Type 1 and 4 capsular polysaccharides of Escherichia coli, EPS and most O-antigens of Gram-negative bacteria follow the second pathway, i.e. assembly of the repeating unit in the cytoplasm and polymerization thereof in the periplasm combined with translocation outside the cell. This pathway is called the Wzx/Wzy-dependent pathway, as it requires the Wzx flippase and Wzy polymerase,Pepstatin A unlike systems involving ABCtransporters or synthase proteins. Polysaccharide biosynthesis is initiated by glycosyltransferases involved in the assembly of the repeating unit on the lipid carrier undecaprenyl pyrophosphate. Complete subunits are then translocated to the periplasmic face of the inner membrane by the Wzx flippase and then polymerized by the Wzy polymerase to the extent that is regulated by the Wzc co-polymerase. Proteins engaged in EPS synthesis in R. leguminosarum bv. trifolii are encoded within the chromosomal Pss-I region. The region comprises genes encoding glycosyltransferases,Rilmenidine Phosphate a putative flippase, a polymerase, a co-polymerase, and an outer membrane channel protein. The functions of several glycosyltransferases encoded within the region were previously dissected. Glucosyl-IP-transferase PssA is the priming glycosyltransferase initiating the assembly of the octasaccharide EPS unit by the transfer of UDP-glucose to the undecaprenyl phosphate lipid carrier attached to the cytoplasmic membrane. In the subsequent step, a glucuronosyl- glucosyl transferase composed of PssD and PssE catalyses the addition of a glucuronic acid residue. The addition of the second glucuronic acid is mediated by the glucuronosyl-b-1,4glucuronosyltransferase PssC. The outcome of mutations in pssA, pssD, pssE, and pssC is pleiotropic and in addition to abolishing the capacity to synthesize EPS, it affects the level of synthesis of several cellular proteins. PssL is homologous to Wzx and was proposed to function as a flippase that translocates EPS subunits to the outer leaflet of the inner membrane. PssT is homologous to Wzy and serves as a polymerase of EPS subunits; the pssT mutant produced EPS with a greater amount of highmolecular-weight EPS than the wild type. Polymerization of polysaccharides is influenced by a protein assigned to a family of polysaccharide co-polymerases that are distinguished by their common membrane topology with a large periplasmic loop flanked by two transmembrane segments.