PMID:14522970

Citation	Nesper, J, Hill, CM, Paiment, A, Harauz, G, Beis, K, Naismith, JH and Whitfield, C (2003) Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza. J. Biol. Chem. 278:49763-72
Abstract	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. WzaHis6 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 WzaHis6 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 WzaHis6 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.
Links	PubMed Online version:10.1074/jbc.M308775200
Keywords	Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/physiology; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Bacteriophages/metabolism; Cell Membrane/enzymology; Cell Membrane/metabolism; Cell Membrane/ultrastructure; Cross-Linking Reagents/pharmacology; Crystallography, X-Ray; Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism; Escherichia coli Proteins; Histidine/chemistry; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Microscopy, Electron; Models, Biological; Periplasm/metabolism; Phenotype; Plasmids/metabolism; Polysaccharides/chemistry; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases/chemistry; Protein-Tyrosine Kinases/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:WZA		GO:0015159: polysaccharide transmembrane transporter activity	ECO:0000315:		F		Fig. 2-Wza derivatives were detected in the western immunoblots and the electron micrograph clearly shows capsule assembly on the surface of cells with Wza genes being expressed.	complete CACAO 4895

References

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