Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes.

Abstract

Certain mutations at the glnB locus result in the failure to fully derepress glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] and to convert it to the active nonadenylylated form in response to nitrogen limitation. In these mutants the P11 regulatory protein is altered such that it cannot be converted by uridylyltransferase to the form stimulating deadenylylation of glutamine synthetase by adenylyltransferase. Additional mutations as well as insertions of transposon Tn5 at the gInB site result in the loss of P11. The loss of PI, does not prevent adenylylation and deadenylylation of glutamine synthetase but reduces the rates of these reactions. Cells lacking PII have a high level of glutamine synthetase even when they are grown with an excess of ammonia and the enzyme is highly adenylylated. The results suggest that the PII protein plays a role, independent of its effect on adenylylation, in the regulation of the level of glutamine synthetase. The biosynthetic activity of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] in enteric bacteria is progressively decreased by the specific covalent attachment of adenylyl groups to each of its 12 subunits (1). This reversible modification is catalyzed by the enzyme adenylyltransferase (ATase). Adenylylation is stimulated by the small (44,000 dalton) regulatory protein PITA (2). Deadenylylation is stimulated by PIID, a uridylylated form of PIIA. The interconversion of PIIA and PIID is catalyzed by uridylyltransferase (UTase) and uridylyl-removing enzyme(s).Glutamine stimulates the adenylylation reaction as well as the conversion of PID to PITA; 2-ketoglutarate inhibits adenylylation but stimulates the deadenylylation reaction and the conversion of PITA to PIID. Glutamine inhibits the latter two reactions. Thus, the relative amounts of PITA and PIID and the adenylylation state of glutamine synthetase are determined in a reciprocal manner by the concentrations of these two metabolites; these in turn are determined by the availability of nitrogen in the cell. When growth is limited by the nitrogen source, the level of 2-ketoglutarate is high, the level of glutamine is low, and the adenylylation state of glutamine synthetase is low. When growth is limited by some other component, such as carbon, glutamine is high, 2-ketoglutarate is low, and the adenylylation state of glutamine synthetase is high (3). In addition, the synthesis of glutamine synthetase is regulated by the availability of nitrogen. During nitrogen-limited growth the level of glutamine synthetase is high, whereas during carbonlimited growth the level of glutamine synthetase is low (3,4).We have studied the regulation of the level and the adenyl- MATERIALS AND METHODS Bacterial Strains. All strains are phage Pl-sensitive derivatives (5) of K. aerogenes MK-1 (6). Media have been described (7).Genetic Techniques. Generalized transduction was carried out as described by Goldberg et al. (5) with phage P1clrLO0CM (obtained from B. Tyler). Lysates of donor st...

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli

“…In agreement with the regulation of ammonia assimilation which is described above and generally accepted, Foor et al (1980) showed that in a Klebsiella aerogenes glnB transposon-insertion mutant, the rate of GS-AMP deadenylylation after removal of ammonia was strongly decreased when compared to the wild type. The same was found in a Klebsiella pneumoniae glnB insertion mutant (W. C. van Heeswijk, and D. Molenaar, unpublished).…”

“…The fact that this dependence was incomplete (Foor et al, 1980) suggests the existence of a second P II protein in this closely related organism, albeit with a lower activity than the GlnK protein described in this study.…”

PIISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

“…The product of glnB was finally identified as P II by Foor et al, who showed that in the original K. aerogenes glnB mutant, P II was altered so that it could not be uridylylated in nitrogenlimited conditions and consequently could not stimulate dead-enylylation of GS by Atase (64,65). A second class of glnB mutations resulted in loss of P II , and these glnB null mutants were still able to adenylylate and deadenylylate GS, although the rates of these reactions were reduced.…”

Characterization of Escherichia coli glnL mutations affecting nitrogen regulation

“…The figure is modeled after a portion of Fig. 8 of reference 41. consequently prevent the expression of the Ntr regulon (3,4,6,10,11,19,20). Suppressor mutations that restore expression of the Ntr regulon have been found in ginL, ginB, and ginG (6,11,19,20,33,43).…”

“…8 of reference 41. consequently prevent the expression of the Ntr regulon (3,4,6,10,11,19,20). Suppressor mutations that restore expression of the Ntr regulon have been found in ginL, ginB, and ginG (6,11,19,20,33,43). Previous results have suggested that the suppressor mutations in glnL and ginB affect the NRII-PI phosphatase activity, whereas the suppressor mutations in glnG result in altered NRI proteins that are able to activate glnA and Ntr expression in the absence of phosphorylation or that activate transcription much more efficiently than does wild-type NR,-phosphate after infrequent phosphorylation by other cross-talking cellular kinases (29).…”