Topological analysis of the membrane-bound glucosyltransferase, MdoH, required for osmoregulated periplasmic glucan synthesis in Escherichia coli.
The MdoH protein is essential for synthesis of the osmoregulated periplasmic glucans, known as membrane-derived oligosaccharides (MDOs), in Escherichia coli. Mutants lacking MdoH are deficient in a glucosyltransferase activity assayed in vitro. The MdoH protein is the product of the second gene of an operon, and it has been shown to span the cytoplasmic membrane. The MdoH protein comprises 847 amino acids and is poorly expressed as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have experimentally measured the topological organization of MdoH within the membrane by construction of fusions to beta-lactamase as a reporter. Analysis of 51 different MdoH-beta-lactamase fusions suggested that the MdoH protein crosses the cytoplasmic membrane eight times, with the N and C termini in the cytoplasm. Moreover, a 310-amino-acid domain is present in the cytoplasm between the second and third transmembrane segments. It was deduced from the measurement of the MDO biosynthetic activity of truncated or fused MdoH proteins that almost all the C-terminal residues are necessary for this activity. The model of the MdoH protein in the membrane suggests that this protein could be directly involved in the translocation of nascent polyglucose chains to the periplasmic space.
Debarbieux
L
L
Laboratoire de Chimie Biologique, UMR111 du CNRS, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Bohin
A
A
Bohin
J P
JP
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002451:Cell Compartmentation; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005936:Glucans; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019897:Periplasm; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D014882:Water-Electrolyte Balance; D001618:beta-Lactamases / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005936:Glucans; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C089831:opgH protein, E coli; D005964:Glucosyltransferases; D019286:Endopeptidase K; D001618:beta-Lactamases