A Systems Biology Approach Uncovers Cellular Strategies Used by Methylobacterium extorquens AM1 During the Switch from Multi- to Single-Carbon Growth

Abstract

BackgroundWhen 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 FindingsThis 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/SignificanceThis 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.

XoxF Is Required for Expression of Methanol Dehydrogenase in Methylobacterium extorquens AM1

“…In a previous study, when wild-type chemostat-grown cells were transitioned from succinate growth to methanol growth, the expression levels of both xoxF genes increased, while the expression levels of the mxa genes decreased (33). To determine how the growth of the xoxF mutant strains would respond when the carbon source was switched from succinate to methanol, cells were grown with succinate to an OD of 0.6, washed, and resuspended in methanol medium.…”

“…To create a high-level-expression vector, the promoter region of RMQ02531/META1_3512, which was shown in previous microarray experiments to be highly expressed and constitutive in all available array data (29,33; E. Skovran, unpublished data), was amplified and cloned into the AclI site of pCM62, creating pAP3. The xox1 operon was amplified and inserted into the EcoRI and KpnI sites of pAP3, creating pES500.…”

“…Cells and cell extracts for enzyme assays were prepared by using a French press as previously described (33). A minimum of 3 biological replicates was used to assay methanol dehydrogenase and catechol dioxygenase (XylE).…”

Linking Post-Translational Modifications and Variation of Phenotypic Traits

“…Among them, metabolic networks involve series of interconnected chemical reactions catalyzed by enzymes, allowing the transformation of input substrates into intermediate or final metabolites. These networks play an essential role in an organism's growth, reproduction, and ability to maintain cell integrity and to respond to environmental changes (1,2). The metabolic fluxes, as well as the metabolite concentrations, are governed by the activity of the enzymes, which depends on three types of factors: kinetic parameters, enzyme abundance, and activation state of the enzyme.…”

“…traits characterizing the lifespan of the organism such as growth or survival. Quantitative proteomics based on two-dimensional electrophoresis (2-DE) 1 is well adapted for this purpose, because the different isoforms of a protein often have different electrophoretic mobility, resulting in distinguishable spots. We applied quantitative proteomics to Saccharomyces cerevisiae alcoholic fermentation (AF), a central metabolic pathway exploited for millennia in three important human food-processes: beer and wine production (39 -41), and bread leavening (42).…”