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 perip lasmic glucans, known as membrane-derived oligosaccharides (MDOs), in Escherichia coli. Mutants lacking MdoH are deficient in a glucosyltran sferase 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 cytopl asmic membrane. The MdoH protein comprises 847 amino acids and is poor ly expressed as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have experimentally measured the topological organ ization 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 membr ane eight times, with the N find C termini in the cytoplasm. Moreover, a 310-amino-acid domain is present in the cytoplasm between the secon d and third transmembrane segments. It was deduced from the measuremen t of the MDO biosynthetic activity of truncated or fused MdoH proteins that almost all the C-terminal residues are necessary for this activi ty. The model of the MdoH protein in the membrane suggests that this p rotein could be directly involved in the translocation of nascent poly glucose chains to the periplasmic space.