Coordination of Polymerization, Chain Termination, and Export in Assembly of the Escherichia coli Lipopolysaccharide O9a Antigen in an ATP-binding Cassette Transporter-dependent Pathway

Abstract

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 ⌬wbdD 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.

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

“…Two of these target to the membrane independently, whereas the third is absolutely dependent on interaction with a membrane-bound chain terminating protein (47). In the O9a system, there are no detectable inter-glycosyltransferase interactions.…”

Antigenic Variation among Bordetella

“…Interestingly, WbdD interacts with WbdA, and this appears to coordinate chain elongation and chain termination (29). Immediately downstream of wzt in the MO149 wbm cluster is a putative methyltransferase-encoding gene (wbmOO).…”

Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase fromEscherichia coliO9a

“…JX235676) was cloned into pBAD24 as described previously (Clarke et al, 2009). The same cloning strategy was used for the WbdD556 sequence.…”

“…WbdD from Escherichia coli O9a contains two enzymatic domains: a methyltransferase (MTase) domain and a kinase domain (Clarke et al, 2004). WbdD stops polymerization of the chain by phosphorylating and then methylating the phosphate on the terminal sugar (Clarke et al, 2009(Clarke et al, , 2011. The C-terminus of WbdD contains an amphipathic helix that locates WbdD at the cytoplasmic face of the inner membrane, as well as several predicted coiled-coil motifs ( Fig.…”

“…The C-terminus of WbdD contains an amphipathic helix that locates WbdD at the cytoplasmic face of the inner membrane, as well as several predicted coiled-coil motifs ( Fig. 1a; Clarke et al, 2004) in a region that interacts with the sugar polymerase WbdA (Clarke et al, 2009), generating a complex for regulating chain extension and termination.…”