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J. Biol. Chem., Vol. 278, Issue 50, 49763-49772, December 12, 2003

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Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE OUTER MEMBRANE LIPOPROTEIN Wza^*

Jutta Nesper, Chris M. D. Hill, Anne Paiment¶, George Harauz, Konstantinos Beis||, James H. Naismith||**, and Chris Whitfield

From the Department of Microbiology, University of Guelph, Guelph, Ontario N1G2W1, Canada, the Department of Molecular Biology & Genetics, University of Guelph, Guelph, Ontario N1G2W1, Canada, and the ^||Centre for Biomolecular Sciences, North Haugh, The University, St. Andrews, Fife KY16 9ST, Scotland, United Kingdom

The late steps in assembly of capsular polysaccharides (CPS) and their translocation to the bacterial cell surface are not well understood. The Wza protein was shown previously to be required for the formation of the prototype group 1 capsule structure on the surface of Escherichia coli serotype K30 (Drummelsmith, J., and Whitfield, C. (2000) EMBO J. 19, 57–66). Wza is a conserved outer membrane lipoprotein that forms multimers adopting a ringlike structure, and collective evidence suggests a role for these structures in the export of capsular polymer across the outer membrane. Wza was purified in the native form and with a C-terminal hexahistidine tag. Wza_His6 was acylated and functional in capsule assembly, although its efficiency was slightly reduced in comparison to the native Wza protein. Ordered two-dimensional crystals of Wza_His6 were obtained after reconstitution of purified multimers into lipids. Electron microscopy of negatively stained crystals and Fourier filtering revealed ringlike multimers with an average outer diameter of 8.84 nm and an average central cavity diameter of 2.28 nm. Single particle analysis yielded projection structures at an estimated resolution of 3 nm, favoring a structure for the Wza_His6 containing eight identical subunits. A derivative of Wza (Wza*) in which the original signal sequence was replaced with that from OmpF showed that the native acylated N terminus of Wza is critical for formation of normal multimeric structures and for their competence for CPS assembly, but not for targeting Wza to the outer membrane. In the presence of Wza*, CPS accumulated in the periplasm but was not detected on the cell surface. Chemical cross-linking of intact cells suggested formation of a transmembrane complex minimally containing Wza and the inner membrane tyrosine autokinase Wzc.

Received for publication, August 8, 2003 , and in revised form, September 29, 2003.

^* This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) (to C. W.) and from the Natural Sciences and Engineering Research Council of Canada (NSERC) (to G. H.). 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 a CIHR postgraduate studentship.

^** Supported by a Biotechnology and Biological Sciences Research Council (United Kingdom) career development fellowship.

Supported by a Canada Research Chair. To whom correspondence should be addressed. E-mail: cwhitfie{at}uoguelph.ca.

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