Article Figures & Data
Figures
- FIG 1
Metabolites secreted by B. theta. Concentrations of secreted metabolites detected after batch growth in defined minimal glucose medium (mean of 5 biological and 5 technical replicates, n = 25).
- FIG 2
Secretion fluxes of organic acids and amino acids in defined minimal medium. Numbers represent percent mole carbon fluxes (not shown, CO2 inferred, 4.6%). Gray outlined circles represent undetected intracellular metabolic nodes. Black outlined circles indicate secreted metabolites. Shading is proportional to concentration in culture medium.
- FIG 3
Effect of metabolic feedback inhibition on growth. (a) Population doubling time of cultures on defined minimal medium supplemented with acetate (n = 8 biological replicates, P < 0.01 versus 0 mM, r2 = 0.94). (b) Population doubling time of cultures on defined minimal medium supplemented with formate (n = 5 biological replicates, P < 0.01 versus 0 mM). (c) Final optical densities of cultures with and without supplementation of 10 mM acetate (Ac) and 10 mM formate (Fo) (n = 5 biological replicates, P > 0.05 versus 0 mM). (d) Concentrations of secreted metabolites with increasing acetate supplementation (means from 5 biological and 5 technical replicates, n = 25). P values are shown in Table S1 in the supplemental material. Curves were fit according to parabolic functions (a) or least-squares regression (b and d). Error bars may be obscured by symbols.
- FIG 4
Effect of acetate feedback inhibition on secretion fluxes. (a) Concentrations of secreted metabolites with increasing acetate supplementation (means from 5 biological and 5 technical replicates, n = 25). P values are shown in Table S2. Error bars may be obscured by symbols. (b) Change in secretion fluxes with increasing acetate supplementation (means from 5 biological and 5 technical replicates, n = 25). P values are shown in Table S3. (c) Effect of acetate feedback inhibition (10 mM) mapped onto a metabolic network. Red, decreased secretion. Shading is proportional to flux magnitude. Gray outlined circles represent undetected intracellular metabolic nodes. asp, aspartate.
- FIG 5
Effect of formate supplementation on acetate feedback inhibition. (a) Change in organic acid secretion with and without supplementation of 10 mM acetate or a combination of 10 mM acetate (Ac) and 10 mM formate (Fo). (b) Change in secreted amino acids with and without supplementation. (c) Effect of 10 mM formate supplementation on 10 mM acetate feedback inhibition mapped to the metabolic network. Green, increased secretion; red, decreased secretion; gray, no significant difference between 10 mM acetate versus 10 mM acetate and 10 mM formate conditions. Shading is proportional to flux magnitude. Gray outlined circles represent undetected intracellular metabolic nodes. asp, aspartate. P values for data in panels a and b are shown in Table S4. Error bars may be too small to see.
- FIG 6
Simulation of the effect of feedback inhibition on metabolism. (a) Predicted biomass in high-glucose (HG) and low-glucose (LG) medium as acetate (Ac) and formate (Fo) concentrations were varied from 0 mM to 10 mM. (b) Predicted effect of acetate and/or formate on exchange fluxes in HG medium. (c) Predicted effect of acetate on exchange fluxes in LG medium. (d) Predicted effect of formate on exchange fluxes in LG medium. Green, increased exchange flux; red, decreased exchange flux; gray, no net change in exchange flux due to metabolic rerouting; white, no change predicted.
Tables
- TABLE 3
Acetate suppression coefficients of secreted metabolites
TABLE S1
Student’s t test P values for metabolomics data in Fig. 3d. P values are given for each metabolite measured in spent medium as acetate is supplemented from 0 to 10 mM. Download Table S1, DOCX file, 0.1 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
NMR metabolomics. (a) PCA scores plot generated from 1D 1H NMR spectra from spent culture medium (red, n = 25) and uninoculated medium (blue, n = 5). Ellipses correspond to the 95% confidence interval for a normal distribution. (b) Back-scaled loadings plot generated from a validated OPLS-discriminant analysis (DA) model (P value < 1 × 10−37) comparing the spent culture medium and uninoculated medium 1D 1H NMR datasets. Positive peaks indicate an increase in spent culture medium and negative peaks indicate a decrease in spent culture medium. Positive peaks labeled and added in as inlets are numbered as follows: 15, formate; 13 and 14, histidine at 7.12 and 7.96 ppm; 12, hematin; 11, glucose; 2 and 10, alanine at 1.48 and 3.83 ppm; 8, cysteine at 3.05, 3.09, and 3.99 ppm; 9, cysteine at 3.20, 3.42, and 4.11 ppm; 7, asparagine at 2.97 ppm; 6, succinate; 5, glutathione at 2.50 ppm; 4, acetate; 3, lactate at 1.33 ppm. Peaks colored red are scaled 4× and colored green are scaled 2× to their actual intensities as depicted in the full-width spectrum in blue. Download FIG S1, PDF file, 1.8 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Student’s t test P values for metabolomics data in Fig. 4a. P values are given for the change in concentration of each metabolite measured in spent medium as acetate is supplemented from 0 to 10 mM. Download Table S2, DOCX file, 0.1 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S3
Student’s t test P values for metabolomics data in Fig. 4b. Slope and Pearson correlation coefficient (r2) values are given for the change in concentration of each metabolite measured in spent medium as acetate is supplemented from 0 to 10 mM. Download Table S3, DOCX file, 0.1 MB.
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FIG S2
Theoretical modeling of the effect of acetate supplementation on the observed secreted metabolites. Models assume a fraction of supplemented acetate is converted to secreted metabolite x.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S4
Student’s t test P values for metabolomics data in Fig. 5a and b. P values are given for the change in concentration of each metabolite measured in spent medium in the absence and presence of 10 mM acetate and 10 mM formate supplementation. Download Table S4, DOCX file, 0.1 MB.
Copyright © 2020 Catlett et al.
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MOVIE S1
Animated secretion fluxes of organic acids and amino acids in defined minimal medium. Numbers represent percent mole carbon fluxes (not shown, CO2 inferred, 4.6%). Gray outlined circles represent undetected intracellular metabolic nodes. Black outlined circles indicate secreted metabolites. Shading is proportional to concentration in culture medium from 0% (red circles) to yellow (50%) to green (100%) carbon flux. Download Movie S1, GIF file, 0.1 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
MOVIE S2
Animated effect of acetate feedback inhibition on secretion fluxes. The effect of acetate feedback inhibition (10 mM) mapped onto a metabolic network. Gray outlined circles represent undetected intracellular metabolic nodes. Black outlined circles indicate secreted metabolites. Shading is proportional to change in carbon flux compared to no acetate control from 0% (red circles) to yellow (50%) to green (100%). Download Movie S2, GIF file, 0.1 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
MOVIE S3
Animated effect of formate supplementation on acetate feedback inhibition. The effect of formate (10 mM) and acetate feedback inhibition (10 mM) mapped onto a metabolic network. Gray outlined circles represent undetected intracellular metabolic nodes. Black outlined circles indicate secreted metabolites. Shading is proportional to concentration in culture medium from 0% (red circles) to yellow (50%) to green (100%) carbon flux. Download Movie S3, GIF file, 0.1 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
DATA SET S1
Model parameters and output flux data for high-glucose and low-glucose FBA models are included in an Excel spreadsheet. Download Data Set S1, XLSX file, 0.1 MB.
Copyright © 2020 Catlett et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
