Glucan metabolism in mycobacterium and streptomyces

Miah, Farzana (2014) Glucan metabolism in mycobacterium and streptomyces. Doctoral thesis, University of East Anglia.

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α-Glucans are typically used for carbon storage in bacteria, however, they are also a major component of the mycobacterial capsule. In this context, glucans have been implicated in the evasion of recognition by macrophages. A novel α-glucan synthesis pathway, called the GlgE
pathway, is a potential source of the capsular glucan in mycobacteria. The pathway converts the disaccharide trehalose into α-glucan by the action of four enzymes: TreS, Pep2, GlgE and GlgB. Functional redundancy between other glucan synthesis pathways has made
characterising the GlgE pathway difficult in mycobacteria. The genes encoding the enzymes of the pathway are found in 14% of sequenced bacterial genomes, suggesting the pathway is relatively wide-spread amongst bacteria. α-Glucans have also been isolated in a number of
streptomycetes, which are distant relatives of mycobacteria. In this study Streptomyces venezuelae was used to show for the first time that the GlgE pathway is responsible for glucan synthesis in vivo, as was predicted by the annotations in the genome. A ΔglgE mutant was devoid of α-glucan and instead accumulated α-maltose 1-phosphate, which was associated with a developmental phenotype. The spores produced by the ΔglgE strain had
normal levels of trehalose and had compromised resistance to abiotic stresses, with the exception of desiccation resistance, which was comparable to wild-type. In this study, another mutant from S. venezuelae ΔotsA was also investigated, which was hypothesised to be feeding the substrate trehalose into the GlgE pathway from glucose. However, under normal laboratory growth conditions, no phenotype was observed and the strain was able to accrue α-glucan. Surprisingly, a developmental phenotype arose when ΔotsA was grown on a galactose carbon source. This developmental phenotype was associated with the accumulation of GDP-glucose. Recombinant S. venezuelae OtsA was produced heterologously and the substrate preference for this enzyme was found to be GDP-glucose, as predicted by
the phenotypic study of the ΔotsA strain. The crystal structure of S. venezuelae OtsA was solved to a resolution of 1.95 Å and the unusual substrate specificity was rationalised by comparison with a solved Escherichia coli OtsA structure, which has substrate specificity for UDP-glucose, and a pseudo-glycosyltransferase VldE, which uses GTP during catalysis. Four key resides were identified as being important for the substrate specificity of S. venezuelae OtsA: Ser345, Phe342, Glu341 and Asp340. Finally, TreS was found to be stereospecific for the α anomer of maltose, the appropriate anomer for Pep2, which confirms the prediction of an α-retaining mechanism.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2593 not found.
Date Deposited: 29 Jan 2015 12:18
Last Modified: 12 Dec 2015 01:38


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