Functional Analysis of Conserved Gene Products Involved in Assembly ofEscherichia coliCapsules and Exopolysaccharides: Evidence for Molecular Recognition between Wza and Wzc for Colanic Acid Biosynthesis

Abstract

Group 1 capsular polysaccharides (CPSs) of Escherichia coli and some loosely cell-associated exopolysaccharides (EPSs), such as colanic acid, are assembled by a Wzy-dependent polymerization system. In this biosynthesis pathway, Wza, Wzb, and Wzc homologues are required for surface expression of wild-type CPS or EPS. Multimeric complexes of Wza in the outer membrane are believed to provide a channel for polymer export; Wzc is an inner membrane tyrosine autokinase and Wzb is its cognate phosphatase. This study was performed to determine whether the Wza, Wzb, and Wzc proteins for colanic acid expression in E. coli K-12 could function in the E. coli K30 prototype group 1 capsule system. When expressed together, colanic acid Wza, Wzb, and Wzc could complement a wza-wzb-wzc defect in E. coli K30, suggesting conservation in their collective function in Wzy-dependent CPS and EPS systems. Expressed individually, colanic acid Wza and Wzb could also function in K30 CPS expression. In contrast, the structural requirements for Wzc function were more stringent because colanic acid Wzc could restore translocation of K30 CPS to the cell surface only when expressed with its cognate Wza protein. Chimeric colanic acid-K30 Wzc proteins were constructed to further study this interaction. These proteins could restore K30 biosynthesis but were unable to couple synthesis to export. The chimeric protein comprising the periplasmic domain of colanic acid Wzc was functional for effective K30 CPS surface expression only when coexpressed with colanic acid Wza. These data highlight the importance of Wza-Wzc interactions in group 1 CPS assembly.Escherichia coli capsules (comprising capsular polysaccharides [CPSs]) are acidic polysaccharide layers surrounding and tightly associated with the surface of the cell. They are recognized virulence determinants and act by increasing adherence to host tissues and conferring resistance to phagocytosis. In some cases, the host immune response against the CPS is impaired because the capsule structure mimics host cell components. More than 80 distinct capsular or K antigens in E. coli have been described, and these are classified into four groups on the basis of genetic and biosynthetic criteria (61). These capsule groups provide prototypes for CPSs and the loosely associated exopolysaccharides (EPSs) produced by many other bacteria.Group 1 capsules and related polysaccharides are found in pathogens of humans and animals and in plant-associated bacteria, where they enhance virulence or mediate symbiosis. Group 1 CPSs and related EPSs are assembled by a Wzydependent polymerization system. The early stages of synthesis are identical to those of lipopolysaccharide biosynthesis, where the action of the Wzy-dependent system is best understood (reviewed in reference 48). In the present model, lipid-linked repeat units are built up on undecaprenol diphosphate at the cytoplasmic face of the inner membrane by the sequential activity of glycosyltransferases. The lipid-linked repeat units are then flipped to the perip...

Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins

“…Therefore, the arrangement of secondary structure elements indicates that CpsC has a fold similar to the base domain of PCP1 proteins, with a rather short ahelical hairpin instead of the long a-helical domain of PCP1s. CpsC is found in Gram-positive organisms that do not face the challenge of exporting polysaccharides across a second membrane, perhaps accounting for its lack of a domain protruding far from the membrane, as previously noted [20,33].…”

Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

“…It is proposed that a water-filled channel would ensure that the polar amino acid side chains and the hydroxyl groups of the polymer make hydrogen bonds, essentially lubricating the channel (26). This would explain the observed lack of specificity of representative OPX channels for a particular polysaccharide repeat-unit structure (91). The smallest internal diameter (R4; 17 Å) is still sufficient to accommodate a polymer in an extended conformation.…”

“…Another close homolog, Wza from the colanic acid EPS system of E. coli, is also a member of OPX group A. The colanic acid Wza homolog can functionally replace its homolog in the K30 biosynthesis pathway (91). Colanic acid is found in many E. coli isolates but not in those with group 1 CPS (47); it is essentially a highly regulated version of a group 1 CPS that is activated by environmental cues, mostly signals outside the host.…”

“…Like Wza, Wzc is also essential for export and assembly of CPS (27,83,91,117). PCP proteins were initially identified in lipopolysaccharide (LPS) O-antigen biosynthesis systems in which PCP-1 representatives (often encoded by wzz genes) are involved in the determination of chain length (89) (see below).…”

The UDP-glucose Dehydrogenase of Escherichia coli K-12 Displays Substrate Inhibition by NAD That Is Relieved by Nucleotide Triphosphates

“…K30 CPS production was monitored by immunoblotting of proteinase K-treated whole-cell lysates (39). SDS-PAGE and immunoblotting with anti-K30 antibodies (40) were performed as described previously (41).…”