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J. Biol. Chem., Vol. 280, Issue 30, 27604-27612, July 29, 2005

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The Role of Galacturonic Acid in Outer Membrane Stability in Klebsiella pneumoniae^*

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

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph Ontario N1G 2W1, Canada and ^¶Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada

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 gla_KP. Gla_KP is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The gla_KP gene was inactivated and the structure of the mutant lipopolysaccharide 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 -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 E. coli and Salmonella.

Received for publication, May 5, 2005 , and in revised form, May 31, 2005.

^* This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Bacterial Diseases Network (to C.W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by postgraduate scholarships from Natural Sciences and Engineering Research Council of Canada and Canadian Institute of Health Research.

^|| Holder of a Canada Research Chair. To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Tel.: 519-824-4120 (ext. 53361); Fax: 519-837-1802; E-mail: cwhitfie{at}uoguelph.ca.

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