scholarly journals Novel Formaldehyde-Activating Enzyme inMethylobacterium extorquens AM1 Required for Growth on Methanol

2000 ◽  
Vol 182 (23) ◽  
pp. 6645-6650 ◽  
Author(s):  
Julia A. Vorholt ◽  
Christopher J. Marx ◽  
Mary E. Lidstrom ◽  
Rudolf K. Thauer
Keyword(s):  
Biological Macromolecules ◽  
Methylotrophic Bacteria ◽  
Methylobacterium Extorquens ◽  
Metabolic Intermediate ◽  
Carbon Compounds ◽  
Methylotrophic Growth ◽  
Special Challenge ◽  
Encoding Gene

ABSTRACT 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 α-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 CO2. 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.

Adaptation and acclimatization to formaldehyde in methylotrophs capable of high-concentration formaldehyde detoxification

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2615-2622 ◽  
Author(s):  
Rotsaman Chongcharoen ◽  
Thomas J. Smith ◽  
Kenneth P. Flint ◽  
Howard Dalton
Keyword(s):  
Industrial Effluents ◽  
Formaldehyde Concentration ◽  
Growth Substrate ◽  
Methylobacterium Extorquens ◽  
High Concentration ◽  
Metabolic Intermediate ◽  
Formaldehyde Removal ◽  
Methylotrophic Growth ◽  
High Concentrations ◽  
Formaldehyde Metabolism

Formaldehyde is a highly toxic chemical common in industrial effluents, and it is also an intermediate in bacterial metabolism of one-carbon growth substrates, although its role as a bacterial growth substrate per se has not been extensively reported. This study investigated two highly formaldehyde-resistant formaldehyde utilizers, strains BIP and ROS1; the former strain has been used for industrial remediation of formaldehyde-containing effluents. The two strains were shown by means of 16S rRNA characterization to be closely related members of the genus Methylobacterium. Both strains were able to use formaldehyde, methanol and a range of multicarbon compounds as their principal growth substrate. Growth on formaldehyde was possible up to a concentration of at least 58 mM, and survival at up to 100 mM was possible after stepwise acclimatization by growth at increasing concentrations of formaldehyde. At such high concentrations of formaldehyde, the cultures underwent a period of formaldehyde removal without growth before the formaldehyde concentration fell below 60 mM, and growth could resume. Two-dimensional electrophoresis and MS characterization of formaldehyde-induced proteins in strain BIP revealed that the pathways of formaldehyde metabolism, and adaptations to methylotrophic growth, were very similar to those seen in the well-characterized methanol-utilizing methylotroph Methylobacterium extorquens AM1. Thus, it appears that many of the changes in protein expression that allow strain BIP to grow using high formaldehyde concentrations are associated with expression of the same enzymes used by M. extorquens AM1 to process formaldehyde as a metabolic intermediate during growth on methanol.

scholarly journals Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

2008 ◽  
Vol 190 (14) ◽  
pp. 5057-5062 ◽  
Author(s):  
Gregory J. Crowther ◽  
George Kosály ◽  
Mary E. Lidstrom
Keyword(s):  
Branch Point ◽  
Condensation Reaction ◽  
Carbon Assimilation ◽  
Methylobacterium Extorquens ◽  
Metabolic Fluxes ◽  
Carbon Compounds ◽  
Methylotrophic Growth ◽  
Methylene Tetrahydrofolate ◽  
Direct Condensation

ABSTRACT In serine cycle methylotrophs, methylene tetrahydrofolate (H4F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H4F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H4F to generate methylene H4F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H4F via H4F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H4F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H4F and formate are unable to grow on methanol, suggesting that this route for methylene H4F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H4F synthesis pathway through formate. These results suggested that the methylene H4F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1.

scholarly journals Genome of Methylobacillus flagellatus, Molecular Basis for Obligate Methylotrophy, and Polyphyletic Origin of Methylotrophy

2007 ◽  
Vol 189 (11) ◽  
pp. 4020-4027 ◽  
Author(s):  
Ludmila Chistoserdova ◽  
Alla Lapidus ◽  
Cliff Han ◽  
Lynne Goodwin ◽  
Liz Saunders ◽  
...  
Keyword(s):  
Formate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Methylococcus Capsulatus ◽  
Methylobacterium Extorquens ◽  
Circular Chromosome ◽  
Transport Chain ◽  
Carbon Compounds ◽  
Formaldehyde Oxidation ◽  
Global Carbon ◽  
Single Circular Chromosome

ABSTRACT Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.

scholarly journals Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

2010 ◽  
Vol 192 (7) ◽  
pp. 1813-1823 ◽  
Author(s):  
Yoko Okubo ◽  
Song Yang ◽  
Ludmila Chistoserdova ◽  
Mary E. Lidstrom
Keyword(s):  
Metabolic Networks ◽  
Alternative Pathway ◽  
Malate Synthase ◽  
Alternative Route ◽  
Methylobacterium Extorquens ◽  
Facultative Methylotroph ◽  
Metabolic Versatility ◽  
Carbon Compounds ◽  
Glyoxylate Metabolism ◽  
Lyase Activity

ABSTRACT Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C1 and C2 compounds, showing that both modes of growth have this pathway in common. However, growth on C2 compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/β-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C2 compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and 13C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/β-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C2 compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H4F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C2 compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.

scholarly journals Structure ofMethylobacterium extorquensmalyl-CoA lyase: CoA-substrate binding correlates with domain shift

2017 ◽  
Vol 73 (2) ◽  
pp. 79-85 ◽  
Author(s):  
Javier M. González ◽  
Ricardo Marti-Arbona ◽  
Julian C.-H. Chen ◽  
Clifford J. Unkefer
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Structural Alignment ◽  
Methylobacterium Extorquens ◽  
Terminal Domain ◽  
Domain Motion ◽  
Citrate Lyase ◽  
Carbon Compounds ◽  
Binding Alignment ◽  
Aspartate Residue

Malyl-CoA lyase (MCL) is an Mg2+-dependent enzyme that catalyzes the reversible cleavage of (2S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL fromMethylobacterium extorquensAM1 with bound Mg2+is presented. Structural alignment with the closely relatedRhodobacter sphaeroidesmalyl-CoA lyase complexed with Mg2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. This domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.

scholarly journals Purification, Overproduction, and Partial Characterization of β-RFAP Synthase, a Key Enzyme in the Methanopterin Biosynthesis Pathway†

2002 ◽  
Vol 184 (16) ◽  
pp. 4442-4448 ◽  
Author(s):  
Joseph W. Scott ◽  
Madeline E. Rasche
Keyword(s):  
Biosynthetic Pathway ◽  
Methanogenic Archaea ◽  
Methylotrophic Bacteria ◽  
Methylobacterium Extorquens ◽  
Gene Encoding ◽  
Methanosarcina Thermophila ◽  
Inorganic Pyrophosphate ◽  
Sulfate Reducing ◽  
Key Enzyme

ABSTRACT Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme β-ribofuranosylaminobenzene 5′-phosphate synthase (β-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form β-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding β-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of β-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative β-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the β-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential β-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.

scholarly journals tRNA Is the Source of Low-Level trans-Zeatin Production in Methylobacterium spp.

2002 ◽  
Vol 184 (7) ◽  
pp. 1832-1842 ◽  
Author(s):  
Robbin L. Koenig ◽  
Roy O. Morris ◽  
Joe C. Polacco
Keyword(s):  
High Performance ◽  
De Novo ◽  
Zeatin Riboside ◽  
Methylotrophic Bacteria ◽  
Methylobacterium Extorquens ◽  
Culture Filtrates ◽  
Heat Treated ◽  
Soil Isolate ◽  
Gene Mutant ◽  
Leaf Surfaces

ABSTRACT Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.

scholarly journals Methylotrophic Metabolism Is Advantageous for Methylobacterium extorquens during Colonization of Medicago truncatula under Competitive Conditions

2005 ◽  
Vol 71 (11) ◽  
pp. 7245-7252 ◽  
Author(s):  
Abdoulaye Sy ◽  
Antonius C. J. Timmers ◽  
Claudia Knief ◽  
Julia A. Vorholt
Keyword(s):  
Medicago Truncatula ◽  
Fluorescent Protein ◽  
Selective Advantage ◽  
Plant Colonization ◽  
Methylotrophic Bacteria ◽  
Bacterial Cells ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Model Legume ◽  
Green Fluorescent

ABSTRACT Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.

scholarly journals A Novel Moderately Thermophilic Facultative Methylotroph within the Class Alphaproteobacteria

2021 ◽  
Vol 9 (3) ◽  
pp. 477
Author(s):  
Tajul Islam ◽  
Marcela Hernández ◽  
Amare Gessesse ◽  
J. Colin Murrell ◽  
Lise Øvreås
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Methanol Oxidation ◽  
Draft Genome ◽  
Methanol Dehydrogenase ◽  
Methylotrophic Bacterium ◽  
Rrna Gene ◽  
Methylotrophic Bacteria ◽  
Moderately Thermophilic ◽  
Carbon Compounds

Methylotrophic bacteria (non-methanotrophic methanol oxidizers) consuming reduced carbon compounds containing no carbon–carbon bonds as their sole carbon and energy source have been found in a great variety of environments. Here, we report a unique moderately thermophilic methanol-oxidising bacterium (strain LS7-MT) that grows optimally at 55 °C (with a growth range spanning 30 to 60 °C). The pure isolate was recovered from a methane-utilizing mixed culture enrichment from an alkaline thermal spring in the Ethiopia Rift Valley, and utilized methanol, methylamine, glucose and a variety of multi-carbon compounds. Phylogenetic analysis of the 16S rRNA gene sequences demonstrated that strain LS7-MT represented a new facultatively methylotrophic bacterium within the order Hyphomicrobiales of the class Alphaproteobacteria. This new strain showed 94 to 96% 16S rRNA gene identity to the two methylotroph genera, Methyloceanibacter and Methyloligella. Analysis of the draft genome of strain LS7-MT revealed genes for methanol dehydrogenase, essential for methanol oxidation. Functional and comparative genomics of this new isolate revealed genomic and physiological divergence from extant methylotrophs. Strain LS7-MT contained a complete mxaF gene cluster and xoxF1 encoding the lanthanide-dependent methanol dehydrogenase (XoxF). This is the first report of methanol oxidation at 55 °C by a moderately thermophilic bacterium within the class Alphaproteobacteria. These findings expand our knowledge of methylotrophy by the phylum Proteobacteria in thermal ecosystems and their contribution to global carbon and nitrogen cycles.

scholarly journals Potentials, Utilization, and Bioengineering of Plant Growth-Promoting Methylobacterium for Sustainable Agriculture

2021 ◽  
Vol 13 (7) ◽  
pp. 3941
Author(s):  
Cong Zhang ◽  
Meng-Ying Wang ◽  
Naeem Khan ◽  
Ling-Ling Tan ◽  
Song Yang
Keyword(s):  
Plant Growth ◽  
Positive Impact ◽  
Methylotrophic Bacteria ◽  
Plant Growth Promoting Bacteria ◽  
Random Mutation ◽  
Sustainable Solutions ◽  
Carbon Compounds ◽  
Plant Growth Promoting ◽  
Promote Plant Growth ◽  
Growth Promoting

Plant growth-promoting bacteria (PGPB) have great potential to provide economical and sustainable solutions to current agricultural challenges. The Methylobacteria which are frequently present in the phyllosphere can promote plant growth and development. The Methylobacterium genus is composed mostly of pink-pigmented facultative methylotrophic bacteria, utilizing organic one-carbon compounds as the sole carbon and energy source for growth. Methylobacterium spp. have been isolated from diverse environments, especially from the surface of plants, because they can oxidize and assimilate methanol released by plant leaves as a byproduct of pectin formation during cell wall synthesis. Members of the Methylobacterium genus are good candidates as PGPB due to their positive impact on plant health and growth; they provide nutrients to plants, modulate phytohormone levels, and protect plants against pathogens. In this paper, interactions between Methylobacterium spp. and plants and how the bacteria promote crop growth is reviewed. Moreover, the following examples of how to engineer microbiomes of plants using plant-growth-promoting Methylobacterium are discussed in the present review: introducing external Methylobacterium spp. to plants, introducing functional genes or clusters to resident Methylobacterium spp. of crops, and enhancing the abilities of Methylobacterium spp. to promote plant growth by random mutation, acclimation, and engineering.

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