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J. Biol. Chem., Vol. 279, Issue 21, 22469-22476, May 21, 2004

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Topological Analysis of Glucosyltransferase GtrV of Shigella flexneri by a Dual Reporter System and Identification of a Unique Reentrant Loop^*

Haralambos Korres and Naresh K. Verma

From the School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra ACT 0200, Australia

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 1,3 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 -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.

Received for publication, February 6, 2004 , and in revised form, March 15, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 61-2-61252666; Fax: 61-2-61250313; E-mail: Naresh.Verma{at}anu.edu.au.

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