O antigen is the major cell surface antigen of gram-negative bacteria, and the genes responsible for its synthesis are located in a single gene cluster. The wzx (rbfX) gene, which is characteristic of the major class of O-antigen gene clusters, encodes a hydrophobic protein with 12 potential transmembrane segments. We demonstrate that a wzx mutant accumulates undecaprenol pyrophosphate-linked O units which appear to be on the cytoplasmic side of the cytoplasmic membrane, suggesting that the wzx gene encodes a flippase for O-unit translocation across that membrane.In enteric bacteria, the lipid component of the outer layer of the cell wall consists of lipopolysaccharide (LPS), which forms a selective barrier between the environment and the cell. An LPS molecule consists of three regions: lipid A, oligosaccharide core, and O-antigen chain. Lipid A, also called endotoxin, is the hydrophobic portion of the molecule which anchors it on the outer membrane. There is little variation found in this part of LPS among different species of enteric bacteria. The mechanisms of LPS biosynthesis have been studied extensively (17,19,21,26). O-antigen synthesis, which is independent of core-lipid A synthesis, starts with assembly of O units by sequential transfer of sugars onto a lipid carrier, undecaprenol phosphate (UndP) (Fig. 1). These reactions occur on the cytoplasmic side of the cytoplasmic membrane. The completed O units are transferred to the periplasmic side of the cytoplasmic membrane and are polymerized from the reducing end by O-antigen polymerase (Wzy) (17). The O-antigen chains are then ligated to the core-lipid A by O-antigen ligase (WaaL), and the complete LPS is translocated to the outer membrane of the bacterial cell. Another mechanism of O-antigen synthesis was established for homopolymer chains (mannan or galactan) of E. coli O9 (9), Klebsiella pneumoniae O1, and Serratia marcescens O16 (32). This mechanism differs from the first in that the O-antigen polymer chains are synthesized entirely by glycosyl transferases on the cytoplasmic side of the cytoplasmic membrane without the involvement of Wzy: the chains are elongated from the nonreducing end, and an ATP-binding cassette (ABC) transporter is required for transport of the complete O polysaccharide across the cytoplasmic membrane (5, 9, 32).The O-antigen gene cluster encodes the enzymes for biosynthesis of nucleotide sugars and the glycosyl transferases for O-unit assembly. The O-antigen gene clusters of several S. enterica serovars have been sequenced and studied in great detail (reviewed by Reeves [23]); those of E. coli serovars O9 (6, 9), O16 (strain K-12) (31), O111 (2), Flexneri (Shigella flexneri) (15, 16), and Dysenteriae (Shigella dysenteriae) (10) and of Yersinia pseudotuberculosis serovar IIA (8) have also been sequenced. The variation in the O-antigen gene clusters reflects the structural variation in O antigens. In general, the sugar pathway genes, such as those for dTDP-rhamnose or GDP-mannose, have a high degree of similarity between differen...
