Limits...
A systems biology approach uncovers cellular strategies used by Methylobacterium extorquens AM1 during the switch from multi- to single-carbon growth.

Skovran E, Crowther GJ, Guo X, Yang S, Lidstrom ME - PLoS ONE (2010)

Bottom Line: This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates.Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA. eskovran@u.washington.edu

ABSTRACT

Background: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap.

Methodology/principal findings: This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.

Conclusions/significance: This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints.

Show MeSH

Related in: MedlinePlus

Pathway schematic depicting changes that occurred in measured metabolites, gene expression, and enzymatic activities for central metabolism during the transition from succinate to methanol growth.A boxed gene/protein name indicates the activity of this enzyme was measured. Red lettering for the protein designation indicates an increase in activity; green, decrease; black, no change. Metabolites appearing more than once are connected by gray lines. Changes are shown for (A) the initial response, time  = 10–30 min with (B) serving as a legend. Graphs depicting changes for pre-methanol addition, time  = 0 min; just prior to/at the start of cell growth, time  = 1–2 h; and exponential cell growth, time  = 3–6 h are included in Figure S1. Reaction descriptions are included in Table S2 along with gene expression intensities, LogRatios, fold changes and p-values. Mesaconyl-CoA, ethylmalonyl-CoA, methylsuccinyl-CoA were measured as free acids. **Color at T = 0 represents the concentration before methanol was added for all metabolites except for methanol which was calculated as 50 mM for the initial T = 0 value.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991311&req=5

pone-0014091-g004: Pathway schematic depicting changes that occurred in measured metabolites, gene expression, and enzymatic activities for central metabolism during the transition from succinate to methanol growth.A boxed gene/protein name indicates the activity of this enzyme was measured. Red lettering for the protein designation indicates an increase in activity; green, decrease; black, no change. Metabolites appearing more than once are connected by gray lines. Changes are shown for (A) the initial response, time  = 10–30 min with (B) serving as a legend. Graphs depicting changes for pre-methanol addition, time  = 0 min; just prior to/at the start of cell growth, time  = 1–2 h; and exponential cell growth, time  = 3–6 h are included in Figure S1. Reaction descriptions are included in Table S2 along with gene expression intensities, LogRatios, fold changes and p-values. Mesaconyl-CoA, ethylmalonyl-CoA, methylsuccinyl-CoA were measured as free acids. **Color at T = 0 represents the concentration before methanol was added for all metabolites except for methanol which was calculated as 50 mM for the initial T = 0 value.

Mentions: With the introduction of global “omics” level tools, it has recently become possible to investigate multiple layers of an organism's metabolic network during a condition of study. However, the power of these tools can also be a detriment, generating large amounts of data that can often be difficult to integrate and understand as a whole. To facilitate insights and infer meaning regarding the multi-leveled changes and adaptations that the metabolic network of M. extorquens AM1 undergoes during the transition from succinate- to methanol-growth, diagrams were constructed that visually compile and summarize each level of data obtained in relation to central metabolism. The initial response to methanol addition (time  = 10–30 min) is shown in Figure 4A with 4B serving as a legend. Diagrams depicting the metabolic state prior to methanol addition (time = 0 min), response just prior to/at the start of cell growth (1–2 h) and during log phase cell growth (3–6 h) are included in Figure S1. These diagrams depict information about gene expression intensities (arrow thickness) and fold changes (number of arrow heads), changes in measured metabolite concentrations (color shadings), and enzymatic activities (color shadings of boxed protein names), providing insight into both the metabolic changes themselves and the level at which those changes occurred. While only semi-quantitative due to possible differing labeling and hybridization efficiencies that could occur during microarray experiments, information on gene intensities is provided since the intensity data aid in pathway interpretation of possible carbon flow. Analysis of fold changes alone can be misleading if for example, no change in expression occurs for a gene, yet expression of that gene is high, or if a gene has a significant change in expression but expression is extremely low.


A systems biology approach uncovers cellular strategies used by Methylobacterium extorquens AM1 during the switch from multi- to single-carbon growth.

Skovran E, Crowther GJ, Guo X, Yang S, Lidstrom ME - PLoS ONE (2010)

Pathway schematic depicting changes that occurred in measured metabolites, gene expression, and enzymatic activities for central metabolism during the transition from succinate to methanol growth.A boxed gene/protein name indicates the activity of this enzyme was measured. Red lettering for the protein designation indicates an increase in activity; green, decrease; black, no change. Metabolites appearing more than once are connected by gray lines. Changes are shown for (A) the initial response, time  = 10–30 min with (B) serving as a legend. Graphs depicting changes for pre-methanol addition, time  = 0 min; just prior to/at the start of cell growth, time  = 1–2 h; and exponential cell growth, time  = 3–6 h are included in Figure S1. Reaction descriptions are included in Table S2 along with gene expression intensities, LogRatios, fold changes and p-values. Mesaconyl-CoA, ethylmalonyl-CoA, methylsuccinyl-CoA were measured as free acids. **Color at T = 0 represents the concentration before methanol was added for all metabolites except for methanol which was calculated as 50 mM for the initial T = 0 value.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991311&req=5

pone-0014091-g004: Pathway schematic depicting changes that occurred in measured metabolites, gene expression, and enzymatic activities for central metabolism during the transition from succinate to methanol growth.A boxed gene/protein name indicates the activity of this enzyme was measured. Red lettering for the protein designation indicates an increase in activity; green, decrease; black, no change. Metabolites appearing more than once are connected by gray lines. Changes are shown for (A) the initial response, time  = 10–30 min with (B) serving as a legend. Graphs depicting changes for pre-methanol addition, time  = 0 min; just prior to/at the start of cell growth, time  = 1–2 h; and exponential cell growth, time  = 3–6 h are included in Figure S1. Reaction descriptions are included in Table S2 along with gene expression intensities, LogRatios, fold changes and p-values. Mesaconyl-CoA, ethylmalonyl-CoA, methylsuccinyl-CoA were measured as free acids. **Color at T = 0 represents the concentration before methanol was added for all metabolites except for methanol which was calculated as 50 mM for the initial T = 0 value.
Mentions: With the introduction of global “omics” level tools, it has recently become possible to investigate multiple layers of an organism's metabolic network during a condition of study. However, the power of these tools can also be a detriment, generating large amounts of data that can often be difficult to integrate and understand as a whole. To facilitate insights and infer meaning regarding the multi-leveled changes and adaptations that the metabolic network of M. extorquens AM1 undergoes during the transition from succinate- to methanol-growth, diagrams were constructed that visually compile and summarize each level of data obtained in relation to central metabolism. The initial response to methanol addition (time  = 10–30 min) is shown in Figure 4A with 4B serving as a legend. Diagrams depicting the metabolic state prior to methanol addition (time = 0 min), response just prior to/at the start of cell growth (1–2 h) and during log phase cell growth (3–6 h) are included in Figure S1. These diagrams depict information about gene expression intensities (arrow thickness) and fold changes (number of arrow heads), changes in measured metabolite concentrations (color shadings), and enzymatic activities (color shadings of boxed protein names), providing insight into both the metabolic changes themselves and the level at which those changes occurred. While only semi-quantitative due to possible differing labeling and hybridization efficiencies that could occur during microarray experiments, information on gene intensities is provided since the intensity data aid in pathway interpretation of possible carbon flow. Analysis of fold changes alone can be misleading if for example, no change in expression occurs for a gene, yet expression of that gene is high, or if a gene has a significant change in expression but expression is extremely low.

Bottom Line: This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates.Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA. eskovran@u.washington.edu

ABSTRACT

Background: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? Methylobacterium extorquens is a bacterium capable of growth on both multi- and single-carbon compounds. These different modes of growth utilize dramatically different central metabolic pathways with limited pathway overlap.

Methodology/principal findings: This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate growth (predicted to be energy-limited) to methanol growth (predicted to be reducing-power-limited), analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylene tetrahydrofolate dehydrogenase as one control point. This "downstream priming" mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints.

Conclusions/significance: This multi-level approach has resulted in new insights into the metabolic strategies carried out to effect this shift between two dramatically different modes of growth and identified a number of potential flux control and regulatory check points as a further step toward understanding metabolic adaptation and the cellular strategies employed to maintain metabolic setpoints.

Show MeSH
Related in: MedlinePlus