An alternative role of FoF1-ATP synthase in Escherichia coli: synthesis of thiamine triphosphate

Abstract

In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate yielding pyruvate. Here we show that in intact bacteria ThTP is synthesized from free thiamine diphosphate (ThDP) and P(i), the reaction being energized by the proton-motive force (Δp) generated by the respiratory chain. ThTP production is suppressed in strains carrying mutations in F(1) or a deletion of the atp operon. Transformation with a plasmid encoding the whole atp operon fully restored ThTP production, highlighting the requirement for F(o)F(1)-ATP synthase in ThTP synthesis. Our results show that, under specific conditions of nutritional downshift, F(o)F(1)-ATP synthase catalyzes the synthesis of ThTP, rather than ATP, through a highly regulated process requiring pyruvate oxidation. Moreover, this chemiosmotic mechanism for ThTP production is conserved from E. coli to mammalian brain mitochondria.

Figure 1. ThTP is formed from ThDP.

The bacteria (BL21 or CV2) were grown overnight in LB medium, transferred to minimal M9 medium and incubated at 37°C. Thiamine derivatives were determined at zero time and 1 h after addition of glucose (10 mM). In both strains, the ThDP content decreases after 1 h, but the total amount [ThDP] + [ThTP] remains constant. The results are expressed as mean ± SD for 3 experiments (*, p < 0.05; **, p < 0.01; one-way ANOVA followed by the Dunnett post-test for comparison with ThDP levels at t = 0).

Figure 2. Effect of metabolic inhibitors and anoxia on the ThTP content of BL21 cells.

The bacteria were grown overnight in LB medium, transferred to minimal M9 medium and incubated 20 min at 37°C either in the absence of substrate or in the presence of D-glucose (10 mM) or L-lactate (10 mM). In each case, the control experiment was carried out in the presence of O₂. For growth in the absence of oxygen, the bacteria were incubated in sterile tubes with screw caps (Greiner Bio-One BVBA/SPRL) and the culture was sparged with N₂ for 1 min and the tubes were hermetically closed before incubation. N₂: oxygen replaced by nitrogen; KCN: 1 mM cyanide was added in the presence of O₂; IAA: 1 mM iodoacetete was added in the presence of O₂. (**, p < 0.01; *, p < 0.05: two-way ANOVA followed by the Dunnett test for comparisons with the respective control, Means ± SD, n = 4).

Figure 3. Dose-dependent effects of CCCP and DCCD on intracellular ThTP content in the E. coli BL21 strain.

The bacteria were grown overnight in LB medium, transferred to minimal M9 medium containing 10 mM D-glucose and incubated (37°C, 20 min) in the presence of CCCP (a) or DCCD (b) at the concentrations indicated. Stock solutions of CCCP and DCCD were made in dimethyl sulfoxide and used at a final solvent concentration of 1%. (Means ± SD, n = 3).

Figure 4. Effect of CCCP on intracellular ThTP levels in the E. coli CV2 strain.

The bacteria were grown overnight in LB medium and transferred to a minimal M9 medium containing 10 mM L-lactate either at 25 or at 37°C. CCCP (50 μM) was added after 1 h. (Means ± SD, n = 3).

Figure 5. ThTP synthesis in E. coli mutants carrying mutations in the α subunit of F₁ (AN120 and AN718) or in the a subunit of F_o (AN382).

The wild-type (MG1655) and the mutants were grown overnight in LB medium (37°C, 250 rpm). For the mutant strains, streptomycin (200 μg/ml) was added to the medium. The bacteria were transferred to M9 medium and incubated (1 h at 37°C) in the absence of glucose (control) or in the presence of glucose (10 mM) with or without CCCP (50 μM). (Means ± SD, n = 3).

Figure 6. ThTP synthesis in the DK8 strain and the DK8 strain containing the whole atp operon.

The DK8(Δunc) strain was grown overnight in LB medium in the presence of 30 mg/l tetracycline (37°C, 250 rpm). For the DK8 (pBWU13unc) strain, the medium also contained in addition 100 mg/l ampicillin. Then, the bacteria were transferred to M9 medium containing 10 mM of either D-glucose or L-lactate and incubated at 37°C. (Means ± SD, n = 3).

Figure 7. Dependence of ThTP synthesis on external phosphate concentration in intact P_i-depleted bacteria.

The bacteria (wild-type MG1655 or CF5802) were grown overnight in LB medium, transferred to minimal M9 medium devoid of P_i (replaced by chloride) and preincubated for 4 h at 37°C to deplete them of endogenous P_i. Then, glucose (10 mM) and Na₂HPO₄ (at the concentrations indicated) were added and ThTP was determined after 20 min. (Means ± SD, n = 3).

Figure 8. Mechanism and regulation of ThTP synthesis in E. coli.

Under conditions of amino acid starvation and in the presence of an energy substrate yielding pyruvate, a hypothetical activator is formed (presumably from acetyl-CoA). This would shift F₁ from the normal conformation (catalyzing ATP synthesis or hydrolysis) to a ThTP synthase conformation, binding ThDP or ThTP rather then ADP or ATP. Both ATP and ThTP synthesis are energized by the proton-motive force generated by the respiratory chain.