EXPRESSION OF THE CAPSULAR K5 POLYSACCHARIDE OF ESCHERICHIA-COLI - BIOCHEMICAL AND ELECTRON-MICROSCOPIC ANALYSES OF MUTANTS WITH DEFECTS INREGION-1 OF THE K5 GENE-CLUSTER

The gene cluster of the capsular K5 polysaccharide, a representative o f group II capsular antigens of Escherichia coli, has been cloned prev iously, and three regions responsible for polymerization and surface e xpression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Regi on 1 has now been sequenced, and five open reading frames (kpsEDUCS) h ave been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and 1. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In thi s study, we characterized region 1 mutants by immunoelectron microscop y, membrane-associated polymerization activity, cytoplasmic CMP-2-keto -3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of t heir K5 polysaccharides. Certain mutations within region 1 not only ef fected polysaccharide transport (lack of region 1 gene products) but a lso impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain le ngth. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was fo und with extracellular and periplasmic polysaccharide and not with cyt oplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO sy nthetase) showed that CMP-KDO is engaged not in initiation of polymeri zation but in translocation of the polysaccharide.