Effect of glutamine on enzymes of nitrogen metabolism in Bacillus subtilis

Abstract

An earlier study of the regulation of glutamate synthase (GOGAT) in Bacillus subtilis (Deshpande et al., Bichem. Biophys. Res. Commun. 95:55--60, 1980) revealed an inverse relationship between the specific activity of this essential ammonia-assimilatory enzyme and the intracellular pool of glutamine: GOGAT activity decreased when the internal glutamine concentration reached or exceeded 2.5 mM. This finding prompted the present investigation of the intracellular events linking glutamine formation to the regulation of GOGAT. A growing culture of B. subtilis was shifted from glutamate plus NH+4 medium (high GOGAT activity) to glutamate medium (low GOGAT activity). At various times after the shift, the intracellular concentrations of aspartate, glutamate, glutamine, alanine, and NH+4 and the activities of GOGAT and glutamine synthetase (GS) were measured. After 30 min, the only significant pool level change was an eightfold increase in glutamine, which paralleled a 2- to 3-fold increase in GS activity. Approximately 15 min after the glutamine pool reached its peak, GOGAT activity began to decrease and eventually declined 2.5-fold. In contrast, when B. subtilis was shifted from glutamate medium to glutamate plus NH+4 medium, there was a 1- to 2-h lag before the glutamine pool and GS activity approached a steady state. As a result, GOGAT activity was low until the concentration of glutamine dropped below 2.5 mM. We propose that glutamine is an important regulatory element in the control of GOGAT activity and that one form of GOGAT regulation involves enzyme inactivation. In addition, these results indicate that glutamine is neither a corepressor nor a feedback inhibitor of GS.

Sensing of Nitrogen Limitation by Bacillus subtilis: Comparison to Enteric Bacteria

“…However, it is clear that a low glutamine pool is not required for high levels of glnA expression (e.g., results for wild-type B. subtilis grown on GABA or urea in batch culture [ Table 2]), glnA mutant strain B276 grown on ammonia in batch culture [ Table 6], and a Salmonella gdh mutant strain grown under ammonia-sufficient conditions in the chemostat [ Table 4]). Schreier et al (42), Deshpande et al (9), and Fisher and Sonenshein (15) have all reported that glnA expression in B. subtilis can be high independent of a drop in the glutamine pool (12,36). Potential roles of other metabolites in controlling glnA transcription in both Bacillus and Salmonella remain to be elucidated, as do the roles of GS itself and the TnrA product in B. subtilis (5,12,36,38,43,49).…”

In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli

“…The filter was washed with 75 ml of M9 salts and placed in ice-cold perchloric acid for 20 min to lyse the cells. The perchlorate was precipitated with potassium carbonate as described by Deshpande et al (7) to provide a cell-free supernatant for quantification of intracellular components. Urocanate concentrations were determined by high-pressure liquid chromatography analysis on an Altex Ultrasphere-ODS reverse-phase column.…”

Ammonium-assimilating enzymes and erythromycin formation in Saccharolypospora erythrea.

“…Ammonium assimilation proceeds via GS/GOGAT or GDH in enteric bacteria (25) whereas in some gram-positive organisms GDH is absent (8). In actinomycetes, we found examples that resembled the enteric bacteria in ammonium assimilation enzymes.…”