Serotype conversion (O-antigen glucosylation) in Shigella flexneri is mediated by temperate bacteriophages, which encode a three-gene cluster that contains gtrA, gtrB, and gtr([type]). Sequence analysis has revealed that gtrA and gtrB are conserved and readily interchangeable between serotypes. The gtr([type]) is unique in each serotype and responsible for specifically mediating conversion by the addition of a glucosyl group to the O-antigen units. Analysis of the GtrA and GtrB amino acid sequence using computer prediction programs indicated that GtrA and GtrB have four and two transmembrane segments, respectively. The topology model of GtrA was analyzed by constructing consecutive sandwich fusions using a dual reporter PhoA/LacZ at predetermined positions targeting each of the 3 cytoplasmic and 2 periplasmic hypothetical loops. The topology of GtrB was determined by constructing C-terminal truncated fusions of GtrB to full-length PhoA and LacZ by a PCR-mediated method. These approaches revealed that GtrA consists of four transmembrane segments with both the N-terminal and C-terminal ends in the cytoplasm. Accordingly, GtrB consists of two transmembrane segments with both ends also in the cytoplasm. Furthermore, membrane anchorage of the extended N-terminal end of GtrB was found to be important in catalysis. This study completes the topology of all three proteins (GtrA, GtrB, and the gtr([type]): GtrV) involved in the glucosyltransferase activity that results in serotype conversion of S. flexneri. A model is proposed showing how both O-antigen synthesis and modification take place in S. flexneri. (C) 2005 Elsevier Inc. All rights reserved.