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Journal of Bacteriology, April 2007, p. 2618-2628, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01905-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide{triangledown}

Jason Lehrer,1,{dagger} Karen A. Vigeant,1,{dagger},{ddagger} Laura D. Tatar,1 and Miguel A. Valvano1,2*

Infectious Diseases Research Group, Siebens-Drake Medical Research Institute, Departments of Microbiology and Immunology,1 Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada2

Received 18 December 2006/ Accepted 15 January 2007

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 Km and Vmax values for UDP-GlcNAc, Mg2+, and Mn2+ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg2+, 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Dental Sciences Building 3014, University of Western Ontario, London, ON N6A 5C1, Canada. Phone: (519) 661-3427. Fax: (519) 661-3499. E-mail: mvalvano{at}uwo.ca.

{triangledown} Published ahead of print on 19 January 2007.

{dagger} J.L. and K.A.V. contributed equally to this work.

{ddagger} Present address: Bioniche Life Sciences Inc., Belleville, ON K8N 1E2, Canada.

Journal of Bacteriology, April 2007, p. 2618-2628, Vol. 189, No. 7
0021-9193/07/$08.00+0     doi:10.1128/JB.01905-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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