Figure 1.
Batch cultivation of M. bovis BCG and ▵icl1 in a bioreactor.
A 10% late log phase culture of M. bovis BCG (black line) or ▵icl1 (red line) was inoculated into the bioreactor and optical density at 600 nm was monitored for 4 d prior to switching to chemostat mode. Average values and standard deviations are shown from two independent experiments.
Figure 2.
Continuous culture of M. bovis BCG and ▵icl1 at a dilution rate of 0.01 h−1 (td = 69.3 h).
A 10% late log phase culture of M. bovis BCG (A) and ▵icl1 (B) was inoculated into the bioreactor. The cultures were operated as batch until day 4. Continuous culture was then started at a dilution rate of 0.01 h−1 Samples were removed and assayed for biomass (red line). CO2 production in the waste gas was measured using a dual tandem sensor gas analyser (green line) and filtered supernatants were assayed for glycerol (blue line). The results are representative of two experiments.
Figure 3.
Continuous culture of M. bovis BCG and ▵icl1 at a dilution rate of 0.03 h−1 (td = 23.1 h).
A 10% late log phase culture of M. bovis BCG (black line) or ▵icl1 (red line) was inoculated into the bioreactor. The cultures were operated as batch until day 4. Continuous culture was then started at a dilution rate of 0.03 h−1. Samples were removed and assayed for biomass (circles). CO2 production in the waste gas was measured using a dual tandem sensor gas analyser (squares). The results are representative of two experiments.
Table 1.
Steady state characteristics of wild type and ▵icl strains of M.bovis BCG (BCG) and wild type M. tuberculosis (MTB) grown in a glycerol-limited chemostat.
Table 2.
In vitro enzyme activities in crude cell extracts of glycerol limited chemostat cultures of Mycobacterium bovis BCG.
Figure 4.
Metabolic network of the central metabolism of Mycobacterium bovis BCG.
Glycolysis/gluconeogenesis (green), oxidative pentose phosphate pathway (orange), tricarboxylic acid cycle (TCA, pink), anaplerotic reactions (blue), and biosynthesis (grey). Standard abbreviations are used for the amino acids. Metabolite abbreviations: ACCOA, acetyl-CoA; ACE, acetate; CHO, chorismate; E4P, d-erythrose 4-phosphate; F6P, d-fructose 6-phosphate; FBP, d-fructose 1,6-bisphosphate; FUM, fumarate; G6P, d-glucose 6-phosphate; GA3P, glyceraldehyde 3-phosphate; GLX, glyoxylate; GLYC, glycerol; ICIT, isocitrate/citrate; KIV, 2-oxoisovalerate; MALOAA, l-malate-oxaloacetate; OXG, 2-oxoglutarate; R5P,α- d -ribose 5-phosphate/l -ribulose 5-phosphate/l-xylulose 5-phosphate; PEP, phosphoenolpyruvate; PGA, 2-phospho- d -glycerate/3-phospho- d-glycerate; PYR, pyruvate; S7P, sedoheptulose 7-phosphate; SUC, succinate; SUCCOA, succinyl-CoA. Enzyme abbreviations: CS: citrate synthase; ENO: enolase; FBA: fructose-bisphosphate adolase; FBP: fructose-bisphosphatase; FUM:fumurase; GAPA: glyceraldehyde 3-phosphate dehydrogenase; GND: 6-phosphogluconate dehydrogenase; ICL: isocitrate lyase; ICDH: isocitrate dehydrogenase NADP-dependent; KOR/KGD: α-ketoglutarate ferredoxin oxidoreductase/α-ketoglutarate decarboxylase;MEZ/PCA: malic enzyme/pyruvate carboxylase: MDH: malate dehydrogenase; MS: malate synthase; PCK: phosphoenolpyruvate carboxykinase; PDH: pyruvate dehydrogenase; PGI: glucose phosphate isomerase; PYK: pyruvate kinase;SDH: succinate dehydrogenase; SCS: Succinyl CoA synthetase; TAL: transaldolase; TKT1/2: transketolase. Explicit names are also given in Table S6. The picture was generated with Omix, an editor for biochemical networks and visualization tool [http://www.13cflux.net/omix].
Figure 5.
In vivo flux distribution of M. bovis BCG in glycerol limited continuous culture at fast growth rate (td = 23 h).
Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes (Table S5) are indicated on each flux arrow. The abbreviations are as in Figure 4.
Figure 6.
Metabolic flux map corresponding to solution A for M. bovis BCG in glycerol limited continuous culture at slow growth rate (td = 69 h).
Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Mass spectral data of proteinogenic amino acids from steady state chemostat cultures grown on a mixture of 20% [U-13C] glycerol and 80% unlabeled glycerol and the physiological data of Table 1 were used to generate the two alternative flux estimations. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes (Table S5) are indicated on each flux arrow. The abbreviations are as in Figure 4.
Figure 7.
Metabolic flux map B for M. bovis BCG in glycerol limited continuous culture at slow growth rate (td = 69 h).
Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Mass spectral data of proteinogenic amino acids from steady state chemostat cultures grown on a mixture of 20% [U-13C] glycerol and 80% unlabeled glycerol and the physiological data of Table 1 were used to generate the two alternative flux estimations. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes (Table S5) are indicated on each flux arrow. The abbreviations are as in Figure 4.
Figure 8.
Metabolic flux map corresponding to solution A for M. tuberculosis in glycerol limited continuous culture at slow growth rate (td = 69 h).
Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Both of the presented flux maps are able to describe the 13C labeling profiles of proteinogenic amino acids and measured extracellular flux values from steady state cultures of M. tuberculosis at slow growth rate. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes (Table S5) are indicated on each flux arrow. The abbreviations are as in Figure 4.
Figure 9.
Metabolic flux map B for M. tuberculosis in glycerol limited continuous culture at slow growth rate (td = 69 h).
Flux values were normalized to the specific glycerol uptake rate which was arbitrarily given the value of 100. Both of the presented flux maps are able to describe the 13C labeling profiles of proteinogenic amino acids and measured extracellular flux values from steady state cultures of M. tuberculosis at slow growth rate. Arrows are pointing in the net flux direction and the width of each line is proportional to the underlying flux value. Numerical values of estimated fluxes (Table S5) are indicated on each flux arrow. The abbreviations are as in Figure 4.
Figure 10.
Schematic of the GAS pathway which is characterised by flux through the glyoxylate shunt and anaplerotic reactions for oxidation of pyruvate and succinyl CoA synthetase for the generation of succinyl CoA. The substrate abbreviations are as in Figure 4.