Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide

Abstract

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K(m) and V(max) values for UDP-GlcNAc, Mg(2+), and Mn(2+) suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg(2+), possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

FIG. 1.

Expression of parental WecA and mutant protein derivatives. Total membranes were prepared from strain MV501 (VW187 wecA::Tn10) transformed with various plasmids containing several versions of wecA (Table 1), as follows: lane 1, WecA(pKV1) (wecA_FLAG-5×His); lane 2, pBAD-His vector control; lane 3, D90E protein, pKV2; lane 4, D90N protein, pKV3; lane 5, D91E protein, pKV4; lane 6, D91N protein, pKV5; lane 7, D159E protein, pKV10; lane 8, D159N protein, pKV11. Each lane contained 4 μg of protein. Membranes were transferred to a nitrocellulose membrane and reacted with anti-FLAG monoclonal antibodies. The molecular mass standards were myosin (206 kDa), β-galactosidase (119 kDa), bovine serum albumin (91 kDa), ovalbumin (51 kDa), and carbonic anhydrase (34.7 kDa).

FIG. 2.

Complementation of O7 LPS synthesis in strain MV501 (VW187 wecA::Tn10) by parental WecA and mutant derivatives. LPS samples were obtained from E. coli MV501 (VW187 wecA::Tn10) transformed with various plasmids. Lane 1, pBAD-His; lane 2, WecA(pKV1) (wecA_FLAG-5×His); lane 3, D90E protein, pKV2; lane 4, D90N protein, pKV3; lane 5, D91E protein, pKV4; 6 lane, D91N protein, pKV5; lane 7, D156E protein, pKV8; lane 8, D156N protein, pKV9; lane 9, D159E protein, pKV10; lane 10, D159N protein, pKV11. LPS was separated by Tricine-SDS-PAGE, which was followed by silver staining. Loading was normalized by determining the amount of KDO in the inner core.

FIG. 3.

Detection of Und-P-P-GlcNAc product conversion by thin-layer chromatography. Membranes of E. coli CLM37 harboring various plasmids were assayed for transferase activity under standard conditions with ¹⁴C-labeled UDP-GlcNAc, and the lipid-linked material was extracted with 1-butanol and processed as described in Materials and Methods. Lane 1, ¹⁴C-labeled UDP-GlcNAc, no membranes; lane 2, membranes from plasmidless CLM37, no ¹⁴C-labeled UDP- GlcNAc; lane 3, membranes from CLM37(pVK9) expressing WecA-D156N_FLAG-5×His; lane 4, membranes from CLM37(pVK3) expressing WecA-D90N_FLAG-5×His; lane 5, membranes from CLM37(pVK11) expressing WecA-D159N_FLAG-5×His; lane 6, membranes from CLM37(pVK1) expressing WecA_FLAG-5×His. The dotted line indicates the loading point.

FIG. 4.

Quantitative immunoblotting to determine the amounts of WecA in total membrane preparations. The relative amounts of FLAG molecules in the samples were determined by comparison with known amounts (0.4 to 3.2 ng) of purified BAP-FLAG, which were included on the same gel. The graph is a plot of the relative pixel densities of the BAP-FLAG bands, which were calculated with the program ImageJ, versus the amounts of purified protein loaded in the lanes. The inset shows the blot with the different amounts of BAP-FLAG (left four lanes) and WecA_FLAG-5×His (right four lanes). The amount of WecA_FLAG-5×His was deduced from the standard curve.

FIG. 5.

Transfer of GlcNAc-1-P to Und-P. All assays were carried out at 37°C for 15 min in triplicate. One unit of enzyme activity was defined as 10⁻³ pmol of GlcNAc incorporated/min/mg protein. (A) UDP-GlcNAc-dependent activity of WecA_FLAG-5×His in the presence of excess Mg²⁺ (16.7 mM) and Mn²⁺ (3.1 mM). (B) Mg²⁺- and Mn²⁺-dependent enzyme activity of WecA_FLAG-5×His. Assays were carried out at 37°C for 15 min with a fixed concentration of UDP-GlcNAc (131.4 pmol), 40 μg of total membranes from an MV501(pKV1) preparation, and different concentrations (0 to 3 mM) of Mg²⁺ and Mn²⁺.

FIG. 6.

(A) Expression of WecA_FLAG-7×His. Total membranes were prepared from strain MV501 (VW187 wecA::Tn10) transformed with various plasmids containing several versions of wecA (Table 1). Lane 1, WecA_FLAG-7×His, pJL1; lane 2, WecA_{FLAG-7×His-Cys}, cysteineless version of WecA encoded by pJL7; lane 3, WecA_FLAG-5×His, pKV1; lane 4, vector control, pBAD-His. Each lane contained 4 μg of protein. Membranes were transferred to a nitrocellulose membrane and reacted with anti-FLAG monoclonal antibodies. The molecular mass standards were myosin (206 kDa), β-galactosidase (119 kDa), bovine serum albumin (91 kDa), ovalbumin (51 kDa), and carbonic anhydrase (34.7 kDa). (B) Complementation of O7 LPS synthesis in strain MV501 (VW187 wecA::Tn10). LPS samples were obtained from E. coli MV501 (VW187 wecA::Tn10) transformed with various plasmids. Lane 1, vector control, pBAD-His; lane 2, WecA_FLAG-5×His, pKV1; lane 3, WecA_{FLAG-7×His-Cys}, cysteineless version of WecA encoded by pJL7. LPS was separated by Tricine-SDS-PAGE, which was followed by silver staining. Loading was normalized by determining the amount of KDO in the inner core.

FIG. 7.

Topological model of WecA. The model was originally derived by using the TMHHM computer program (49) and was experimentally refined based on the results of the substituted cysteine accessibility experiments (see Fig. 6). Boldface numbers indicate cytosolic loops 1 to 5. The residues spanning predicted transmembrane segments are enclosed in boxes. The individual residues with a gray background are the residues that, when replaced by cysteine, are protected by MTSET from biotin maleimide labeling. The circled residues with a white background are the residues that, when replaced by cysteine, are resistant to labeling by biotin maleimide. The residues with a black background are the residues that, when replaced by cysteine, are labeled with biotin maleimide irrespective of MTSET pretreatment. Squares indicate residues at which cysteine replacement affects the function of WecA, as determined by complementation of O7 LPS synthesis in E. coli MV501. Circles indicate residues at which cysteine replacement did not compromise WecA function. The dotted circle indicates Asp217. The asterisks indicate additional residues important for WecA function, which were identified in previous studies (3, 4). The question marks indicate residues where the experimental and predicted topologies do not agree.

FIG. 8.

Sulfhydryl labeling accessibility of cysteine replacements in WecA_FLAG-7×His. The top panel shows the results obtained with biotin maleimide labeling. The middle panel shows the results of biotin maleimide labeling with MTSET-pretreated samples. The lower panel shows Western blots with the FLAG monoclonal antibody. WecA-Cys is the cysteineless WecA protein encoded by pJL7. Each of the replacement mutants is indicated by its short designation (see Fig. 7 for the location of each residue in the topological model of WecA). Biotin maleimide labeling of S362C in the presence and absence of MTSET was done in a different experiment. Lane M contained molecular mass markers.

FIG. 9.

WecA localizes in discrete membrane domains: fluorescence microscopy of bacteria expressing WecA_GFP. (Inset) Punctate pattern of fluorescence that is limited to the circumference of bacterial cells. The micrograph was taken with a ×100 oil immersion objective, resulting in a magnification of ×1,000.

FIG. 10.

Complementation of O7 LPS expression in E. coli MV501 by plasmids encoding the cysteine-substituted derivatives of cysteineless WecA_FLAG-7×His, as determined with silver-stained polyacrylamide gels. WecA-Cys is the cysteineless WecA protein encoded by pJL7. Each of the replacement mutants is indicated by its short designation (see Fig. 7 for the location of each residue in the topological model of WecA).