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

Overview of flux changes through methylotrophic central metabolism during the transition from succinate to methanol growth.Dissimilatory steps are shown in green, assimilatory steps in brown. Methylene-H4F is abbreviated as meH4F.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991311&req=5

pone-0014091-g003: Overview of flux changes through methylotrophic central metabolism during the transition from succinate to methanol growth.Dissimilatory steps are shown in green, assimilatory steps in brown. Methylene-H4F is abbreviated as meH4F.

Mentions: In M. extorquens AM1, methanol is first oxidized to the toxic metabolite, formaldehyde, then to formate. Formate serves as a branch point and can either be oxidized to CO2 producing reducing power, or be converted to methylene-H4F, which enters the central metabolic carbon assimilation pathways including the serine cycle, the EMC pathway, the PHB cycle and a portion of the TCA cycle, where essential intermediates are produced for cell growth [1]–[2], [21] (Figure 3). These pathways were analyzed in detail along with two core pathways involved in multi-carbon growth, the TCA cycle and the pentose-phosphate pathway. After methanol was added to succinate-grown cultures, distribution of carbon flow was assessed using 4 methods: growth curve analysis, measurement of flux to CO2 using 14C-labeled methanol, measurement of methanol, formaldehyde and formate in the culture supernatant and measurement of assimilatory pathway metabolites (described below). Growth did not occur until between 1–2 h post transition (Figure 1A), yet flux of methanol to CO2 increased significantly within the first hour, prior to growth (Figure 1B). Formaldehyde and formate concentrations in the supernatant increased until about 2 h, when levels began to decrease (Figure 1C), suggesting that before active growth occurred, both compounds were excreted and then as the cells began to divide, excretion decreased. For the first hour, approximately 1/3 of the carbon from methanol oxidation was in these two pools, the remainder in CO2. Note that the peak concentrations, 50 and 450 µM, respectively, are not toxic for M. extorquens AM1 [26]–[28]. By 2 h, the total flux to formaldehyde, formate, and CO2 of the culture (∼32 nmol min−1 [ml at 1 OD]−1) was about ¾ of the full flux measured in methanol-grown cells (41.5 nmol min−1 [ml at 1 OD]−1; [21]). These data suggest that the lag in growth was due to a block in formate assimilation, not production (summarized in Figure 3).


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)

Overview of flux changes through methylotrophic central metabolism during the transition from succinate to methanol growth.Dissimilatory steps are shown in green, assimilatory steps in brown. Methylene-H4F is abbreviated as meH4F.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014091-g003: Overview of flux changes through methylotrophic central metabolism during the transition from succinate to methanol growth.Dissimilatory steps are shown in green, assimilatory steps in brown. Methylene-H4F is abbreviated as meH4F.
Mentions: In M. extorquens AM1, methanol is first oxidized to the toxic metabolite, formaldehyde, then to formate. Formate serves as a branch point and can either be oxidized to CO2 producing reducing power, or be converted to methylene-H4F, which enters the central metabolic carbon assimilation pathways including the serine cycle, the EMC pathway, the PHB cycle and a portion of the TCA cycle, where essential intermediates are produced for cell growth [1]–[2], [21] (Figure 3). These pathways were analyzed in detail along with two core pathways involved in multi-carbon growth, the TCA cycle and the pentose-phosphate pathway. After methanol was added to succinate-grown cultures, distribution of carbon flow was assessed using 4 methods: growth curve analysis, measurement of flux to CO2 using 14C-labeled methanol, measurement of methanol, formaldehyde and formate in the culture supernatant and measurement of assimilatory pathway metabolites (described below). Growth did not occur until between 1–2 h post transition (Figure 1A), yet flux of methanol to CO2 increased significantly within the first hour, prior to growth (Figure 1B). Formaldehyde and formate concentrations in the supernatant increased until about 2 h, when levels began to decrease (Figure 1C), suggesting that before active growth occurred, both compounds were excreted and then as the cells began to divide, excretion decreased. For the first hour, approximately 1/3 of the carbon from methanol oxidation was in these two pools, the remainder in CO2. Note that the peak concentrations, 50 and 450 µM, respectively, are not toxic for M. extorquens AM1 [26]–[28]. By 2 h, the total flux to formaldehyde, formate, and CO2 of the culture (∼32 nmol min−1 [ml at 1 OD]−1) was about ¾ of the full flux measured in methanol-grown cells (41.5 nmol min−1 [ml at 1 OD]−1; [21]). These data suggest that the lag in growth was due to a block in formate assimilation, not production (summarized in Figure 3).

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