Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum

Abstract

Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/alpha-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

FIG. 1.

Glycolysis and the rapid interchange between the carboxyl group of pyruvate and CO₂. (A) Overview of active and inactive pathways. Glycolysis operates normally through phosphoenolpyruvate with the Entner-Doudoroff pathway inactive (see Fig. S1 in the supplemental material). The reaction catalyzed by pyruvate ferredoxin oxidoreductase (PFOR) is partially, but not fully, reversible. GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate. (B) Dynamic incorporation of uniformly ¹³C-labeled glucose (100%) into glycolysis intermediates. Glycolysis intermediates through phosphoenolpyruvate were labeled rapidly and completely. Pyruvate, however, appeared in roughly equimolar amounts in its fully labeled form and in an unexpected form with two ¹³C carbons. Environmental CO₂ was ∼99% nonlabeled. The x axis represents minutes after the switch from unlabeled to [U-¹³C]glucose medium, and the y axis represents the fraction of the observed compound of the indicated isotopic form. (C) Steady-state labeling patterns of phosphoenolpyruvate and pyruvate obtained from cells grown in [3-¹³C]glucose (100%), [4-¹³C]glucose (100%), or [1,2-¹³C]glucose (100%). In [3-¹³C]glucose or [4-¹³C]glucose, about half of the phosphoenolpyruvate was labeled but only about a quarter of pyruvate was labeled. In contrast, growth in [1,2-¹³C]glucose resulted in identical labeling patterns for phosphoenolpyruvate and pyruvate. Environmental CO₂ was ∼99% nonlabeled. These results indicate that the ¹³C label in pyruvate is specifically lost from the carboxyl carbon. (D) The fraction of [1-¹³C]pyruvate increased with increasing amounts of NaH¹³CO₃ added to the medium. Cells were fed unlabeled glucose throughout, and labeling of upstream glycolysis intermediates was minimal or nonexistent (not shown). This experiment was performed in liquid closed-vessel cultures. The data, in conjunction with those in panels B and C, indicate exchange of the carboxyl carbon of pyruvate with carbon dioxide. (E) [U-¹³C]acetate was assimilated and incorporated into acetyl phosphate and acetyl-CoA. Pyruvate, however, remained unlabeled. The data suggest that the reaction catalyzed by PFOR is not fully reversible. The error bars in panels B through E show standard deviations (SD) (n = 2 to 4 independent experiments).

FIG. 2.

Complete bifurcated TCA cycle in C. acetobutylicum. (A) The diagram represents the proposed bifurcated TCA cycle in C. acetobutylicum. α-Ketoglutarate is produced from oxaloacetate and acetyl-CoA via citrate. Succinate can be produced reductively from fumarate or oxidatively from α-ketoglutarate. Gray boxes show the fate of the carbons in the incoming acetyl group from acetyl-CoA, and dotted boxes show the fate of the carbons in the carboxyl group from pyruvate. The unusual stereospecificity of citrate synthesis was confirmed by MS/MS analysis (see Fig. S5 in the supplemental material). Panels B and C show the dynamic incorporation of [U-¹³C]glucose (100%) and [U-¹³C]acetate (in the presence of nonlabeled glucose) into TCA metabolites and glutamate. The x axis represents minutes after the switch from unlabeled to ¹³C-labeled medium, and the y axis represents the fraction of the observed compound of the indicated isotopic form. There was no detectable labeling of oxaloacetate, malate, or fumarate in the [U-¹³C]acetate experiments (not shown). These results are consistent with a bifurcated TCA cycle in which oxaloacetate flows to succinate both through citrate/α-ketoglutarate and via malate/fumarate as shown in panel A. Panels D and E show the long-term labeling patterns of TCA metabolites when cells are grown in glucose minimal medium supplemented with [U-¹³C]aspartate or with [U-¹³C]glutamate. These data corroborate the results obtained for panels B and C and the existence of a bifurcated TCA cycle. AKG, α-ketoglutarate. In all experiments, the environmental CO₂ comprised 5% of the anaerobic gaseous environment and was ∼99% nonlabeled. In panels B through D, the error bars show SD (n = 2 to 4 independent experiments).

FIG. 3.

One-carbon metabolism in C. acetobutylicum. (A) Proposed network of one-carbon metabolism in C. acetobutylicum. Blue arrows highlight the major production routes for glycine, serine, and one-carbon units (C₁ folates). The fate of the carbons originating from pyruvate is highlighted by gray and dotted boxes. (B) Dynamic incorporation of [U-¹³C]glucose (100%) into the amino acids serine, glycine, and threonine. The labeling patterns observed in glycine indicate that its primary route of production is via threonine and not serine. (C) The synthesis of glycine from threonine was confirmed by growing cells on [U-¹³C]glucose plus nonlabeled aspartate and observing that both the threonine and glycine pools are largely nonlabeled while the serine pool remains largely labeled. (D) Cells grown in [1,2-¹³C]glucose (100%) showed less than a 5% label in C₁ units, even though the precursor carbon in glycine (methylene group highlighted in gray in panel A) is ∼50% labeled. (E) Correlation between the labeled fractions of the carboxylic acid carbon of pyruvate and the labeled fractions of C₁ units across diverse labeling experiments. The addition of unlabeled aspartate to cells growing in [U-¹³C]glucose (100%), which results in the production of unlabeled glycine (C), does not affect labeling of C₁ units. (F) Cells grown with increasing concentrations of NaH¹³CO₃ showed increasing labeling of C₁ units that closely followed the labeling in the carboxyl group of pyruvate but not the labeling of CO₂ present in the medium. The fraction of labeled CO₂ medium was determined based on labeling of CO₂ assimilated into pyrimidines. In panels B through E, the environmental CO₂ comprised 5% of the anaerobic gaseous environment and was ∼99% nonlabeled. In panel F, the experiments were performed in liquid closed-vessel cultures to minimize the interchange between atmospheric ¹²CO₂ and NaH¹³CO₃.

FIG. 4.

Quantitation of fluxes in central metabolism. (A) Ordinary differential equation (ODE) model fitting (lines) to the [U-¹³C]glucose dynamic labeling data (error bars) for three representative metabolites. Complete results are in Fig. S8 in the supplemental material. (B) Metabolic fluxes identified from the ODE model. Arrow sizes indicate absolute values (in logarithmic scale) of net fluxes. The fluxes shown are median values of 1,000 sets of identified fluxes, whose distributions are plotted in Fig. S9 in the supplemental material. The flux from succinyl-CoA into succinate is a combination of the flux through succinyl-CoA synthetase (∼25% median contribution) and the fluxes through the methionine and lysine biosynthesis pathways that are coupled with the conversion of succinyl-CoA into succinate (∼75% median contribution). Hexose-P, combined pools of glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, combined pools of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate; 3PG, combined pools of glycerate-3-phosphate and glycerate-2-phosphate; PEP, phosphoenolpyruvate; Pentose-P, combined pools of ribose-5-phosphate, xylulose-5-phosphate, and ribulose-5-phosphate; OAA, oxaloacetate; αKG, α-ketoglutarate; SucCoA, succinyl-CoA; Asp, aspartate; Glu, glutamate; Gln, glutamine.