Contrasting in vitro and in vivo methanol oxidation activities of lanthanide-dependent alcohol dehydrogenases XoxF1 and ExaF from Methylobacterium extorquens AM1
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ABSTRACT:
ORGANISM(S): Methylorubrum extorquens AM1
PROVIDER: GSE125593 | GEO | 2019/02/01
REPOSITORIES: GEO
Similar Datasets
Project description:Methylobacterium extorquens AM1 on methanol
2012-02-28 | PRD000446 | Pride
Project description:In order to provide information about the gene expression response that occurs when cells experience a change in carbon source, succinate limited chemostat cultures of Methylobacterium extorquens AM1 were grown to and maintained at an OD of ~0.63, transferred to flasks and methanol was added. Cells were harvested for RNA extraction at time: 0 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr post transition. At 30 min, a no methanol addition sample was extracted as a carbon starvation control. These data were used in conjunction with flux, enzymatic and metabolite measurements to assess the changes in central metabolism during this transition. Abstract from manuscript: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate to methanol growth by the methylotrophic bacterium Methylobacterium extorquens, 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 methylenetetrahydrofolate 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.
2011-08-01 | GSE22031 | GEO
Project description:In order to provide information about the gene expression response that occurs when cells experience a change in carbon source, succinate limited chemostat cultures of Methylobacterium extorquens AM1 were grown to and maintained at an OD of ~0.63, transferred to flasks and methanol was added. Cells were harvested for RNA extraction at time: 0 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr post transition. At 30 min, a no methanol addition sample was extracted as a carbon starvation control. These data were used in conjunction with flux, enzymatic and metabolite measurements to assess the changes in central metabolism during this transition. Abstract from manuscript: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate to methanol growth by the methylotrophic bacterium Methylobacterium extorquens, 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 methylenetetrahydrofolate 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. Gene expression in succinate limited chemostat cultures was compared to gene expression in cells transferred to flasks before and after methanol addition. As a control, a time = 0 sample (RNA prepared from cells harvested directly from the chemostat) was compared to a time = 0 sample immediately obtained after the cells were transferred to flasks, before methanol was added in order to identify changes due to flask transfer. A carbon starvation control was also done comparing expression from time = 0 (chemostat cells) to cells transferred to flasks for 30 min with no carbon source added. Two biological replicates each with two techinal replicates (dye swap) were analyzed for time = 0 (chemostat) vs 10 min, 30 min, 1 hr and 2 hr after methanol addition. One biological replicate with two technical replicates (dye swap) were analyzed for time = 0 (chemostat) vs time = 0 (flask transfer), and time = 0 (chemostat) vs time = 4 hr, 6 hr and 30 min no methanol addition.
2011-07-31 | E-GEOD-22031 | biostudies-arrayexpress
Project description:Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) from Methylobacterium extorquens AM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol- and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9-12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation of xox1 and downregulation of mxa genes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation of fdh4 formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in the fae mutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy.
| S-EPMC6414531 | biostudies-literature
Project description:Methylobacterium extorquens AM1 is a facultative methylotrophic Alphaproteobacterium and has been subject to intense study under pure methylotrophic as well as pure heterotrophic growth conditions in the past. Here, we investigated the metabolism of M. extorquens AM1 under mixed substrate conditions, i.e., in the presence of methanol plus succinate. We found that both substrates were co-consumed, and the carbon conversion was two-thirds from succinate and one-third from methanol relative to mol carbon. (13)C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO(2) for energy generation. However, a portion of the methanol entered biosynthetic reactions via reactions specific to the one-carbon carrier tetrahydrofolate. In contrast, succinate was primarily used to provide precursor metabolites for bulk biomass production. This work opens new perspectives on the role of methylotrophy when substrates are simultaneously available, a situation prevailing under environmental conditions.
| S-EPMC3486813 | biostudies-literature
Project description:Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.
| S-EPMC111405 | biostudies-literature
Project description:Engineered Methylobacterium methanol batch exponential
2014-12-22 | GSE34662 | GEO
Project description:Beijerinckiaceae bacterium RH AL1 was grown exponentially in with methanol (1% [v/v]) as carbon source and lanthanum (1µM) as necessary growth supplement. Harvested biomass was subjected to RNA extraction, mRNA-enrichment and Illumina sequencing library preparation for subsequent RNA-Seq analysis. The scope of this gene expression analysis was to validate the expression of genes linked to pathways that are involved in C1-metabolism.
2019-08-08 | E-MTAB-8184 | biostudies-arrayexpress
Project description:The nucleotide and deduced amino acid sequence of a novel small (beta) subunit of methanol dehydrogenase of Methylobacterium extorquens AM1 (previously Pseudomonas AM1) has been determined. Work with the whole protein has shown that is has an alpha 2 beta 2 configuration.
| S-EPMC1138755 | biostudies-other
Project description:The goal of this study was to use microarrays to identify genes differentially regulated under conditions of formaldehyde stress relative to two other stress conditions (oxidative, osmotic) in an effort to identify genes that might be involved in a formaldehyde-specific stress response, rather than a general stress response, in the model methylotroph Methylobacterium extorquens AM1.
2015-01-01 | GSE15631 | GEO