Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens

Abstract

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA(O9a) contains two separable and functionally active domains, whereas WbdA(O8) possesses three. In WbdC(O9a) and WbdB(O9a), substitution of the first Glu of the EX(7)E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX(7)E motif of the N-terminal WbdA(O9a) domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA(O9a) domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA(O9a) possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.

FIGURE 1.

Structures the E. coli O8, O9 and O9a polymannose O-PSs. Each polysaccharide contains four structural regions, the primer, adaptor, repeat unit domain, and terminal modification, which are represented in the schematic in the context of the und-PP-linked biosynthetic intermediate. GlcpNAc is represented by a blue square and Manp by a green circle according to the nomenclature used by the Consortium for Functional Glycomics. The enzymes responsible for the formation of each part of the glycan are identified in parentheses.

FIGURE 2.

Multiple-sequence alignment of the mycobacterial PimA α-mannosyltransferase and the O9a and O8 WbdA mannosyltransferase domains. Conserved residues are highlighted in black, and similar residues are highlighted in gray. The Glu residues of the EX₇E motifs are highlighted in red. WbdA^O9a Arg⁸⁰ involved in the WbdA^O9 C80R O9 → O9a seroconversion is highlighted in cyan. Alignments were performed using the ClustalW2 server (28, 29).

FIGURE 3.

Predicted domains of the O8 and O9a mannosyltransferases. A shows WbdC^O8/O9a and WbdB^O8/O9a, which both contain a single mannosyltransferase domain. WbdA^O9a (B) contains two putative mannosyltransferase domains, and WbdA^O8 (C) possesses three. Domain predictions were identified using the NCBI Conserved Domain Database (24). Cloned regions encoding individual domains of the WbdA homologs are identified.

FIGURE 4.

Structural models of N-WbdA^O9a and C-WbdA^O9a compared with the mycobacterial PimA mannosyltransferase. A and B show an overlay of the N-WbdA^O9a model (purple) and C-WbdA^O9a model (magenta) with PimA (gray), respectively. C and D show expanded views of the corresponding modeled active sites. Glu residues of the WbdA EX₇E motifs are colored green. Arg⁸⁰ is involved in the Wbd^O9 C80R O9 → O9a seroconversion (76) and is highlighted in orange in the N-WbdA^O9a model. The Glu residues of the EX₇E motif in PimA are in black, and GDP-Manp is shown in cyan. The first Glu residue of the EX₇E motif is in a position to hydrogen-bond with the Manp moiety of GDP-Manp. The second Glu residue of the motif is in a position to hydrogen-bond with the ribose moiety of GDP-Manp.

FIGURE 5.

WbdA^O9a contains two separable domains. The results show mutant complementation experiments with CWG1105 (ΔwbdA^O9a) expressing N-WbdA^O9a (pWQ590), C-WbdA^O9a (pWQ591), or N-WbdA^O9a and C-WbdA^O9a combined (pWQ590 + pWQ591). Top, silver-stained SDS-polyacrylamide gel of LPS samples from whole-cell lysates; bottom, the corresponding Western immunoblot using O9a-specific antiserum. Native O-PS biosynthesis was restored only when both WbdA^O9a domains were present.

FIGURE 6.

WbdA^O8 contains three separable domains. The results show mutant complementation experiments with CWG1104 (ΔwbdA^O8) expressing WbdA^O8-D1+D2 (pWQ595), WbdA^O8-D2+D3 (pWQ596), WbdA^O8-D1+D3 (pWQ593 + pWQ594), or WbdA^O8-D1+D2+D3 (pWQ592 + pWQ596 or pWQ594 + pWQ595). Top, silver-stained SDS-polyacrylamide gel of LPS samples from whole-cell lysates; bottom, the corresponding Western immunoblot using O8-specific antiserum. Native O-PS biosynthesis was restored only when all three WbdA^O8 domains were present.

FIGURE 7.

Mutagenesis of Glu residues in the EX₇E motifs of WbdC^O9a and WbdB^O9a. The results show mutant complementation experiments with CWG1010 (ΔwbdC^O9a) expressing WbdC^O9a (pWQ575), WbdC^O9a E275A (pWQ583), or WbdC^O9a E283A (pWQ584) (A and B) and CWG1009 (ΔwbdB^O9a) expressing WbdB^O9a (pWQ576), WbdB^O9a E294A (pWQ583), or WbdB^O9a E302A (pWQ584) (C and D). Top panels, silver-stained SDS-polyacrylamide gel of LPS samples from whole-cell lysates; bottom panels, the corresponding Western immunoblots using O9a-specific antiserum. O-PS biosynthesis was severely impaired when only the first Glu residues of the EX₇E motif were substituted in WbdC^O9a and WbdB^O9a. IPTG, isopropyl β-d-1-thiogalactopyranoside.

FIGURE 8.

Differential effects of mutations in the EX₇E motifs of WbdA^O9a. The results show gene complementation experiments of CWG1105 (ΔwbdA^O9a) expressing WbdA^O9a E317A (pWQ597), WbdA^O9a E325A (pWQ598), WbdA^O9a E750A (pWQ599), or WbdA^O9a E758A (pWQ630). A, silver-stained SDS-polyacrylamide gel of LPS samples from whole-cell lysates; B, the corresponding Western immunoblot using O9a-specific antiserum. The E317A and E325A mutants were unable to support biosynthesis of O-PS, whereas the E750A and E758A mutant forms resulted in the production of O-PS with an altered structure and antigenicity.

FIGURE 9.

CWG901 (ΔwbdA^O9a) cells expressing WbdA^O9a E750A produce a (poly-)α-(1→2)-Manp O-PS. Shown is the ¹H NMR spectrum for purified LPS substituted with O-PS generated by E. coli CWG901 transformed with pWQ599.

FIGURE 10.

Analysis of the in vitro products generated by N-WbdA^O9a using synthetic acceptors. The reaction products were separated by thin layer chromatography (A), using the fluorescein tag on the acceptors for detection. Note that C-WbdA^O9a did not modify either acceptor; nor did its inclusion change the product profile obtained with N-WbdA^O9a. The products generated by N-WbdA^O9a with Acceptor B were examined by MALDI MS (B), revealing a series of incrementally sized products that differ by the addition of one Manp residue. a.u., arbitrary units.

FIGURE 11.

N-WbdA^O9a transfers Manp residues in α-(1→2) linkages to Acceptor B. A, ¹H NMR spectrum; B, gCOSY spectrum; C, tROESY spectrum.