Experimental identification and quantification of glucose metabolism in seven bacterial species

Abstract

The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, gamma-proteobacteria, and alpha-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively.

FIG. 1.

Master reaction network that was used as the basis for net-flux analysis. Metabolites in bold were precursors for amino acid biosynthesis, and metabolites in boxes were extracellular substrates or products. Doubled-headed arrows indicate reactions assumed to be reversible. Abbreviations: S7P, sedoheptulose-7-P; Acetyl-CoA, acetyl coenzyme A.

FIG. 2.

In vivo carbon flux distribution in Z. mobilis (A), P. fluorescens (B), S. meliloti (C), and A. tumefaciens (D). All fluxes were normalized to the glucose uptake rate that is given at the top of each panel, and the widths of the arrows are scaled to the relative percentages of flux. Fluxes below 2.6% of the glucose uptake rate are represented by nonscaled hairlines. Possible cyclic fluxes through the ED pathways of P. fluorescens and S. meliloti are not resolved by the data and are not shown. Generally, the 95% confidence intervals were between 5 and 10% for the major fluxes. Larger confidence intervals were estimated for reactions with low flux. Abbreviations: G6P, glucose-6-P; 6PG, 6-P-gluconate; F6P, fructose-6-P; P5P, pentose-5-P; E4P, erythrose-4-P; S7P, sedoheptulose-7-P; G3P, glyceraldehyde-3-P; OGA, 2-ketoglutarate; PPP, PP pathway; Acetyl-CoA, acetyl coenzyme A.

FIG. 3.

Possible reactions that introduce C₁ fragments into serine, methionine, and histidine (31). Abbreviations: AICAR-P, 5-aminoimidazole-4-carboxamide-1-ribotide; THFPG, tetrahydrofolylpolyglutamate; 10-Fo-THF, 10-formyl-tetrahydrofolyl; 5,10-Me-THF, 5,10-methyl-tetrahydrofolyl; 3PG, 3-P-glycerate; AcCoA, acetyl coenzyme A.

FIG. 4.

Time courses of OD₆₀₀ (squares) and concentrations of extracellular glucose (circles), gluconate (triangles), and 2-ketogluconate (diamonds) in P. fluorescens.

FIG. 5.

Glucose uptake in P. fluorescens. The following fluxes (shown by arrows) are involved in glucose uptake: direct glucose uptake (r₁), membrane-bound glucose dehydrogenase (r₂), gluconate dehydrogenase (r₃), uptake of gluconate (r₄), and 2-ketogluconate (r₅). The experimentally determined extracellular glucose decrease rate, a, is defined as r₁ plus r₂; the total carbon uptake rate by the cell, b, is defined as r₁ plus r₄ plus r₅; and the total gluconate-2-ketogluconate uptake rate, g, is defined as r₄ plus r₅.

FIG. 6.

In vivo carbon flux distribution in Paracoccus versutus (A), R. sphaeroides (B), E. coli (C), and B. subtilis (D). All fluxes were normalized to the glucose uptake rates that are given at the top of each panel, and the widths of the arrows are scaled to the relative fluxes, expressed as percentages of the glucose uptake rates. Fluxes below 2.6% are represented by nonscaled hairlines. Generally, the 95% confidence intervals were between 5 and 10% for the major fluxes. Larger confidence intervals were estimated for reactions with low flux. Abbreviations: G6P, glucose-6-P; 6PG, 6-P-gluconate; F6P, fructose-6-P; FBP, fructose-1,6-bisphosphate; P5P, pentose-5-P; G3P, glyceraldehyde-3-P; OGA, 2-ketoglutarate; PPP, PP pathway; Acetyl-CoA, acetyl coenzyme A.