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Most microorganisms do not produce sialic acid (sialate), andthose that do appear to use a biosynthetic mechanism distinct frommammals. Genetic hybrids of nonpathogenic, sialate-negative laboratory Escherichia coli K-12 strains designed for the de novo synthesis of the polysialic acid capsulefrom E. coli K1 proved useful in elucidating the geneticsand biochemistry of capsule biosynthesis. In this article we proposea dynamic model of sialometabolism to investigate the effects ofbiosynthetic neu (N-acetylneuraminicacid) and catabolic nan (N-acylneuraminate) mutationson the flux of intermediates through the sialate synthetic pathway.Intracellular sialate concentrations were determined by high pHanion exchange chromatography with pulsed amperometric detection.The results indicated that a strain carrying a null defect in thegene encoding polysialyltransferase (neuS) accumulated > 50times more CMP–sialic acid than the wild type when strainswere grown in a minimal medium supplemented with glucose and casaminoacids. Metabolic accumulation of CMP–sialic acid dependedon a functional sialic acid synthase (neuB), as shownby the inability of a strain lacking this enzyme to accumulate adetectable endogenous sialate pool. The neuB mutantconcentrated trace sialate from the medium, indicating its potentialvalue for quantitative analysis of free sialic acids in complexbiological samples. The function of the sialate aldolase (encodedby nanA) in limiting intermediate flux throughthe synthetic pathway was determined by analyzing free sialate accumulationin neuA (CMP–sialic acid synthetase) nanA double mutants. The combined results demonstratehow E. coli avoids a futile cycle in which biosyntheticsialate induces the system for its own degradation and indicatethe feasibility of generating sialooligosaccharide precursors throughtargeted manipulation of sialate metabolism. [ABSTRACT FROM PUBLISHER]