Publications

• Coordination of polymerization, chain termination, and export in assembly of the Escherichia coli lipopolysaccharide O9a antigen in an ATP-binding cassette transporter-dependent pathway.

  Clarke B.R., Greenfield L.K., Bouwman C., Whitfield C.

  The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for O-PS synthesis and export by the ATP-binding cassette transporter-dependent pathway. Comparable systems are widespread in Gram-negative bacteria. The polymannose O9a O-PS is assembled on a polyisoprenoid lipid intermediate by mann ... >> More

  The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for O-PS synthesis and export by the ATP-binding cassette transporter-dependent pathway. Comparable systems are widespread in Gram-negative bacteria. The polymannose O9a O-PS is assembled on a polyisoprenoid lipid intermediate by mannosyltransferases located at the cytoplasmic membrane, and the final polysaccharide chain length is determined by the chain terminating dual kinase/methyltransferase, WbdD. The WbdD protein is tethered to the membrane via a C-terminal region containing amphipathic helices located between residues 601 and 669. Here, we establish that the C-terminal domain of WbdD plays an additional pivotal role in assembly of the O-PS by forming a complex with the chain-extending mannosyltransferase, WbdA. Membrane preparations from a DeltawbdD mutant had severely diminished mannosyltransferase activity in vitro, and no significant amounts of the WbdA protein are targeted to the membrane fraction. Expression of a polypeptide comprising the WbdD C-terminal region was sufficient to restore both proper localization of WbdA and mannosyltransferase activity. In contrast to WbdA, the other required mannosyltransferases (WbdBC) are targeted to the membrane independent of WbdD. A bacterial two-hybrid system confirmed the interaction of WbdD and WbdA and identified two regions in the C terminus of WbdD that contributed to the interaction. Therefore, in the O9a assembly export system, the WbdD protein orchestrates the critical localization and coordination of activities involved in O-PS chain extension and termination at the cytoplasmic membrane. << Less

  J. Biol. Chem. 284:30662-30672(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan.

  Clarke B.R., Richards M.R., Greenfield L.K., Hou D., Lowary T.L., Whitfield C.

  The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located a ... >> More

  The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [γ-(33)P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-(3)H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit. << Less

  J. Biol. Chem. 286:41391-41401(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Nonreducing terminal modifications determine the chain length of polymannose O antigens of Escherichia coli and couple chain termination to polymer export via an ATP-binding cassette transporter.

  Clarke B.R., Cuthbertson L., Whitfield C.

  The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis. This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread amon ... >> More

  The chain length of bacterial lipopolysaccharide O antigens is regulated to give a modal distribution that is critical for pathogenesis. This paper describes the process of chain length determination in the ATP-binding cassette (ABC) transporter-dependent pathway, a pathway that is widespread among Gram-negative bacteria. Escherichia coli O8 and O9/O9a polymannans are synthesized in the cytoplasm, and an ABC transporter exports the nascent polymer across the inner membrane prior to completion of the LPS molecule. The polymannan O antigens have nonreducing terminal methyl groups. The 3-O-methyl group in serotype O8 is transferred from S-adenosylmethionine by the WbdD(O8) enzyme, and this modification terminates polymerization. Methyl groups are added to the O9a polymannan in a reaction dependent on preceding phosphorylation. The bifunctional WbdD(O9a) catalyzes both reactions, but only the kinase activity controls chain length. Chain termination occurs in a mutant lacking the ABC transporter, indicating that it precedes export. An E. coli wbdD(O9a) mutant accumulated O9a polymannan in the cytoplasm, indicating that WbdD activity coordinates polymannan chain termination with export across the inner membrane. << Less

  J. Biol. Chem. 279:35709-35718(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.