Regulation of two forms of glutamine synthetase in Escherichia coli by the ammonia content of the growth medium.

Addition of ammonium salts to E. coli cells grown on a medium with glutamate as the sole nitrogen source is followed by rapid inactivation of glutamine synthetase (GS) I1 The active form of the enzyme (GSa) and the inactive form (GSb) have been isolated.2 Both forms behave similarly in the analytical ultracentrifuge, electrophoresis, and ion exchange chromatography. 2 In a cell-free system GSa is converted to GSb by an "inactivating enzyme"3 in the presence of adenosine 5'-triphosphate (ATP), Mg2+, and glutamine.4 The inactivating enzyme catalyzes an adenylylation of GSa5-7 in the presence of glutamine or certain related metabolites as allosteric effectors.8 Kingdon and Stadtman9' 10 also reported the isolation of two forms of glutamine synthetase from E. coli cells grown on a medium containing either glutamate or ammonia, respectively, as sole nitrogen source. Both forms show characteristic differences in their responses to feedback inhibitors. In the present paper it will be shown that our GSa preparation behaves like the "ammonia preparation" and the GSb preparation like the "glutamate preparation" of Kingdon and Stadtman. As will be shown, in the late logarithmic or in the stationary growth phase, the "ammonia medium" contains no more ammonia, whereas the "glutamate medium" contains large amounts of ammonia. There is a striking parallelism between the appearance or disappearance of ammonia in the culture medium and the formation of GSb or GSa. The "ammonia preparation" of Kingdon and Stadtman is isolated from cultures with very low ammonia concentrations, and the "glutamate preparation" from cultures with high concentrations of ammonia. Both our previous data and those of Kingdon and Stadtman therefore suggest that ammonia controls the ratio GSa: GSb by causing formation of GSb from GSa. Results.-Growth (as recorded by optical density at 650 mM), specific activity of glutamine synthetase, ratio of glutamine synthetase to glutamyltransferase (S/T), and the concentration of ammonia in the media of batch cultures of E. coli are shown in Figure 1. The left side of the figure represents a culture in a medium with ammonia as the sole nitrogen source, whereas in the experiment shown on the right glutamate was the sole nitrogen source. The composition of the media as well as the other growth conditions were those of Kingdon and Stadtman.9 The specific activity of glutamine synthetase (i.e., GSa) increases sharply as ammonia disappears from the ammonia culture and decreases in a similar manner as ammonia appears in the glutamate culture. The ratio S/T decreases with the appearance of ammonia in the glutamate culture and shows high values in the ammonia culture during and after disappearance of ammonia. The ratio S/T is high in GSa and low in GSb.2, 11 The observed changes