The wbnH gene of Escherichia coli O86:H2 encodes an α-1,3-N-acetylgalactosaminyl transferase involved in the O-repeating unit biosynthesis
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) [F−ompT hsdSBgal 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.
References (36)
- et al.
The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat
J. Biol. Chem.
(1999) - et al.
Investigation of the structural requirements in the lipopolysaccharide core acceptor for ligation of O antigens in the genus Salmonella: WaaL ⧹“Ligase⧹” is not the sole determinant of acceptor specificity
J. Biol. Chem.
(2004) - et al.
Substrate analogues to study cell-wall biosynthesis and its inhibition
Curr. Opin. Chem. Biol.
(2002) - et al.
Identification of a UDP-Gal:GlcNAc-R galactosyltransferase activity in Escherichia coli VW187
Bioorg. Med. Chem. Lett.
(2005) - et al.
Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen
J. Biol. Chem.
(2003) - et al.
Biosynthesis of enterobacterial common antigen in Escherichia coli. In vitro synthesis of lipid-linked intermediates
J. Biol. Chem.
(1987) - et al.
Current trends in capsular polysaccharide biosynthesis of Streptococcus pneumoniae
Res. Microbiol.
(2000) - et al.
Molecular biology of the capsular genes of Streptococcus pneumoniae
FEMS Microbiol. Lett.
(1997) - et al.
Remarkable structural similarities between diverse glycosyltransferases
Chem. Biol.
(2002) - et al.
Lipopolysaccharide endotoxins
Annu. Rev. Biochem.
(2002)
Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit
Microbiology (Reading, United Kingdom)
An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase
J. Bacteriol.
A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917
J. Bacteriol.
Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8
J. Bacteriol.
Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)
Mol. Microbiol.
Isolation of rfb gene clusters directing the synthesis of O polysaccharides consisting of mannose homopolymers and serological analysis of lipopolysaccharides
Microbiol. Immunol.
Genetic analysis of Escherichia coli O9 rfb: identification and DNA sequence of phosphomannomutase and GDP-mannose pyrophosphorylase genes
Microbiology (Reading, United Kingdom)
A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing
Infect. Immun.
Cited by (32)
Biosynthesis of Bacterial Polysaccharides
2021, Comprehensive Glycoscience: Second EditionPhosphopolyprenols, their glycosyl esters and analogs: Strategy of chemical synthesis, applications and perspectives
2020, Studies in Natural Products ChemistryCitation Excerpt :The methanolic eluate contained sodium salt of PhOUn-PP-GlcNAc [42]. PhOUn-PP-GalNAc was obtained similar to the procedure [50] and finally purified by reverse-phase C18 column chromatography in a yield of 60% [59]. PhOUn-PP-GlcNAc and PhOUn-PP-GalNAc were also obtained later by coupling of the corresponding components at the same conditions followed by purification of protected diphosphates by preparatory-scale TLC on silica gel and deacetylation [60].
Gut microbiota have blood types as human
2018, Science BulletinBiochemical characterization of the novel α-1, 3-galactosyltransferase WclR from Escherichia coli O3
2016, Carbohydrate ResearchCitation Excerpt :WclR is specific for the axial 4-hydroxyl of the GlcNAc and the phospholipids in the acceptor substrate. The requirement for the phospholipids linked to GalNAc or GlcNAc in the acceptors appears to be a characteristic of the second enzyme in the O-antigen assembly pathway, which includes WclR.11,21–23 This study enhanced our understanding of function and specificity of bacterial GTs.
Defining function of lipopolysaccharide O-antigen ligase waal using chemoenzymatically synthesized substrates
2012, Journal of Biological ChemistryCitation Excerpt :Accordingly, an alternative methodology was employed for obtaining the O-unit-PP-Und molecule. Specifically, we exploited a chemoenzymatic approach in which the chemically synthesized GalNAc-PP-Und precursor was elaborated to the E. coli O86 pentasaccharide repeating unit via successive enzymatic glycosylation using four glycosyltransferases cloned from E. coli O86 following the established protocols (7, 19–21) (Fig. 2). Some evidence has demonstrated that bacterial glycosyltransferases acting on Und-linked acceptors are relaxed in their recognition of lipid moiety (21, 27, 28).
- 1
These two authors contributed equally to this work.