Biosynthesis and export of the D-Galactan I O-antigen of Klebsiella pneumoniae
D-galactan I is an O-antigenic polysaccharide with the disaccharide repeat-unit structure [[right arrow]3-[beta]-D-Gal' f'-(1[right arrow]3)-[alpha]-D-Gal'p'-(1[right arrow]]. This glycan is part of the lipopolysaccharide found in several Gram-negative bacteria, including several 'Klebsiella pneumoniae' O serotypes. One example, 'K. pneumoniae' O2a provides the prototype bacterium for these studies on the biosynthesis and export of D-galactan I. D I is synthesized in the cytoplasm by dedicated galactosyltransferases and is exported to the periplasm via an ATP binding cassette-transporter. In the periplasm, the lipopolysaccharide molecule is completed and then translocated to the cell surface. Work reported in this doctoral thesis investigated the interaction between the biosynthesis and export phases in the assembly of the serotype O2a lipopolysaccharide. A model was established which shows important differences to the previously characterized 'Escherichia coli' O9a prototype. In 'E. coli' O9a, the C-terminus of Wzt determines specificity for the polymannose O-polysaccharide by recognizing a unique structural element at the terminus of the nascent chain. In the 'K. pneumoniae ' O2a model, D-galactan I chain length is determined by coupling chain extension to transport. To further investigate the coordination of the assembly system, the functions of the essential galactosyltransferases and molecular interactions between these enzymes were examined. It was established that three galactosyltransferases are essential for D-galactan I biosynthesis and these enzymes form a multi-enzyme complex. These studies form a framework for investigation of the assembly of diverse glycans structures in other bacteria.