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Title: The metabolism of amino sugars
Author: Bates, C. J.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1964
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The incorporation and distribution of radioactivity from 1-14C-D-glucosamine, N-acetyl-1-14C-D-glucosamine and 1-14C-N-acetyl-D-glucosamine in growing cultures of Bacillus subtilis NCTC 1379 has been investigated. The radioactivity from N-acetyl-1-14C-D-glucosamine is incorporated rapidly and the rate of incorporation is proportional to the external concentration over a wide range. Both compounds are incorporated more rapidly in cells pregrown in the presence of amino sugars than in cells pregrown without amino sugar. The incorporation of 1-14C-D-glucosamine is greatly reduced by D-glucose, but very little affected by N-acetyl-D-glucosamine, N-propionyl-D-glucosamine or N-formyl-D-glucosamine in the growth medium. The incorporation of N-acetyl-1-14C-D-glucosamine is not greatly reduced by D-glucose, but severely affected by N-propionyl-D-glucosamine and N-formyl-D-glucosamine. Radioactivity from 1-14C-D-glucosamine and N-acetyl-1-14C-D-glucosamine is incorporated mainly into the hot trichloracetic acid fraction and residue fraction of Bacillus subtilis, whereas that from 1-14C-N-acetyl-D-glucosamine is incorporated into all the fractions. Evidence is presented that the acetyl group of N-acetyl-D-glucosamine is liberated or exchanged during incorporation of the glucosamine moiety. The distribution of radioactivity from labelled amino sugars in Escherichia coli is somewhat different from that obtained in Bacillus subtilis. When the fractions obtained from Bacillus subtilis grown with 1-14C-D-glucosamine or N-acetyl-D-14C-D-glucosamine are subjected to acid hydrolysis, a high proportion of the radioactivity can be recovered in compounds behaving like glucosamine, galactosamine and muramic acid; most of the muramic acid is present in the residue fraction. The overall ratios in terms of radioactivity are: glucosamine (14) : galactosamine (1) : muramic acid (4). Addition of 6-azauracil to the growth medium results in a small increase in amino sugar-containing material in the acid-soluble fraction of the cells. 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (deaminating), E.C. (Gm-6-P deaminase) and 2-acetylamino-2-deoxy-D-glucose-6-phosphate N-acetyl hydrolase (AcGm-6-P deacetylase) are induced by N-acetyl-D-glucosamine in Bacillus subtilis and in Escherichia coli, but not by many structurally related amino sugars in Bacillus subtilis. L-glutamine-D-fructose-6-phosphate aminotransferase, E.C. (Gm-6-P synthetase) is repressed by N-acetyl-D-glucosamine in Bacillus subtilis and Escherichia coli and by D-glucosamine, N-propionyl-D-glucosamine and N-formyl-D-glucosamine in Bacillus subtilis. Other structurally related amino sugars were found not to repress this enzyme in Bacillus subtilis.

All the amino sugars which produce control effects are metabolised by the growing cells; a gratuitous inducer or repressor was not found. If D-glucose is added to a culture of Bacillus subtilis growing in the presence of N-acetyl-D-glucosamine, the induction of Gm-6-P deaminase and that of AcGm-6-P deacetylase are reduced from 20-30 fold to 1-3 fold. Likewise the repression of Gm-6-P synthetase is reduced from tenfold to twofold. If D-glucose is added to a culture of Bacillus subtilis growing in the presence of D-glucosamine, N-formyl-D-glucosamine or N-propionyl-D-glucosamine, the repression of Gm-6-P synthetase is abolished. Glucose has a much smaller effect on the induction of Gm-6-P deaminase and AcGm-6-P deacetylase by N-acetyl-D-glucosamine in Escherichia coli. It does, however, reduce the rate of disappearance of N-acetyl-D-glucosamine from the growth medium and the rate of growth of this organism on N-acetyl-D-glucosamine as sole nitrogen source. Benzyl penicillin, 2,6 dimethoxybenzamide penicillin and 6-azauracil have no appreciable effect on the specific activity Gm-6-P deaminase, AcGm-6-P deacetylase or Gm-6-P synthetase in Bacillus subtilis. When grown beyond the end of logarithmic phase in broth + glutamate medium, the specific activity of Gm-6-P deaminase in Bacillus subtilis is increased. If glucose is present in the growth medium, this increase is not observed, but instead some material behaving like an acylated amino sugar accumulates in the growth medium. Methods for the partial purification of Gm-6-P deaminase from Bacillus subtilis are described. L-valine-sRNA ligase (AMP), E.C. was shown to be present in extracts of Bacillus subtilis. Attempts to demonstrate incorporation of radioactivity from 1-14C-DL-valine and synthesis of Gm-6-P deaminase in a cell-free system were unsuccessful. Kinases responsible for the phosphorylation of D-glucosamine and of N-acetyl-D-glucosamine were demonstrated in extracts of Bacillus subtilis and Escherichia coli. The products formed form radioactive amino sugars by Bacillus subtilis extracts were purified and their properties examined. In Bacillus subtilis, both kinases are induced by N-acetyl-D-glucosamine and by D-glucosamine, whereas in Escherichia coli they are constitutive. In both organisms the kinase which phosphorylates D-glucosamine appears to be separate from that which phosphorylates N-acetyl-D-glucosamine. The activity of acetyl CoA : 2-amino-2-deoxy-D-glucose-1-phosphate N-acetyl transferase is slightly lower in extracts of Escherichia coli grown on N-acetyl-D-glucosamine than in extracts of cells grown on D-glucose. The activity of D-glucose-6-phosphate ketol-isomerase E.C. is the same in both extracts.

Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available