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Papers In Press, published online ahead of print June 1, 2005
J. Biol. Chem, 10.1074/jbc.M504987200
Submitted on May 5, 2005
Revised on May 31, 2005
Accepted on June 1, 2005

The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae

Emilisa Frirdich, Catrien Bouwman, Evgeny Vinogradov, and Chris Whitfield

Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1

Corresponding Author: cwhitfie{at}uoguelph.ca

In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in glaKP. GlaKP is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The glaKP gene was inactivated and the structure of the mutant LPS was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme ß-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in Escherichia coli and Salmonella.


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