Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

Zhang, Meng; Fitzgerald, Kelly A.; Lidstrom, Mary E.

doi:10.1099/mic.0.28243-0

Cited by 11 publications

(10 citation statements)

References 33 publications

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“…The results described above suggested that Ecm was functioning as a control point during the transition from growth on succinate to growth on ethylamine, restricting the consumption of ethylmalonyl-CoA and the production of downstream intermediates during the lag period of the transition. To test this model, we overrode the control point by overexpressing ecm from a strong promoter (the mxaF promoter) that is highly expressed during growth on both succinate and ethylamine (16,37). Ecm activity increased 4-fold (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1

et al. 2015

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The metabolism of one-and two-carbon compounds by the methylotrophic bacterium Methylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-type M. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts for ecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressing ecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation.T he function of central carbon metabolism is to generate essential metabolic intermediates and the energy and reducing power necessary to convert them to biomass. Metabolism must be fine-tuned to maintain cell homeostasis, balancing the production and consumption of metabolic intermediates and preventing a lethal accumulation of toxic compounds. The regulation of enzymes that control this balance is vital to the cell, particularly when challenged with an environmental perturbation such as a sudden change in the growth substrate. Identifying metabolic control points leads to a better understanding of fundamental physiology and can suggest physiological significance, such as preventing a toxic intermediate from accumulating or balancing the growth rate and growth yield. In addition, characterization of the metabolic strategies employed to effectively navigate an environmental perturbation provides valuable insight for strain manipulation and applied goals.Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on single-carbon (C 1 ) compounds such as methanol and methylamine, multicarbon compounds such as succinate (C 4 ), and two-carbon compounds such as ethylamine and acetate (C 2 ) (1-3). M. ...

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Section: Resultsmentioning

confidence: 99%

Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1

et al. 2015

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“…The lacZ gene of pCM132 was deleted and replaced by a 33-bp multiple cloning site to generate pHC41. The promoter-probe plasmid, pHC42, was generated by cloning a 734-bp PCR fragment containing the ribosome binding site (RBS) of the fae gene (encoding formaldehyde-activating enzyme) of Methylobacterium and the reporter GFPuv gene from pKF133 into pHC41 [73],[74]. A 51-bp synthetic fragment containg the constitutive promoter P tac was inserted upstream of this reporter to make pHC62.…”

Section: Methodsmentioning

confidence: 99%

Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

2009

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Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences.

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“…Also, MxcQ and MxcE, another putative two component regulatory system, are required for the expression of mxaF, the gene for the large subunit of methanol dehydrogenase (MDH) (37). Further, a methanol-inducible promoter with a multi-A tract sequence upstream of mxaF is essential for the expression of mxaF (22,39). In Paracoccus denitrificans, genes involved in methanol oxidation are located in the mxa gene cluster (38).…”

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confidence: 99%

MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

Park

Kim

2011

J Bacteriol

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Mycobacterium sp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol:N,N-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for methanol oxidation. The second open reading frame (mdoR) upstream of, and running divergently from, the mdo gene was identified as a gene for a TetR family transcriptional regulator. The N-terminal region of MdoR contained a helix-turn-helix DNA-binding motif. An electrophoretic mobility shift assay (EMSA) indicated that MdoR could bind to a mdo promoter region containing an inverted repeat. The mdoR deletion mutant did not grow on methanol, but growth on methanol was restored by a plasmid containing an intact mdoR gene. In DNase I footprinting and EMSA experiments, MdoR bound to two inverted repeats in the putative mdoR promoter region. Reverse transcription-PCR indicated that the mdoR gene was transcribed only in cells growing on methanol, whereas ␤-galactosidase assays showed that the mdoR promoter was activated in the presence of methanol. These results indicate that MdoR serves as a transcriptional activator for the expression of mdo and its own gene. Also, MdoR is the first discovered member of the TetR family of transcriptional regulators to be involved in the regulation of the methanol oxidation, as well as to function as a positive autoregulator.Methylotrophic bacteria use reduced carbon compounds containing no carbon-carbon bonds as sole carbon and energy sources (4). The enzyme for the oxidation of methanol to formaldehyde in methanol-oxidizing bacteria is highly expressed in cells growing on methanol, indicating the expression of the gene for this enzyme is regulated according to the presence of methanol (19).The regulation of methanol oxidation in Gram-negative bacteria is known to be variable. At least 26 genes are required for the oxidation of methanol in Methylobacterium extorquens AM1 (23, 37). MxaB, a putative two-component response regulator, and MxbD and MxbM, a putative sensor-regulator pair, are involved in the regulation of methanol oxidation in this bacterium (35,36). Also, MxcQ and MxcE, another putative two component regulatory system, are required for the expression of mxaF, the gene for the large subunit of methanol dehydrogenase (MDH) (37). Further, a methanol-inducible promoter with a multi-A tract sequence upstream of mxaF is essential for the expression of mxaF (22, 39). In Paracoccus denitrificans, genes involved in methanol oxidation are located in the mxa gene cluster (38). A two-component system consisting of MxaY, a putative histidine kinase, and MxaX, a putative response regulator, is involved in the control of MDH expression (11). Another two-component system consisting of FlhR and FlhS also regulates methanol oxidation in Paracoccus denitrificans (12). However, little is known about the regulation of the genes responsible for the oxidation of methanol in Grampositive bacteria. It is only known that the gene for NADdependent MDH in Bacillus methanolicus strain MGA3 is upregulated in cells growing...

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Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

Cited by 11 publications

References 33 publications

Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1

Ethylmalonyl Coenzyme A Mutase Operates as a Metabolic Control Point in Methylobacterium extorquens AM1

Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

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