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 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.

FIGURE 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 Table 1).

FIGURE 2.

Complementation of wbdD mutations with genes encoding His₆-WbdD and His₆-WbdD_1–600. LPS from proteinase K-treated whole cell lysates was separated by SDS-PAGE and visualized by silver staining. The relative migration distances of lipid A-core OS and O-PS-substituted LPS (S-LPS) are shown at the left. The LPS of the parental E. coli O9a strain CWG634 contains O-PS. The mutants CWG635 (wbdD::aacC1) and CWG900 (ΔwbdD) were devoid of O-PS. CWG635 and CWG900 were transformed with plasmids encoding either full-length His-WbdD (pWQ470) or His-WbdD_1–600 (pWQ471) and grown to mid-exponential phase in LB containing 0.1% (w/v) d-mannose.

FIGURE 3.

In vitro synthesis of the O9a mannan in membranes from E. coli CWG634 and from CWG900 (ΔwbdD) overexpressing His₆-WbdD and its truncated derivatives. The membranes were prepared from bacteria grown to mid-exponential phase in M9 minimal medium containing 0.4% glycerol and 0.01% (w/v) l-arabinose. The substrate (GDP-[¹⁴C]mannose) was used to measure [¹⁴C]mannose incorporation into membrane-bound polysaccharide at 30 °C. The reaction was stopped at time points throughout 30 min of incubation. ■, CWG634; □, CWG900[pBAD24]; ●, CWG900[pWQ470] (His₆-WbdD); *, CWG900[pWQ471] (His₆-WbdD_1–600); ♦, CWG900[pWQ472] (His₆-WbdD_475–708). The results shown are the means from three independent experiments. The error bars represent ± S.E.

FIGURE 4.

The C-terminal region of WbdD binds to the cytoplasmic membrane. CWG900 (ΔwbdD) cells overexpressing His₆-WbdD or truncated His₆-tagged derivatives were grown in LB containing 0.001% (w/v) l-arabinose. The cell lysates were separated into soluble and membrane-containing fractions by centrifugation. Western immunoblots of the fractions were probed with an anti-His₅ monoclonal antibody to detect the recombinant WbdD proteins. The sizes of the detected polypeptides were consistent with the predicted values for His₆-WbdD (82.6 kDa), His₆-WbdD_1–600 (69.5 kDa), and His₆-WbdD_475–708 (28.4 kDa), respectively. L, cleared cell lysate; S, soluble fraction; M, membrane fraction.

FIGURE 5.

Secondary structure prediction of the WbdD C-terminal region. The amino acid sequence comprises residues 475–708 at the WbdD C terminus. The gray rectangles shown below the sequence represent α-helices (H1–H10) predicted using the PSIpred application. Helical wheel diagrams are shown for the putative α-helices H5–H8 within the membrane-binding region (MB). Note the distribution of positively charged (basic) amino acids to one side of each helix. H6, H7, and H8 also contain hydrophobic faces and are potentially amphipathic helices. Gray circles in the helical wheel diagrams represent empty positions and are results of α-helical sequences that were shorter than the window size within the helical wheel drawing application. Regions predicted to form coiled-coil structures are represented by open rectangles. TIR1 and TIR2 indicate transferase interactive regions. Deletion of either TIR1 or TIR2 abrogates WbdD-WbdA interaction.

FIGURE 6.

Membrane localization of the WbdB and WbdC mannosyltransferases is not dependent on the presence of WbdD. CWG634 (wild-type O9a PS) and CWG900 (ΔwbdD) overexpressing WbdB-FLAG (pWQ474) (A) or WbdC-FLAG (pWQ473) (B) were grown in LB containing 0.02% (w/v) l-arabinose. The cell lysates were separated into soluble and membrane-containing fractions, and the fusion proteins were detected in Western blots using an anti-FLAG monoclonal antibody as a probe. The sizes of the detected polypeptides were consistent with the predicted values for WbdB-FLAG (44.6 kDa) and WbdC-FLAG (43.5 kDa), respectively. L, cleared cell lysate; S, soluble fraction; M, membrane fraction.

FIGURE 7.

WbdD is essential for targeting the mannosyltransferase, WbdA, to the cytoplasmic membrane. A, Western blot of subcellular fractions from the E. coli O9a parental strain, CWG634, and mutant derivatives. The blot was probed with anti-WbdA antiserum to detect chromosomally expressed WbdA. B and C, Western immunoblot showing the subcellular location of WbdA in CWG900 (ΔwbdD) overexpressing His₆-WbdD and its truncated derivatives. Plasmid-encoded His₆-WbdD polypeptides were induced with 0.001% (w/v) l-arabinose, and chromosomally expressed WbdA was detected using anti-WbdA antiserum. In C, His₆-WbdD was detected using anti-His₅ monoclonal antibody. The sizes of the detected polypeptides were consistent with the predicted values for WbdA (94.2 kDa), His₆-WbdD_1–686 (79.8 kDa), and His₆WbdD_1–669 (78.0 kDa), respectively. L, cleared cell lysate; S, soluble fraction; M, membrane fraction.

FIGURE 8.

In vivo interaction between the WbdD O-PS chain terminator and the WbdA mannosyltransferase. An adenylate cyclase-based bacterial two-hybrid assay was used to detect protein-protein interaction between T25-WbdD (pWQ486) and T18-WbdA (pWQ487) in the reporter strains E. coli BTH101 (K-12 cyaA) and E. coli CWG910 (O9a cyaA). A, BTH101 transformants after growth for 36 h at 30 °C on LB, IPTG, X-gal agar. Blue colonies indicate cAMP-dependent induction of the lac operon. B, CWG910 transformants after growth for 36 h at 30 °C on LB, IPTG, maltose, neutral red agar. Red colonies indicate induction of the maltose utilization operon. Zip, leucine zipper domain from yeast GCN4. C, lysates from BTH101 expressing T18-WbdA together with T25 fusions of WbdD or its truncated derivatives were assayed for β-galactosidase activity. The values presented are the mean activities (Miller units) ± S.E. from three independent experiments.