Coordination of polymerization, chain termination, and export in assembly of the Escherichia coli lipopolysaccharide O9a antigen in an ATP-binding cassette transporter-dependent pathway.
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.
Clarke
Bradley R
BR
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Greenfield
Laura K
LK
Bouwman
Catrien
C
Whitfield
Chris
C
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001692:Biological Transport; D004731:Endotoxins / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000378:metabolism; D008364:Mannosyltransferases; D053858:Metabolic Networks and Pathways; D008780:Methyltransferases; D011485:Protein Binding
D018528:ATP-Binding Cassette Transporters; D004731:Endotoxins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; C044101:endotoxin, Escherichia coli; D008780:Methyltransferases; D008364:Mannosyltransferases