The wbnH gene of Escherichia coli O86:H2 encodes an α-1,3-N-acetylgalactosaminyl transferase involved in the O-repeating unit biosynthesis

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Abstract

O-repeating unit biosynthesis is the first committed step in lipopolysaccharide (LPS) biosynthesis in a variety of gram-negative bacteria. The wbnH gene was previously proposed to encode a glycosyltransferase involved in O-repeating unit synthesis in Escherichia coli O86:H2 strain. In this work, we provide biochemical evidence to show that wbnH encodes a N-acetylgalactosaminyl transferase (GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to the GalNAc-pyrophosphate-lipid acceptor. WbnH activity was characterized using a synthetic acceptor substrate GalNAcα-PP-O(CH2)11-OPh. The resulting disaccharide product GalNAc-α-1,3-GalNAcα-PP-O(CH2)11-OPh was analyzed by LC–MS and NMR spectroscopy. Substrate specificity study indicates that pyrophosphate and hydrophobic lipid moiety are structural requirements for WbnH activity. Divalent metal cations are not required for enzyme catalysis, suggesting WbnH belongs to glycosyltransferase GT-B superfamily. Our results complete the characterization of O86 O-unit assembly pathway, and provide the access of chemically defined O-unit substrates for the further investigation of O-antigen biosynthetic mechanism.

Section snippets

Materials and methods

Bacterial strains, plasmids, and materials. Escherichia coli O86:H2 strain was kindly provided by W. F. Vann from Department of Health and Human Service, Food and Drug Administration. E. coli competent cell DH5α [lacZΔM15 hsdR recA] was from Gibco-BRL Life Technology. E. coli competent cell BL21 (DE3) [FompT hsdSB(rB-mB-)gal dcm (DE3)] was from Novagen Inc. (Madison, WI). Expression plasmid pET15b was purchased from Novagen (Carlsbad, CA). Plasmids pKD20 and pKK232-8 were kindly provided by L.

Characterization of wbnH mutant in E. coli O86:H2

To determine whether wbnH is required for O86 LPS biosynthesis, we constructed the wbnH knockout O86 strain by replacing the wbnH with CAT gene using the RED recombination system of phage lambda as described [22], [23]. The replacement was confirmed by PCR amplification and sequencing the region upstream and downstream of the wbnH gene. LPS from mutant and wild-type strains was extracted according to standard procedure and visualized by silver staining. Wild-type E. coli O86 strain, as

Discussions

In the fight against infectious bacterial diseases, the genetics and biochemistry of LPS has been a special but extremely important research field. While great progresses have been made on the mechanism of the biosynthesis of lipid A [26], [27], [28] and bacterial cell wall, our current understanding on the biosynthesis of complex polysaccharides in general and O-polysaccharide in particular has lagged behind to a large extent. Currently, three pathways have been proposed to describe the

Acknowledgments

We thank L. Wang for providing us with plasmids pKD20 and pKK232-8, and W.F. Van for sending E. coli O86:H2 strain. P.G. Wang acknowledges support from an endowed Ohio Eminent Scholar Professorship on Macromolecular Structure and Function in the Department of Biochemistry at the Ohio State University and financial support (R01 AI44040) from National Institute of Health.

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    These two authors contributed equally to this work.

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