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.