Biosynthesis of the lipopolysaccharide core oligosaccharide region of Escherichia coli and Salmonella

The Gram-negative outer membrane is an essential component in bacterial structure, necessary for survival and protection. Lipopolysaccharide (LPS) is an abundant glycolipid that forms the outer-most layer of the outer membrane. The core oligosaccharide (core OS) links lipid A and the distal repeating-unit polysaccharide (O antigen) and together, these components make up the LPS molecule. Genes within the chromosomal waa locus encode the majority of glycosyltransferases required for core OS assembly. This thesis describes research aimed at understanding the structure and function of the waa loci.; The R3 core OS predominates in the LPS from enterohemorrhagic E. coli isolates including O157:H7. The R3 waa locus contains waaD and waaJ, that are predicted to encode glycosyltransferases involved in completion of the core OS. Through determination of structures of the LPS core in precise mutants and biochemical analyses of enzyme activities, WaaJ was shown to be a UDP-glucose:(galactosyl) LPS alpha-1,2-glucosyltransferase and WaaD a UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase. The residue added by WaaJ was identified as the ligation site for O antigen. The gene responsible for a non-stoichiometric alpha-1,7-linked N-acetylglucosamine substituent in inner core was identified on the large virulence plasmid of E. coli O157. This is the first plasmid-encoded core OS biosynthesis enzyme reported in E. coli.; There are two known core structures in Salmonella, represented by serovars Typhimurium and Arizonae IIIa. The waa locus for sv. Typhimurium has been characterized. Here the corresponding locus from sv. Arizonae is described and the molecular basis for the distinctive structures are established. Isolates comprising Salmonella Reference Collections were examined to assess the distribution of the waa locus polymorphic regions in natural populations. These comparative studies shed light on the genetic basis for diversity in the core OS and identified novel structures.; The ligation of O antigen to lipid A-core is a late step in the formation of LPS. To date, there is only one gene product known to be required. The structural requirements in the core OS acceptor for O-antigen ligation are investigated in prototype serovars of Salmonella enterica. The data provide the first indication of a more complicated recognition process involving additional cellular components.