Files
Abstract
The spore-forming Gram-positive bacteria of the genus Bacillus is present in diverse natural environments. Among Bacilli, we show that members of the Bacillus cereus sensu lato including B. cereus, B. anthracis, and B. thuringiensis produce a myriad of glycan structures. This thesis presents the discovery and elucidation of a few biosynthetic pathways leading to the formation of these glycans in spores, flagella, and biofilm. In Bacillus thuringiensis israelensis ATCC 35646, we found flagellin to be O-glycosylated with pseudaminic acid, and biochemically characterized seven enzymes encoded by the operon PSE, that forms the precursor CMP-pseudaminic acid. Among the seven enzymes, the first two showed unusual enzymatic activities, in the conversion of UDP-D-GlcNAc to UDP-6-deoxy-D-GlcNAc-5,6-ene and then to UDP-4-keto-6-deoxy-L-AltNAc. In other bacterial system studied, these two different activities are catalyzed by a single enzyme. This work also identified a novel exopolysaccharide (EPS) produced during spore formation in many members of Bacillus cereus sensu lato species. This EPS abbreviated pZx, may have roles in spore adherence, dispersal, or anti-aggregation, depending on the pH. The chemical structure of this EPS was characterized by NMR and GC-SM and consists of two uncommon acetamido sugars (XylNAc and GlcNAcA) that form a backbone of [-3)XylNAc4OAc(1-3)GlcNAcA4OAc(1-3)XylNAc(1-]n. The biosynthetic operon XNAC for the production of UDP-XylNAc, UDP-GlcNAcA and the glycosyltransferases were shown to be involved in the formation of this EPS. Lastly, this work found that spores of B. cereus ATCC 14579 and ATCC 10876 are decorated with two rare sugar epimers of C3-methyl-6-deoxy-hexoses. We also identified C3CM operon that is involved in the formation of such decoration and elucidated the metabolic pathway that forms CDP-glucose and transforms it in three enzymatic steps to CDP-C3-methyl-6-deoxy-gulose and CDP-C3-methyl-6-deoxy-allose. Subsequently, we used genetic approaches to find that this operon is required for the addition of these sugars to spore glycoproteins. Mutants impaired with genes in the C3CM operon gave spores that germinate faster. Knowledge of the glycans found on spores, flagella and the extracellular matrix provides a foundation for investigating the roles of glycosylation in motility, survival and pathogenicity, and may explain some physiological differences observed between members of the Bacilli species.