Lipopolysaccharide inner core oligosaccharide biosynthesis and outer membrane stability in Escherichia coli and Klebsiella pneumoniae

In Gram-negative bacteria, the outer membrane (OM) is primarily comprised of lipopolysaccharides (LPS). The LPS molecule is involved in mediating interactions between the bacterium and its environment. The regions of the molecule extending further away from the cell surface show a higher amount of structural diversity. LPS can be conceptually divided into three regions: (1) lipid A; (2) the core oligosaccharide (OS) attached to lipid A; and (3) the O polysaccharide. The inner core OS (lipid A proximal) backbone is also well conserved, consisting of 3-deoxy-D- manno-oct-2-ulosonic acid (Kdo) and heptose sugar residues. However, non-stoichiometric substitutions of the basic inner core structure lead to structural variation and microheterogeneity. These include the addition of negatively-charged groups, ethanolamine derivatives, and glycose residues. Modification of heptose residues with phosphate in Escherichia coli and Salmonella has been shown to be involved in maintaining OM stability. However, the biological role(s) of the remaining substitutions is unknown. One of the main objectives of this work was to examine the genetics and biosynthesis of these inner core substitutions in Escherichia coli. The WaaZ and WaaS gene products were shown to be involved in the transfer of a KdoIII and a rhamnose residue, respectively, to the inner core KdoII. Addition of a KdoIII substituent was also shown to produce incomplete core molecules, leading to heterogeneous core structures on the cell surface. A second objective focuses on studying the core region of K. pneumoniae LPS whose structure is quite distinct from that of E. coli and Salmonella. The transferases involved in outer core biosynthesis were characterized. In addition, the role of certain core OS residues in OM integrity was established. K. pneumoniae lacks the phosphate moieties that are involved in E. coli and Salmonella OM stability. Instead, it uses the negative charge provided by galacturonic acid residues. A mutant lacking these galacturonic acid residues shows alterations in OM structure and composition leading to an increase in permeability. This work looked at core OS structural variation in E. coli and K. pneumoniae and established the importance of certain core OS residues in creating core microheterogeneity and in OM integrity.