Wzc failed to crystallise due to the presence of large disordered regions and the overall difficulty of membrane protein crystallisation. A truncated version of Wzc (1-480) without the C-terminal tyrosine kinase domain was crystallised and diffracted to 15 Å in house. A previous study suggested Wza and Wzc form a functional complex (Whitfield, 2006), so Wza was also studied. Since the full-length Wza structure is available (C. Dong et al., 2006), Pulsed electron–electron double resonance spectroscopy (PELDOR) was used to study the conformational change. The PELDOR spectroscopy distance fingerprint of Wza was determined. These data also confirmed that PELDOR is a powerful tool to study large, highly symmetrical membrane proteins and can be used to study other complex membrane protein systems, such as ion channels or transporters. The crystal structure of Wzb the cognate phosphatase of Wzc was determined to 2.2 Å. Also Cps4B, which is a functional homologue of Wzb but has a completely unrelated sequence, was crystallised in two crystal forms. Form I and II Cps4B crystals diffracted to 2.8 Å and 1.9 Å resolution in house, respectively. The full-length WbdD failed to crystallise due to the presence of large disordered regions. Therefore, a shorter construct, WbdD₅₅₆ (1-556) was cloned and crystallised. The structure was determined to 2.2 Å. WbdD is a bifunctional enzyme consisting of a methyltransferase (MTase) and a kinase domain. In order to better understand the function of this protein, a variety of techniques were used, such as the ADP-Glo kinase assay, Nuclear magnetic resonance (NMR) spectroscopy, small angle X-ray scattering (SAXS) and X-ray crystallography. The various findings in the current projects provide meaningful insights towards a better understanding of the CPS biosynthesis and assembly mechanisms, which may contribute to a more intensive study identifying inhibitors and beginning to unravel the mechanism of chain length regulation.