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Biosynthesis of the polysialic acid capsule of Escherichia coli K1: factors influencing cessation of capsule expression at 15/sup 0/C

Thesis/Dissertation ·
OSTI ID:7002168
Initial experiments were designed to determine if increases in unsaturated fatty acids (UFA) that usually occur in cells grown at 15/sup 0/C were related to defects in membrane-associated sialyltransferase (ST) activity at 15/sup 0/C. An E. coli K1 hybrid strain that did not increase UFA levels after growth at 15/sup 0/C due to a mutant fabF gene was constructed. Isogenic strains with and without the fabF defect produced capsule at 33/sup 0/C but not at 15/sup 0/C. Membranous ST complexes isolated from both strains grown at 33/sup 0/C transfered (/sup 14/C)-sialic acid (NeuNAC) from CMP-(/sup 14/C)-NeuNAc to endogeneous acceptors and to exogenous sialyl oligomers. Membranes from 15/sup 0/C grown cells of the fabF/sup +/ strain catalyzed incorporation of (/sup 14/C)NeuNAc from CMP-(/sup 14/C)-NeuNAc to exogenous sialyl oligomers, but required 2-4 h incubation at 33/sup 0/C for endogenous incorporation. Membranes from the fabF mutant strain grown at 15/sup 0/C did not incorporate (/sup 14/C)NeuNAc from CMP-(/sup 14/C)-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 15/sup 0/C on NeuNAc synthesis and initiation of polysialic acid capsule synthesis. These strains accumulated high internal NeuNAc internal NeuNAc at 37/sup 0/C, but NeuNAc was undetectable after growth at 15/sup 0/C. Intracellular NeuNAc levels increased within 10 min. after shift from 15/sup 0/C to 37/sup 0/C even in the presence of rifampicin (100 g ml/sup -1/) or chloramphenicol (100 g ml/sup -1/). Extracts from these strains grown at 15/sup 0/C and 37/sup 0/C lacked NeuNAc synthase activity in 15/sup 0/C assays, but were active in 37/sup 0/C assays. We conclude that NeuNAc synthase is present but nonfunctional at 15/sup 0/C.
Research Organization:
California Univ., Davis (USA)
OSTI ID:
7002168
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550701 -- Microbiology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDE-LYASES
ALDOLASE
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
CARBOHYDRATES
CARBON COMPOUNDS
CARBON-CARBON LYASES
CARBOXYLIC ACIDS
CATABOLISM
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
HYBRIDIZATION
ISOTOPE APPLICATIONS
KINETICS
LIGASES
LYASES
MEMBRANES
METABOLISM
MICROORGANISMS
MONOSACCHARIDES
MUTANTS
ORGANIC ACIDS
ORGANIC COMPOUNDS
POLYMERIZATION
REACTION KINETICS
RESPONSE MODIFYING FACTORS
SACCHARIDES
SIALIC ACID
SYNTHESIS
TEMPERATURE DEPENDENCE
TRACER TECHNIQUES
TRANSFERASES