Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

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. Wza His 6 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 His 6 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 His 6 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.In Gram-negative bacteria, macromolecules destined for the cell surface or the extracellular environment must cross both the inner (IM) 1 and the outer membrane (OM). In protein export, where the processes are arguably best understood, secretion systems with varying complexity accomplish the translocation steps; all involve multi-enzyme complexes where an outer membrane protein (channel) is linked directly, or via helper proteins, to IM components.Cell-associated capsular polysaccharides (CPS) and their secreted (cell-free) counterparts, exopolysaccharides (EPS), represent another type of macromolecule that must be transported across the cell envelope. The early steps in assembly of these polymers are reasonably well established. However, there is little understanding of the terminal steps, including the mechanism by which they cross the bacterial cell envelope and the machinery involved.In Escherichia coli, more than 80 antigenically distinct capsular (K) polysaccharide structures are recognized. They are divided into four groups based on the organization of their genetic loci, polymerization mechanisms, and regulation (1). Group 1 and 2 capsules have received the most attention, and they involve biosynthet...

Three-dimensional Structure of Wza, the Protein Required for Translocation of Group 1 Capsular Polysaccharide across the Outer Membrane of Escherichia coli

“…The structure is entirely consistent with a model in which Wza interacts with K antigen synthesis components located at the inner membrane. Preliminary biochemical evidence suggests protein-protein interaction with the inner membrane protein Wzc (13). This would be similar to the interactions of secretins (such as MxiD) with the inner membrane components in type III needle complex structures (45,46).…”

“…This gating may be triggered either by substrate or interaction with another protein. One candidate is Wzc, a protein that can be cross-linked to Wza in a higher order complex (13). This structural information for Wza multimers provides a starting point to investigate the nature of the Wza complex and the impact of protein-protein interactions on its function.…”

“…At a gross structural level, Wza multimers resemble the secretins such as PilQ (18), pIV (19), PulD (20), XcpQ (21), and YscC (22), which export proteins in Neisseria sp., Pseudomonas aeruginosa, Klebsiella oxytoca, P. aeruginosa, and Yersinia sp., respectively. However, these similarities do not extend to the primary sequence features (13). Nevertheless, the higher order structural similarities suggest that detailed studies of one protein will provide information on the biology of the other systems.…”

“…The Wzi ␤-barrel protein acts late in the assembly process and appears to be involved in surface association (but not synthesis) of capsular polymer, although its exact role is unknown (13). Wza proteins are lipoproteins that form multimeric outer membrane complexes that are involved in translocation of the capsular polymer across the outer mem-brane (6,13). Group 2 capsules are synthesized by a heterooligomeric membrane-bound biosynthetic complex on the inner face of the cytoplasmic membrane by the sequential action of glycosyltransferases that elongate the polysaccharide at its non-reducing end (14).…”

“…Two outer membrane proteins are involved in K30 assembly. The Wzi ␤-barrel protein acts late in the assembly process and appears to be involved in surface association (but not synthesis) of capsular polymer, although its exact role is unknown (13). Wza proteins are lipoproteins that form multimeric outer membrane complexes that are involved in translocation of the capsular polymer across the outer mem-brane (6,13).…”