Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli

Abstract

An osmosensitive mutant of Escherichia coli was isolated and shown to harbor two mutations that were together necessary for osmosensitivity. One (ossB) was an insertion mutation in the gltBD operon, which encodes the enzyme glutamate synthase (GOGAT), involved in ammonia assimilation and L-glutamate biosynthesis. The other (ossA) was in the fnr gene, encoding the regulator protein FNR for anaerobic gene expression. Several missense or deletion mutations in fnr and gltBD behaved like ossA and ossB, respectively, in conferring osmosensitivity. A mutation affecting the DNA-binding domain of FNR was recessive to fnr ؉ with respect to the osmotolerance phenotype but was dominant-negative for its effect on expression of genes in anaerobic respiration. Our results may most simply be interpreted as suggesting the requirement for monomeric FNR during aerobic growth of E. coli in high-osmolarity media, presumably for L-glutamate accumulation via the GOGAT-independent pathway (catalyzed by glutamate dehydrogenase [GDH]), but the mechanism of FNR action is not known. We also found that the spoT gene (encoding guanosine 3,5-bispyrophosphate

Evidence for an Arginine Exporter Encoded by yggA ( argO ) That Is Regulated by the LysR-Type Transcriptional Regulator ArgP in Escherichia coli

“…Unless otherwise indicated, supplementation with Arg, Lys, and the dipeptides was done at a concentration of 1 mM. The concentrations of antibiotics used were described previously (38). Trimethoprim was used in nutrient and defined media at 60 and 30 g/ml, respectively.…”

Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective

“…Fifth, in glucose-glutamine medium, considered to yield intracellular nitrogen-limited conditions, GDH was hyperinduced in a GOGAT mutant (53). Sixth, no growth of a GOGAT mutant was observed in a glucose medium with a low (0.4 mM) ammonium concentration, but growth did occur when poorer carbon sources (such as maltose, glycerol, or succinate) were used instead (494). It was argued that C limitation might lead to accumulation of ppGpp, which in its turn might stimulate GDH expression.…”

“…Likewise, it was argued that GOGAT mutants grow slowly on poor organic nitrogen sources, because they are starved for glutamate (53). Later, however, it was demonstrated that with ammonium as the N source, it holds true only when glucose is the carbon source; a minimal medium with poorer C sources (such as glycerol, succinate, or maltose) did support growth at a low ammonium concentration (0.4 mM), where glucose failed to do so (494). Mutants lacking both GOGAT and GDH require externally added glutamate for growth, even in the presence of ample ammonium (593).…”

“…However, whether the mechanism of repression of gdhA in E. coli by Nac E is similar to that of Nac K remains to be determined. The expression of gdhA may also be affected by the global transcriptional regulator FNR during growth in high-osmolarity media, but the underlying mechanism is unknown (494). Finally, gdhA transcription is regulated by ArgP.…”

“…It is activated by Lrp (488)(489)(490)(491), by IHF (490,492), and by GadE, a transcriptional regulator of genes involved in pH homeostasis (493) (see below). In addition, transcription of gltBDF is repressed by Crp-cAMP (484,490), by the fumarate nitrate reductase regulator (FNR) (494), by Nac (32,53), and by ArgR, the arginine repressor (490) (see sections on Nac and Crp-cAMP below).…”