A Novel Outer Membrane Protein, Wzi, Is Involved in Surface Assembly of theEscherichia coliK30 Group 1 Capsule

Abstract

Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface. Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface. Group 1 capsule biosynthesis gene clusters (cps) in E. coli and Klebsiella pneumoniae include a conserved open reading frame, wzi. The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes. Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides. The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence. Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane. Circular dichroism spectroscopy indicates that Wzi consists primarily of a ␤-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution. A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage. However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material. Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure. It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface.

Klebsiella pneumoniae: Going on the Offense with a Strong Defense

“…This locus is present in all capsular types of K. pneumoniae, and different wzi locus sequences are strongly associated with specific K antigens. The wzi gene encodes a surface protein involved in capsule attachment to the outer membrane, where the loss of this protein results in essentially acapsular bacteria (211,212). wzy, also known as orf4, is involved in the polymerization of capsular polysaccharides, while wza, wzc, orf5, and orf6 are involved in their surface assembly (202,213).…”

wzi Gene Sequencing, a Rapid Method for Determination of Capsular Type for Klebsiella Strains

“…The cps locus has a mosaic structure, with a group of six conserved genes (galF, orf2, wzi, wza, wzb, and wzc) at its 5= end. Among these, the wzi gene codes for the outer membrane protein Wzi, which is involved in attachment of the capsular polysaccharide to the outer membrane (27,28). The objective of this study was to evaluate the sequence polymorphisms of the wzi gene for strain typing and for inference of the serotypes of K. pneumoniae clinical isolates.…”

Identification of an Escherichia coli Operon Required for Formation of the O-Antigen Capsule

“…However, in contrast to LPS, G4Cs are composed of a high-molecular-weight polysaccharide attached to the bacterial surface not via lipid but probably by interaction with a specific surface protein(s) (29). We previously showed that EPEC O127:H6 expresses the etk gene, which encodes a protein tyrosine kinase (17).…”