Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza.
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.
Nesper
Jutta
J
Department of Microbiology, University of Guelph, Guelph, Ontario N1G2W1, Canada.
Hill
Chris M D
CM
Paiment
Anne
A
Harauz
George
G
Beis
Konstantinos
K
Naismith
James H
JH
Whitfield
Chris
C
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001435:Bacteriophages / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D019897:Periplasm / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011134:Polysaccharides; C417168:WZC protein, Bacteria; C404158:Wza protein, E coli; D006639:Histidine; D011505:Protein-Tyrosine Kinases; C499615:wzc protein, E coli