LPSs of NCTC 11637-derived mutants in and were methylated and subjected to fast atom bombardment-mass spectrometry (FAB-MS) as previously described (5). RESULTS AND DISCUSSION Characterization of MAbs 4D2 (anti-H type I) and 218/2B3 (anti-Lea). lost both H type I and Ley expression. Inactivation of HP093-HP094 resulted in a transformant expressing Lex and lacking Ley and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that this HP093-HP094 2-fucosyltransferase (2-FucT) gene product is involved in the biosynthesis of both Ley and Lex. Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Lex and Lea expression, which demonstrates that this HP0379 gene product is usually both an 3- and an 4-FucT. Our data provide understanding at the molecular level of how is able to diversify in the host, a requirement likely essential for successful colonization and transmission. The human gastric pathogen displays molecular mimicry with the gastric epithelial cells of the human host (3). Often, lipopolysaccharide (LPS) expresses both Lewis x (Lex) (see Fig. ?Fig.1)1) and Ley human blood group antigens, but strains expressing sialyl-Lex, H type I, Lea, Leb, and the nonfucosylated polylactosamine chain (i-antigen) have been described (16, 17); strains expressing H type 2 have not been found yet. For biosynthesis of Lex/y antigens, the activity of a variety of glycosyltransferases is required: 2- and 3-fucosyltransferases (2- and 3-FucT), 4-galactosyltransferases (4-GalT) and 3-genes, homologues of HP0379 and HP0651 of strain 26695, have been identified (13, 15). The products of these genes have different fine specificities (5). For this reason, the homologues of HP0379 and HP0651 are designated and (the homologue of gene HP093-HP094 in strain 26695) is designated (21, 22). A gene has recently been identified in (the homologue of HP0826 in strain 26695 [14]). It is unknown which gene codes for 3-GalT and 3-GlcNAcT. In vitro, the recombinant gene product is able to form H type I from a synthetic Gal1.3GlcNAc acceptor (22), but formal proof of the involvement of this gene in chroman 1 biosynthesis of H type I in LPS is lacking. Open in a separate window FIG. 1 Structure of Lewis blood group antigens and glycosyltransferases required for biosynthesis. Gal, d-galactose; Fuc, l-fucose; GlcNAc, Lewis antigens is usually their ability to phase vary (4, 5). Phase variation is defined as the high-frequency, reversible switching of phenotype. For instance, a strain expressing Lex may yield phase variants expressing Ley or the i-antigen. Phase variation in other bacteria like and spp. has been shown to contribute to bacterial virulence and host adaptation (13). We have shown that populations of LPS phase variants can be isolated from the human stomach, chroman 1 which provides evidence that LPS phase variation contributes to strain diversity in the human host (6). The molecular mechanism of phase variation in Lex/y has been investigated recently (5). Long homopolymeric C tracts present in the open reading frames of may change length during replication due chroman 1 to DNA slippage (slipped-strand mispairing). The result is usually a reversible (translational) frame switch that leads to either a full-length active gene product (fucosyltransferase enzyme) or an inactive truncated form. Phase variation in H type I or Lea has not been documented yet. In this paper, we show that this H type I and Lea epitopes are also phase variable, and we identify a hitherto unrecognized gene (gene product is capable of acting as an 4-FucT, which is required for Lea biosynthesis, and that the gene product is essential for biosynthesis of H type I. MATERIALS AND METHODS Bacterial strains and LPSs. Strains NCTC 11637, G27, and phase variant 3a have Rabbit Polyclonal to DCC been described before (4, 8, 11). The LPSs of strains J223 (expresses H type I [16]) chroman 1 and UA948 (expresses Lea [16]) were purified as described previously (16). MAbs, synthetic glycoconjugates, and serological procedures. The monoclonal antibodies.