Uptake of N-acetyl-D-mannosamine: an essential intermediate in polysialic acid biosynthesis by Escherichia coli K92

The N-acetyl-D-mannosamine (ManNAc) transport system of Escherichia coli K9 2 was studied when this bacterium was grown in a chemically defined medium containing ManNAc as carbon source. Kinetic measurements,were carried out i n vivo at 37 degrees C in 25 mM phosphate buffer, pH 7.5, Under these condi tions, the uptake rate was linear for at least 15 min and the calculated K- m for ManNAc was 280 mu M. The transport system,vas strongly inhibited by s odium arsenate (97%), potassium cyanide (84%) and 2,4-dinitrophenol (88%) a dded at final concentrations of 1 mM (each). Analysis of bacterial ManNAc p hosphotransferase activity revealed in vitro ManNAc phosphorylation activit y only when phosphoenolpyruvate was present. These results strongly support the notion that ManNAc uptake depends on a specific phosphotransferase sys tem, Study of specificities showed that N-acetylglucosamine and mannosamine specifically inhibited the transport of ManNAc in this bacterium. Analysis of expression revealed that the ManNAc transport system was induced by Man NAc, glucosamine, galactosamine, mannosamine and mannose but not by N-acety lglucosamine or N-acetylgalactosamine. Moreover, ManNAc permease mas subjec t to glucose repression and cAMP stimulation. Full induction of the ManNAc transport system required the simultaneous presence of both cAMP and ManNAc . (C) 1999 Federation of European Biochemical Societies.