Thesis

Nitrogen assimilation in Sporosarcina ureae

Ammonia, glutamate, or glutamine can be utilized as sole source of nitrogen by Sporosarcina ureae. The growth rate is proportional to the concentration of ammonia up to a set value of 0.6 div./h at 84 mM, but concentration independent for glutamate at 0.5 div./h and glutamine at 0.33 div/h. When glutamate or glutamine is supplied as the sole source of carbon, energy, and nitrogen a diphasic type of growth is observed. Glutamate synthase (GOGAT; EC 1.4.1.13) is not detected in extracts of cells grown in tryptic soy yeast (TSY) medium nor in defined media containing acetate and ammonia, glutamate or glutamine; however, glutamine synthetase (GS; EC 6.3.1.2.) exhibits both biosynthetic and transferase activities at basal levels in extracts of cells grown under all growth conditions. A two-fold increase in the GS basal activity is observed in extracts of cells grown in ammonia-limited media. Two distinct glutamate dehydrogenases were detected and separated electrophoretically. One is specific for NAO (NAO-GOH; EC 1.4.1.2) and the other for NAOP (NAOP-GOH; EC 1.4.1.4). The NAO-GOH has an unusually high degree of thermal stability maintaining 95-100% of its activity after incubation for 15 minutes at 75�C; however, it irreversibly loses its activity upon freezing. The NAOP-GOH maintains 100% of its activity upon freezing but cannot survive the treatment at 75�C. The NAO-GOH exhibits increased activity in extracts of cells grown with glutamate as carbon and energy source, whereas the NAOP-GOH activity increases in extracts of cells grown in limiting ammonia concentrations. The Km values for glutamate in extracts of cells grown on TSY at pH 7.7 are 5mM and 30 mM for NADGDH and NADP-GDH, respectively. Two heat-labile glutaminases were separated electrophoretically; glutaminase ? is constitutive and glutaminase ? is repressed in complex media. Possible mechanisms of nitrogen assimilation in this organism are discussed.

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