Lipopolysaccharide synthesis and characterization of a core oligosaccharide kinase essential to outer membrane stability

In 'Escherichia coli' and 'Salmonella enterica', genes for assembly of the lipopolysaccharide (LPS) core region are clustered on the chromosome in three adjacent operons. The central of these operons encodes glycosyltransferases and other enzymes responsible for assembling one of seven distinct core structures. In 'E. coli' with core type R1, there are nine "'waa'" genes in the central core biosynthesis operon. In order to assign function to each of these genes, non-polar mutations were constructed in each, and the resulting mutant LPS core structures were determined by chemical methods. In 'E. coli' and 'S. enterica', phosphoryl substituents in the LPS core region are essential for the formation of a stable outer membrane. Of particular note, therefore, mutations in four genes characterized by the above approach were shown to affect the degree of core phosphorylation. Two of these genes ('waaY' and 'waaP') were presumed to be involved directly in phosphotransfer reactions based on their weak homology to eukaryotic kinases. The remaining two genes ('waaQ ' and 'waaG') were shown to encode glycosyltransferases acting near phosphorylation sites, and are thus likely required to fulfill the substrate specificity requirements of the phosphorylating enzymes. Mutant derivatives with altered core phosphorylation were analyzed for outer membrane stability by novobiocin and SDS sensitivity testing, and were shown to be more susceptible to these agents than the parent strain. In general, decreased core phosphate substitution resulted in greater membrane instability. In particular, mutation of 'waaP', resulting in a core completely devoid of phosphate, caused a dramatic increase in antibiotic susceptibility. To assess how WaaP and LPS core phosphorylation influence the biology of an intracellular pathogen, a 'waaP' mutant was constructed using an invasive strain of 'S. enterica', and this mutant was tested for virulence using mouse models of infection. The results were unequivocal; mutation of 'waaP' completely abrogated virulence. Given these data, the WaaP protein provides a novel potential target for the development of antibiotics. To further characterize WaaP, the protein was purified, and its enzymatic activity was reconstituted 'in vitro '. Purified WaaP was shown to catalyze the incorporation of 33P from [[gamma]-33P]ATP into phosphate-deficient acceptor LPS.

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