Protection of the glutamate pool concentration in enteric bacteria

Abstract

The central nitrogen metabolic circuit in enteric bacteria consists of three enzymes: glutamine synthetase, glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). With the carbon skeleton provided by 2-oxoglutarate, ammonia/ammonium (NH(4)(+)) is assimilated into two central nitrogen intermediates, glutamate and glutamine. Although both serve as nitrogen donors for all biosynthetic needs, glutamate and glutamine play different roles. Internal glutamine serves as a sensor of external nitrogen availability, and its pool concentration decreases upon nitrogen limitation. A high glutamate pool concentration is required to maintain the internal K(+) pool. The configuration of high glutamate and low glutamine pools was disrupted in GOGAT(-) mutants under low NH(4)(+) conditions: the glutamate pool was low, the difference between glutamate and glutamine was diminished, and growth was defective. When a GOGAT(-) mutant was cultured in an NH(4)(+)-limited chemostat, two sequential spontaneous mutations occurred. Each resulted in a suppressor mutant that outgrew its predecessor in the chemostat. The first suppressor overexpressed GDH, and the second also had a partially impaired glutamine synthetase. The result was a triple mutant in which NH(4)(+) was assimilated by two enzymes instead of the normal three and yet glutamate and glutamine pools and growth were essentially normal. The results indicate preference for the usual ratio of glutamate and glutamine and the resilient and compensatory nature of the circuit on pool control. Analysis of other suppressor mutants selected on solid medium suggests that increased GDH expression is the key for rescue of the growth defect of GOGAT(-) mutants under low NH(4)(+) conditions.

Fig. 1.

The central nitrogen metabolic circuit in enteric bacteria. The three-enzyme circuit assimilates NH₄⁺ and produces two central intermediates, glutamine and glutamate. GS catalyzes glutamine synthesis. Glutamate can be synthesized by the action of either GS/GOGAT or GDH, respectively, with high or low affinity for NH₄⁺. The two glutamate molecules shown have no known functional difference.

Scheme 1.

Fig. 2.

GOGAT⁻ SK3062 (ΔgltB824) in an NH₄⁺-limited chemostat. The diluted minimal medium (0.2 × N⁻C⁻DB) contained 2 mM NH₄⁺ as the limiting N-source, and 0.2% glycerol as the C-source. Five dilution rates were chosen from 0.52 h⁻¹ (close to maximal growth rate, doubling time of 81 min, day 2) to 0.25 h⁻¹ (about half of the maximal growth rate, doubling time of 168 min, days 1 and 6), with each interval corresponding to a change in doubling time of ≈20 min. After each overnight equilibration, two to three samples were collected several hours apart. (Upper) Cell doubling time calculated from dilution rate measurement after each sample collection. (Lower) Cell density (○) and free NH₄⁺ concentration in the medium (▴).

Fig. 3.

GDH overexpression with gdhA promoter-up mutations. (A) Isogenic strains SK2633 (wild type, lane 1), SK3111 (gdh-515 zch-1436::Tn10, chemostat suppressor mutation, lane 2), and SK3128 (gdh-51 zch-1436::Tn10, null mutation, lane 3) were grown in nutrient broth. Proteins of lysates from equal amounts of cells were separated by 10% SDS/PAGE and examined by Western blot. (B) Sequences of the −10 and −35 motifs of gdhA promoter in wild type, gdh-515 (from chemostat early suppressor SK3074), and gdh-611 (from plate suppressor FG1143) alleles.

Fig. 4.

Growth of GOGAT⁻ suppressor mutants selected from low NH₄⁺ plates. Cells were grown in N⁻C⁻ medium with 1 mM NH₄⁺ as N-source and 0.2% glycerol as C-source. Batch cultures were inoculated with overnight cultures grown in the same medium except with 5 mM NH₄⁺. (A) S. typhimurium: SK2633 (wild type, ○), SK3062 (ΔgltB824, ×), the chemostat early suppressor SK3074 (ΔgltB824 gdh-515, ■), and the plate suppressors FG1143 (▴) and FG1158 (▾). (B) E. coli: NCM3722 (wild type, ○), FG1079 (gltD::Tn5KAN-I-SceI, ×), and the plate suppressors FG1126 (▴) and FG1127 (▾).