Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.conjugate vaccines ͉ oligosaccharyltransferase ͉ STT3 ͉ glycoengineering

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N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria (28). It has a tripartite structural organization consisting of lipid A, a conserved core oligosaccharide region, and an O-specific polysaccharide chain or O antigen. In the majority of gram-negative bacteria, the core oligosaccharide can be subdivided into an outer core, generally composed of hexoses and hexosamines, and an inner core made of 3-deoxy-D-manno-oct-2-ulosonic acid and L,Dheptose units. LPS plays an important role in maintaining the structural integrity of the bacterial outer membrane by interacting with outer membrane proteins and divalent cations (15), thereby providing a barrier against the entry of toxic hydrophobic compounds into the bacterial cell (27). Escherichia coli mutants defective in the biosynthesis of 3-deoxy-D-manno-oct-2-ulosonic acid are nonviable, whereas those impaired in L,Dheptose synthesis survive in vitro, although they display a pleiotropic phenotype referred to as "deep rough" (17). This phenotype is characterized by an extreme sensitivity to very low concentrations of novobiocin, detergents, and bile salts (32). Deep rough mutants also have defects in F plasmid conjugation and generalized transduction by the bacteriophage P1 (6, 16). Haemophilus influenzae heptose-deficient mutants were found to be serum sensitive and displayed a reduced virulence in vivo (18,36).The complete biosynthesis pathway of the L,D-heptose precursor has not been elucidated. Eidels and Osborn (11) In gram-negative bacteria, functional studies have only been performed for the isomerization reaction and the epimerization step (3,9,26), while the conversion of D,D-heptose 7-phosphate to D,D-heptose 1-phosphate and a functional proof of the activating step have not been demonstrated. The D-sedoheptulose 7-phosphate isomerase activity was described in S. enterica serovar Typhimurium (12), and the corresponding gene, gmhA, has been cloned both from E. coli and from H. influenzae (3, 4). The amino acid sequence of the GmhA polypeptide is highly conserved in different gram-negative bacteria (33). The epimerization step is catalyzed by the WaaD (formerly RfaD) protein (5), which has also been crystallized (8). We * Corresponding author. Mailing address for Paul Messner: Zentrum für Ultrastrukturforschung,