Biosynthesis of the polysialic acid capsule of Escherichia coli K1: factors influencing cessation of capsule expression at 150C

Initial experiments were designed to determine if increases in unsaturated fatty acids (UFA) that usually occur in cells grown at 150C were related to defects in membrane-associated sialyltransferase (ST) activity at 150C. An E. coli K1 hybrid strain that did not increase UFA levels after growth at 150C due to a mutant fabF gene was constructed. Isogenic strains with and without the fabF defect produced capsule at 330C but not at 150C. Membranous ST complexes isolated from both strains grown at 330C transfered [14C]-sialic acid (NeuNAC) from CMP-[14C]-NeuNAc to endogeneous acceptors and to exogenous sialyl oligomers. Membranes from 150C grown cells of the fabF+ strain catalyzed incorporation of [14C]NeuNAc from CMP-[14C]-NeuNAc to exogenous sialyl oligomers, but required 2-4 h incubation at 330C for endogenous incorporation. Membranes from the fabF mutant strain grown at 150C did not incorporate [14C]NeuNAc from CMP-[14C]-NeuNAc under these conditions. We concluded that membrane-associated ST activity is not interrupted by low temperature increases in UFA content. Acapsular mutants derived from E. coli K1 that were defective in NeuNAc catabolism (NeuNAc aldolase) and activation or polymerization were used to examine the effects of growth at 150C on NeuNAc synthesis and initiation of polysialic acid capsule synthesis. These strains accumulated high internal NeuNAc internal NeuNAc at 370C, but NeuNAc was undetectable after growth at 150C. Intracellular NeuNAc levels increased within 10 min. after shift from 150C to 370C even in the presence of rifampicin (100 g ml-1) or chloramphenicol (100 g ml-1). Extracts from these strains grown at 150C and 370C lacked NeuNAc synthase activity in 150C assays, but were active in 370C assays. We conclude that NeuNAc synthase is present but nonfunctional at 150C