Abstract
Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a β-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal β-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures.
Highlights
Cell-surface carbohydrates are essential for the viability and pathogenicity of bacteria
The details of ATP-binding cassette (ABC) transporter architecture vary between systems; the two halves of the transmembrane domain (TMD) and nucleotide binding domains (NBDs) domains can either be identical or encoded by different genes, and the various domains can be present as separate polypeptides or fused
Terminating -Kdo Residue Is Required for O-antigenic polysaccharide (O-PS) Export— Biosynthesis of the O12 OPS requires glycosyltransferase (GT) activity provided by the WbbL and WbbB proteins [26]
Summary
Cell-surface carbohydrates are essential for the viability and pathogenicity of bacteria. A typical O-PS ABC transporter is composed of two identical TMDs (Wzm proteins) and two identical NBDs (Wzt proteins) They export undecaprenyl diphosphate (UndPP)-linked completed O-PS glycans across the cytoplasmic membrane. The terminator dictates glycan chain length and serves as an export signal recognized by the ABC transporter. In this mechanism, synthesis and export are not obligatorily coupled [8]. ABC Transporter-dependent O-Antigen Export hydrate binding module (CBM) appended to the C terminus of the NBD of the cognate ABC transporter [15, 16] As their name implies, CBMs are non-catalytic polysaccharide/oligosaccharide-recognizing subdomains of many glycosidic enzymes and lectins. It is not classified on the CAZy server because Wzt has no modifying activity directed against a carbohydrate substrate
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