Transport and incorporation of N-acetyl-D-glucosamine in Bacillus subtilis

Abstract

Bacillus subtilis 168 has been found to possess a high-affinity transport system for N-acetyl-D-glucosamine (GlcNAc). The Km for uptake was approximately 3.7 μM GlcNAc, regardless of the nutritional background of the cells. Apparent increases in V(max) were noted when the bacteria were grown in the presence of GlcNAc. The uptake of GlcNAc by B. subtilis was highly stereoselective; D-glucose, D-glucosamine, N-acetyl-D-galactosamine, D-galactose, D-mannose, and N-acetylmuramic acid did not inhibit GlcNAc uptake. In contrast, glycerol was an effective inhibitor of [3H]GlcNAc transport and incorporation. Partial inhibition of GlcNAc uptake was observed with azide, fluoride, and cyanide anions, carbonyl cyanide-m-chlorophenyl hydrazone, methyltriphenylphosphonium bromide, N,N'-dicyclohexylcarbodiimide, gramicidin, valinomycin, monensin, and nigericin. Two anions, arsenite and iodoacetate, were potent inhibitors of the uptake of GlcNAc in B. subtilis. Results from paper chromatography showed that there was no intracellular pool of free GlcNAc and that the acetylamino sugar was probably phosphorylated during transport. A modification of the Park-Hancock cell fractionation scheme indicated that cells grown on glycerol or D-glucose incorporated [3H]GlcNAc primarily into the cell wall fraction. When GlcNAc was used as the sole carbon source, label could be demonstrated in fractions susceptible to protease and nuclease, as well as lysozyme, showing that the N-acetylamino sugar was utilized in macromolecular synthesis and energy metabolism.