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Journal of Bacteriology, July 2008, p. 4922-4932, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00160-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of Two β-1,3-Glucosyltransferases from Escherichia coli Serotypes O56 and O152 ^,

Department of Medicine and Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada,¹ TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, People's Republic of China,² Tianjin Key Laboratory of Microbial Functional Genomics, People's Republic of China,³ Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, People's Republic of China,⁴ Department of Chemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada⁵

Received 31 January 2008/ Accepted 6 May 2008

The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-β1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc--PO₃-PO₃-(CH₂)₁₁-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-β1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn²⁺ or Mg²⁺) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

Published ahead of print on 16 May 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

^¶ Both authors contributed equally to the paper.