Topological analysis of glucosyltransferase GtrV of Shigella flexneri by a dual reporter system and identification of a unique reentrant loop.
Lipopolysaccharide, particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-Antigen modification is mediated by glucosyltransferase genes (gtr) encoded by temperate serotype-converting bacteriophages. The gtrV gene encodes the GtrV glucosyltransferase, an integral membrane protein that catalyzes the transfer of a glucosyl residue via an alpha1,3 linkage to rhamnose II of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a. Analysis of the GtrV amino acid sequence using computer prediction programs indicated that GtrV had 9-11 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of GtrV to a dual reporter system composed of alkaline phosphatase and beta-galactosidase. Sandwiched GtrV-PhoA/LacZ fusions were also constructed at predetermined positions. The enzyme activities of cells with the GtrV-PhoA/LacZ fusions and the particular location of the fusions in the gtrV indicated that GtrV has nine transmembrane segments and one large N-terminal periplasmic loop with the N and C termini located on the cytoplasmic and periplasmic sides of the membrane, respectively. The existence of a unique reentrant loop was discovered after transmembrane segment IV, a feature not documented in other bacterial glycosyltransferases. Its potential role in mediating serotype conversion in S. flexneri is discussed.
Korres
Haralambos
H
School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra ACT 0200, Australia.
Verma
Naresh K
NK
D000469:Alkaline Phosphatase / Q000378:metabolism; D001435:Bacteriophages / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D017353:Gene Deletion; D017930:Genes, Reporter; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D019081:O Antigens / Q000737:chemistry; D019897:Periplasm / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D012763:Shigella flexneri / Q000201:enzymology*; D001616:beta-Galactosidase / Q000378:metabolism
D017931:DNA Primers; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D019081:O Antigens; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C487330:GtrV protein, Shigella flexneri; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase