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Until recently, rare-earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca 2+ -dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La 3+ ) is sensed both in a XoxF-dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsis thaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 μg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La 3+ -regulated proteins under defined artificial media conditions. Among these was a REE-binding protein that is encoded next to a gene for a TonB-dependent transporter. The latter was essential for REE-dependent growth on methanol indicating chelator-assisted uptake of REEs., (© 2019 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd.)

The ethylmalonyl-CoA pathway (EMCP) is an anaplerotic reaction sequence in the central carbon metabolism of numerous Proteo- and Actinobacteria. The pathway features several CoA-bound mono- and dicarboxylic acids that are of interest as platform chemicals for the chemical industry. The EMCP, however, is essential for growth on C1 and C2 carbon substrates and therefore cannot be simply interrupted to drain these intermediates. In this study, we aimed at reengineering central carbon metabolism of the Alphaproteobacterium Methylobacterium extorquens AM1 for the specific production of EMCP derivatives in the supernatant. Establishing a heterologous glyoxylate shunt in M. extorquens AM1 restored wild type-like growth in several EMCP knockout strains on defined minimal medium with acetate as carbon source. We further engineered one of these strains that carried a deletion of the gene encoding crotonyl-CoA carboxylase/reductase to demonstrate in a proof-of-concept the specific production of crotonic acid in the supernatant on a defined minimal medium. Our experiments demonstrate that it is in principle possible to further exploit the EMCP by establishing an alternative central carbon metabolic pathway in M. extorquens AM1, opening many possibilities for the biotechnological production of EMCP-derived compounds in future.

Methylotrophy is the ability of organisms to grow at the expense of reduced one-carbon compounds, such as methanol or methane. Here, we used transposon sequencing combining hyper-saturated transposon mutagenesis with high-throughput sequencing to define the essential methylotrophy genome of Methylobacterium extorquens PA1, a model methylotroph. To distinguish genomic regions required for growth only on methanol from general required genes, we contrasted growth on methanol with growth on succinate, a non-methylotrophic reference substrate. About 500,000 insertions were mapped for each condition, resulting in a median insertion distance of five base pairs. We identified 147 genes and 76 genes as specific for growth on methanol and succinate, respectively, and a set of 590 genes as required under both growth conditions. For the integration of metabolic functions, we reconstructed a genome-scale metabolic model and performed in silico essentiality analysis. In total, the approach uncovered 95 genes not previously described as crucial for methylotrophy, including genes involved in respiration, carbon metabolism, transport, and regulation. Strikingly, regardless of the absence of the Calvin cycle in the methylotroph, the screen led to the identification of the gene for phosphoribulokinase as essential during growth on methanol, but not during growth on succinate. Genetic experiments in addition to metabolomics and proteomics revealed that phosphoribulokinase serves a key regulatory function. Our data support a model according to which ribulose-1,5-bisphosphate is an essential metabolite that induces a transcriptional regulator driving one-carbon assimilation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a (13)C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely γ-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of α- and γ-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

In Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ(EcfG), its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ(EcfG) proteins. Here, we analyzed the six σ(EcfG) paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

Bio-based production of dicarboxylic acids is an emerging research field with remarkable progress during the last decades. The recently established synthesis of the ethylmalonyl-CoA pathway (EMCP)-derived dicarboxylic acids, mesaconic acid and (2S)-methylsuccinic acid, from the alternative carbon source methanol (Sonntag et al., Appl Microbiol Biotechnol 98:4533-4544, 2014) gave a proof of concept for the sustainable production of hitherto biotechnologically inaccessible monomers. In this study, substantial optimizations of the process by different approaches are presented. Abolishment of mesaconic and (2S)-methylsuccinic acid reuptake from culture supernatant and a productivity increase were achieved by 30-fold decreased sodium ion availability in culture medium. Undesired flux from EMCP into polyhydroxybutyrate (PHB) cycle was hindered by the knockout of polyhydroxyalkanoate synthase phaC which was concomitant with 5-fold increased product concentrations. However, frequently occurring suppressors of strain ΔphaC lost their beneficial properties probably due to redirected channeling of acetyl-CoA. Pool sizes of the product precursors were increased by exploiting the presence of two cobalt-dependent mutases in the EMCP: Fine-tuned growth-limiting cobalt concentrations led to 16-fold accumulation of mesaconyl- and (2S)-methylsuccinyl-CoA which in turn resulted in 6-fold increased concentrations of mesaconic and (2S)-methylsuccinic acids, with a combined titer of 0.65 g/l, representing a yield of 0.17 g/g methanol. This work represents an important step toward an industrially relevant production of ethylmalonyl-CoA pathway-derived dicarboxylic acids and the generation of a stable PHB synthesis negative Methylobacterium extorquens strain.

Methylotrophy is the ability to use reduced one-carbon compounds, such as methanol, as a single source of carbon and energy. Methanol is, due to its availability and potential for production from renewable resources, a valuable feedstock for biotechnology. Nature offers a variety of methylotrophic microorganisms that differ in their metabolism and represent resources for engineering of value-added products from methanol. The most extensively studied methylotroph is the Alphaproteobacterium Methylobacterium extorquens. Over the past five decades, the metabolism of M. extorquens has been investigated physiologically, biochemically, and more recently, using complementary omics technologies such as transcriptomics, proteomics, metabolomics, and fluxomics. These approaches, together with a genome-scale metabolic model, facilitate system-wide studies and the development of rational strategies for the successful generation of desired products from methanol. This review summarizes the knowledge of methylotrophy in M. extorquens, as well as the available tools and biotechnological applications.

The general stress response of alphaproteobacteria is regulated by a partner-switching mechanism that involves the alternative sigma factor σ(EcfG), the anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. To address the question of how the PhyR-NepR-σ(EcfG) cascade is activated and modulated in Methylobacterium extorquens, a forward genetic screen was applied. The screen identified the single-domain response regulator Mext_0407 as a novel regulatory element in the general stress response of M. extorquens. Analysis of phenotypes and of transcriptional fusions of PhyR-dependent genes shows that the mext_0407 deletion mutant fails to respond to various stresses. Mext_0407 requires the putative phosphorylatable aspartate-64 for its activity in vivo and genetic evidence indicates that Mext_0407 operates upstream of the PhyR-NepR-σ(EcfG) cascade.

Oxalate catabolism is conducted by phylogenetically diverse organisms, including Methylobacterium extorquens AM1. Here, we investigate the central metabolism of this alphaproteobacterium during growth on oxalate by using proteomics, mutant characterization, and (13)C-labeling experiments. Our results confirm that energy conservation proceeds as previously described for M. extorquens AM1 and other characterized oxalotrophic bacteria via oxalyl-coenzyme A (oxalyl-CoA) decarboxylase and formyl-CoA transferase and subsequent oxidation to carbon dioxide via formate dehydrogenase. However, in contrast to other oxalate-degrading organisms, the assimilation of this carbon compound in M. extorquens AM1 occurs via the operation of a variant of the serine cycle as follows: oxalyl-CoA reduction to glyoxylate and conversion to glycine and its condensation with methylene-tetrahydrofolate derived from formate, resulting in the formation of C3 units. The recently discovered ethylmalonyl-CoA pathway operates during growth on oxalate but is nevertheless dispensable, indicating that oxalyl-CoA reductase is sufficient to provide the glyoxylate required for biosynthesis. Analysis of an oxalyl-CoA synthetase- and oxalyl-CoA-reductase-deficient double mutant revealed an alternative, although less efficient, strategy for oxalate assimilation via one-carbon intermediates. The alternative process consists of formate assimilation via the tetrahydrofolate pathway to fuel the serine cycle, and the ethylmalonyl-CoA pathway is used for glyoxylate regeneration. Our results support the notion that M. extorquens AM1 has a plastic central metabolism featuring multiple assimilation routes for C1 and C2 substrates, which may contribute to the rapid adaptation of this organism to new substrates and the eventual coconsumption of substrates under environmental conditions.

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.

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.

Background: Methylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms., Results: In this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl-CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources., Conclusions: This work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products.

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.

Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50 % to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein, and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild-type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant with those of the wild-type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30 %. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared with the wild-type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO(2) production were measured after the addition of methanol or formaldehyde to starved cells. The wild-type, but not the xoxF mutant, accumulated formate upon substrate addition and had a 1 h lag in CO(2) production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.

PhyR represents a novel alphaproteobacterial family of response regulators having a structure consisting of two domains; a predicted amino-terminal extracytoplasmic function (ECF) sigma factor-like domain and a carboxy-terminal receiver domain. PhyR was first described in Methylobacterium extorquens AM1, in which it has been shown to be essential for plant colonization, probably due to its suggested involvement in the regulation of a number of stress proteins. Here we investigated the PhyR regulon using microarray technology. We found that the PhyR regulon is rather large and that most of the 246 targets are under positive control. Mapping of transcriptional start sites revealed candidate promoters for PhyR-mediated regulation. One of these promoters, an ECF-type promoter, was identified upstream of one-third of the target genes by in silico analysis. Among the PhyR targets are genes predicted to be involved in multiple stress responses, including katE, osmC, htrA, dnaK, gloA, dps, and uvrA. The induction of these genes is consistent with our phenotypic analyses which revealed that PhyR is involved in resistance to heat shock and desiccation, as well as oxidative, UV, ethanol, and osmotic stresses, in M. extorquens AM1. The finding that PhyR is involved in the general stress response was further substantiated by the finding that carbon starvation induces protection against heat shock and that this protection is at least in part dependent on PhyR.

A mutant of Methylobacterium extorquens AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [13C]formate, as well as 14C-labeling experiments, demonstrated production of labeled CO2 in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (fdh4A) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO2 formation in growing cultures. Mutation of a small open reading frame (fdh4B) downstream of fdh4A resulted in mutant phenotypes similar to the phenotypes of fdh4A mutants, suggesting that fdh4B is also involved in formate oxidation.

A cryptic plasmid of Methylobacterium extorquens AM1 was found to encode tslI, a truncated luxI homolog. tslI was shown to be expressed and to control transcription of the acyl-homoserine lactone (HSL) synthase gene msaI and thus, indirectly, acyl-HSL production. In addition, tslI was found to positively regulate extracellular polysaccharide production.

Aerial plant surfaces are colonized by diverse bacteria such as the ubiquitous Methylobacterium spp. The specific physiological traits as well as the underlying regulatory mechanisms for bacterial plant colonization are largely unknown. The purpose of this study was to identify proteins produced specifically in the phyllosphere by comparing the proteome of Methylobacterium extorquens colonizing the leaves either with that of bacteria colonizing the roots or with that of bacteria growing on synthetic medium. We identified 45 proteins that were more abundant in M. extorquens present on plant surfaces as compared with bacteria growing on synthetic medium, including 9 proteins that were more abundant on leaves compared with roots. Among the proteins induced during epiphytic growth, we found enzymes involved in methanol utilization, prominent stress proteins, and proteins of unknown function. In addition, we detected a previously undescribed type of two-domain response regulator, named PhyR, that consists of an N-terminal sigma factor (RpoE)-like domain and a C-terminal receiver domain and is predicted to be present in essentially all Alphaproteobacteria. The importance of PhyR was demonstrated through phenotypic tests of a deletion mutant strain shown to be deficient in plant colonization. Among PhyR-regulated gene products, we found a number of general stress proteins and, in particular, proteins known to be involved in the oxidative stress response such as KatE, SodA, AhpC, Ohr, Trx, and Dps. The PhyR-regulated gene products partially overlap with the bacterial in planta-induced proteome, suggesting that PhyR is a key regulator for adaptation to epiphytic life of M. extorquens.

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.

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.

Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C(2), C(3), and C(4) compounds, for example succinate. This study provides a proteomic view of the cellular adaptation of M. extorquens AM1 to growth on methanol and succinate, respectively. Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining. A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established. Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate. This group includes most proteins known to be directly involved in methanol oxidation to CO(2) and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function. Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies.

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C(1) transfers, the tetrahydrofolate (H(4)F) pathway and the tetrahydromethanopterin (H(4)MPT) pathway, and both are required for growth on C(1) compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H(4)F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C(1) compounds. Unlike mutants defective for the H(4)MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [(14)C]methanol to [(14)C]CO(2). Collectively, these data confirm our previous presumptions that the H(4)F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H(4)F pathway in this organism in which it functions to convert formate to methylene H(4)F for assimilatory metabolism.

NAD-dependent formate dehydrogenase (FDH1) was isolated from the alpha-proteobacterium Methylobacterium extorquens AM1 under oxic conditions. The enzyme was found to be a heterodimer of two subunits (alpha1beta1) of 107 and 61 kDa, respectively. The purified enzyme contained per mol enzyme approximately 5 mol nonheme iron and acid-labile sulfur, 0.6 mol noncovalently bound FMN, and approximately 1.8 mol tungsten. The genes encoding the two subunits of FDH1 were identified on the M. extorquens AM1 chromosome next to each other in the order fdh1B, fdh1A. Sequence comparisons revealed that the alpha-subunit harbours putative binding motifs for the molybdopterin cofactor and at least one iron-sulfur cluster. Sequence identity was highest to the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum). The beta-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron-sulfur cluster binding motif. The beta-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiquinone oxidoreductases.

NADP-dependent methylene-H(4)MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP(+) as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A resolution. The enzyme is present as a homotrimer. The alpha,beta fold of the monomer is related to that of methylene-H(4)F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H(4)MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H(4)F dehydrogenases are discussed.

Methylobacterium extorquens AM1 possesses a formyltransferase (Ftr) complex that is essential for growth in the presence of methanol and involved in formaldehyde oxidation to CO(2). One of the subunits of the complex carries the catalytic site for transfer of the formyl group from tetrahydromethanopterin to methanofuran (MFR). We now found via nuclear magnetic resonance-based studies that the Ftr complex also catalyzes the hydrolysis of formyl-MFR and generates formate. The enzyme was therefore renamed Ftr/hydrolase complex (Fhc). FhcA shares a sequence pattern with amidohydrolases and is assumed to be the catalytic site where the hydrolysis takes place.

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran-tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 microM; apparent Km for H4MPT = 30 microM). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of approximately 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.

MtdA catalyzes the dehydrogenation of N(5),N(10)-methylenetetrahydromethanopterin (methylene-H4MPT) with NADP(+) as electron acceptor. In the reaction two prochiral centers are involved, C14a of methylene-H4MPT and C4 of NADP(+), between which a hydride is transferred. The two diastereotopic protons at C14a of methylene-H4MPT and at C4 of NADPH can be seen separately in 1H-NMR spectra. This fact was used to determine the stereospecificity of the enzyme. With (14aR)-[14a-2H(1)]-[14a-13C]methylene-H4MPT as the substrate, it was found that the pro-R hydrogen of methylene-H4MPT is transferred by MtdA into the pro-R position of NADPH.

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.

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1). The Km value for NAD+ was 200 microM and for NADP+ 20 microM. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.