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J. Biol. Chem., Vol. 282, Issue 37, 26939-26947, September 14, 2007

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Biochemical Characterization of MsbA from Pseudomonas aeruginosa^*

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Lipopolysaccharide of Pseudomonas aeruginosa is a major constituent of the outer membrane, and it is composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. Lipid A and core oligosaccharides (OS) are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of the membrane where lipid A-core becomes the acceptor of the O antigens. Here we show that MsbA encoded by pA4997 of the P. aeruginosa genome is a member of the ABC transporter family, but this protein has distinctive features when compared with other MsbA proteins. msbA is an essential gene in this organism since mutation in this gene is lethal to the bacterium. Disruption of the chromosomal msbA was achieved only when a functional copy of the gene was provided in trans. msbA from Escherichiacoli (msbA_Ec) could not cross complement the msbA merodiploid cells of P. aeruginosa. MsbA was expressed and purified, and the kinetic of its ATPase activity is vastly different than that of MsbA_Ec. The activity of MsbA could be selectively stimulated by different truncated versions of core OS of P. aeruginosa LPS. Specifically, phosphate substituents in the lipid A-core are important for stimulating ATPase activity of MsbA. Expression of MsbA_Ec but not MsbA_Pa conferred resistance to erythromycin in P. aeruginosa.

Received for publication, April 6, 2007 , and in revised form, July 18, 2007.

^* This work was supported by operating grants (to J. S. L.) from the Canadian Cystic Fibrosis Foundation (CCFF) and the Canadian Institutes of Health Research (MOP 14687). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Table S1.

¹ Recipient of a CCFF postdoctoral fellowship.

² Holds a Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology. 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. 53823; Fax: 519-837-1802; E-mail: jlam{at}uoguelph.ca.

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