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 ΔwbdD 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.Lipopolysaccharide (LPS)³ is a glycolipid unique to the outer membranes of Gram-negative bacteria. LPS has three structural domains in most bacteria (1). Hydrophobic lipid A is a major component of the outer leaflet of the outer membrane. A short core oligosaccharide (OS) serves as a linker between lipid A and a repeat unit polymer termed the O-polysaccharide (O-PS; O-antigen). The structure of lipid A is conserved among Gram-negative bacteria, whereas limited variability is observed among the core OSs of a given species. For example, five closely related core oligosaccharides have been described for Escherichia coli (2). In contrast, the O-PS structures vary extensively within species. O-PS structural variations include differences in the number and type of sugars in the repeat unit and the nature of the glycosidic linkages within and between repeat units. O-PS variations provide the basis for the O-antigen serotyping system, and there are over 180 O-antigen serogroups proposed for E. coli (3, 4).Lipid A-core OS and O-PS are synthesized independently at the cytoplasmic membrane and are subsequently linked together in the periplasm (reviewed in Ref. 1). O-PS assembly is initiated by transfer of a sugar-1-phosphate from a nucleotide sugar precursor to the 55-carbon lipid acceptor, undecaprenol phosphate. In the majority of E. coli serotypes, the initiating reaction is performed by the GlcNAc:Und-P GlcNAc-1-P transferase, WecA (5, 6). WecA is an integral membrane protein and is also essential for initiating synthesis of the enterobacterial common antigen (7). In E. coli, elongation and export of the undecaprenol-PP-linked intermediate proceeds through one of two fundamentally different O-PS assembly pathways. These pathways have been termed Wzy (polymerase)-dependent and ATP-binding cassette (ABC) transporter-dependent biosynthesis, respectively (reviewed in Ref. 1). In Wzy-dependent O-PS biosynthesis, single repeat units are assembled on the undecaprenol-PP-linked intermediate at the cytoplasmic face of the inner membrane. The lipid-linked repeat units are subsequently reoriented to the periplasm where they are assembled into polysaccharide by a process involving Wzy and a chain length regulator, Wzz. In contrast, in the ABC transporter-dependent pathway, the O-PS is elongated on the undecaprenol-PP-linked intermediate in the cytoplasm by sequential glycosyl transfer. Depending on the system, chain extension is terminated by the addition of a nonreducing terminal residue or by interaction with the ABC transporter (8). Full-length O-PS chains are then translocated across the inner membrane by the ABC transporter. The two O-PS assembly pathways converge at a ligation reaction, which transfers the O-PS from undecaprenol-PP to lipid A-core OS at the periplasmic face of the inner membrane. Once assembled, LPS molecules are shuttled to the outer membrane through a process involving the LptABCDE complex (reviewed in Ref. 9).The polymannose O-PS of E. coli O9a provides a model system for ABC transporter-dependent O-PS biosynthesis. The E. coli O9a PS biosynthesis gene cluster (see Fig. 1A) encodes three GDP-mannose-dependent mannosyltransferases (WbdA, WbdB, and WbdC) that assemble the O-PS on undecaprenol-PP-GlcNAc (10). Structural studies identify terminal capping residues in a number of O-PSs synthesized by the ABC transporter-dependent pathway (11). It has been proposed that the addition of a capping residue to the nonreducing end of the undecaprenol-PP-linked PS serves to regulate O-PS chain length by terminating elongation. In the case of the O9a PS, termination involves methylation and phosphorylation. The chain length of the O9a PS is strictly controlled by the activity of WbdD, and O-PS-substituted LPS molecules expressed on the cell surface exhibit a narrow size distribution. The E. coli O9a WbdD protein contains putative kinase and methyltransferase domains, and these activities have been confirmed in biochemical studies (12). In addition to the role in O-PS chain regulation, methyl and/or phosphoryl modification is required for binding of the O9a PS to the nucleotide-binding component (Wzt) of the ABC transporter (13, 14), a crucial initial step in O-PS export. Unmodified O9a PS does not bind to Wzt, and a wbdD mutant accumulates unmodified polysaccharide in the cytoplasm.Open in a separate windowFIGURE 1.Structure and biosynthesis of the E. coli O9a PS and schematic showing WbdD and mutant derivatives. A, the structure of the O9a PS shows the adaptor region, repeat unit, and terminating residues. The nonreducing end of the O-PS is capped by methylation and phosphorylation, but the nature of the linkage between capping residues and the repeat unit is unknown (11, 12). The O9a-PS biosynthesis and export genes are shown together with the functions of the encoded proteins. B, a linear representation of the wild-type WbdD protein from CWG634 is shown in context with the genomic wbdD mutations in CWG635 and CWG900. The methyltransferase (MTase) and kinase domains are shown within WbdD and have been described previously (12). In CWG635, the chromosomal wbdD ORF was disrupted by replacing a 500-bp SmaI restriction fragment with the aacC1 cassette. A potential ribosomal-binding site, initiation codon, and stop codon are shown and together define an ORF encoding amino acids 501–708 of WbdD. In CWG900, the entire wbdD ORF has been removed from the chromosome. C, a schematic of the truncated WbdD polypeptide derivatives encoded by plasmids used in this study. The numbers shown above the polypeptides refer to amino acid positions in the native WbdD protein. Each polypeptide contained either an N-terminal His₆ tag or the T25 fragment of B. pertussis adenylate cyclase (see plasmids in 15, 16). However, the variability of specific assembly proteins among different biosynthetic systems precludes development of a generalized model for a polysaccharide assembly complex. Here we present data revealing the mechanisms that target the O9a mannosyltransferases to the cytoplasmic membrane and identify essential protein-protein interactions within the biosynthesis complex.