Methylamine Utilization via the N-Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C1 Assimilation and Dissimilation Pathways

Methylotrophs grow on reduced single-carbon compounds like methylamine as the sole source of carbon and energy. In Methylobacterium extorquens AM1, the best-studied aerobic methylotroph, a periplasmic methylamine dehydrogenase that catalyzes the primary oxidation of methylamine to formaldehyde has been examined in great detail. However, recent metagenomic data from natural ecosystems are revealing the abundance and importance of lesser-known routes, such as the N-methylglutamate pathway, for methylamine oxidation. In this study, we used M. extorquens PA1, a strain that is closely related to M. extorquens AM1 but is lacking methylamine dehydrogenase, to dissect the genetics and physiology of the ecologically relevant N-methylglutamate pathway for methylamine oxidation. Phenotypic analyses of mutants with null mutations in genes encoding enzymes of the N-methylglutamate pathway suggested that γ-glutamylmethylamide synthetase is essential for growth on methylamine as a carbon source but not as a nitrogen source. Furthermore, analysis of M. extorquens PA1 mutants with defects in methylotrophy-specific dissimilatory and assimilatory modules suggested that methylamine use via the N-methylglutamate pathway requires the tetrahydromethanopterin (H₄MPT)-dependent formaldehyde oxidation pathway but not a complete tetrahydrofolate (H₄F)-dependent formate assimilation pathway. Additionally, we present genetic evidence that formaldehyde-activating enzyme (FAE) homologs might be involved in methylotrophy. Null mutants of FAE and homologs revealed that FAE and FAE2 influence the growth rate and FAE3 influences the yield during the growth of M. extorquens PA1 on methylamine.     

Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions

Methylobacterium extorquens AM1 has been shown to accumulate polyhydroxyalkanoate (PHA) composed solely of (R)-3-hydroxybutyrate (3HB) during methylotrophic growth. The present study demonstrated that the wild-type strain AM1 grown under Co²⁺-deficient conditions accumulated copolyesters of 3HB and a C₅-monomer, (R)-3-hydroxyvalerate (3HV), using methanol as the sole carbon source. The 3HV unit was supposed to be derived from propionyl-CoA, synthesized via the ethylmalonyl-CoA pathway impaired by Co²⁺ limitation. This assumption was strongly supported by the dominant incorporation of the 3HV unit into PHA when a strain lacking propionyl-CoA carboxylase was incubated with methanol. Further genetic engineering of M. extorquens AM1 was employed for the methylotrophic synthesis of PHA copolymers. A recombinant strain of M. extorquens AM1C_Ac in which the original PHA synthase gene phaC _Me had been replaced by phaC _Ac , encoding an enzyme with broad substrate specificity from Aeromonas caviae, produced a PHA terpolymer composed of 3HB, 3HV, and a C₆-monomer, (R)-3-hydroxyhexanoate, from methanol. The cellular content and molecular weight of the PHA accumulated in the strain AM1C_Ac were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1C_Ac showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding β-ketothiolase and NADPH-acetoacetyl-CoA reductase increased the cellular PHA content and 3HV composition in PHA, although the cell growth on methanol was decreased. This study opens up the possibility of producing practical PHA copolymers with methylotrophic bacteria using methanol as a feedstock.     

Co-Consumption of Methanol and Succinate by Methylobacterium extorquens AM1

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. ¹³C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO₂ 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.     

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

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.     

Development of an Optimized Medium,Strain and High-Throughput Culturing Methods for Methylobacterium extorquens

Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.     

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

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.Methylobacterium extorquens AM1 grows on one-carbon (C₁) compounds using the serine cycle for assimilation (25). This metabolism requires the conversion of acetyl-coenzyme A (CoA) to glyoxylate, which occurs via a novel pathway in which acetyl-CoA is converted to methylsuccinyl-CoA via acetoacetyl-CoA, ß-hydroxybutyryl-CoA, and ethylmalonyl-CoA (30-33). Recently, the steps involved in the conversion of methylsuccinyl-CoA to glyoxylate have been elucidated, and the pathway has been termed the ethylmalonyl-CoA (EMC) pathway (1, 19, 20, 40). Careful labeling measurements coupled to measurements of intermediates has confirmed that, during the growth of M. extorquens AM1 on methanol, methylsuccinyl-CoA is converted to glyoxylate and propionyl-CoA via mesaconyl-CoA and ß-methylmalyl-CoA (40).This finding has raised questions regarding how M. extorquens AM1 grows on two-carbon (C₂) compounds. The pathway involved in the conversion of acetyl-CoA to glyoxylate is known to operate during growth on both C₁ and C₂ compounds, as mutants in genes involved in this conversion are unable to grow on either C₁ or C₂ compounds, and in both cases they are rescued by glyoxylate (11, 15-17, 44). If glyoxylate is produced as an end product of this pathway during C₂ growth, then it must be converted to an intermediate of central metabolism, which has been proposed to involve a malate synthase activity (2, 14-17) (Fig. ​(Fig.1).1). In M. extorquens AM1, the apparent malate synthase activity is carried out in two steps, first by converting acetyl-CoA and glyoxylate to malyl-CoA by malyl-CoA lyase and then by converting malyl-CoA to malate by malyl-CoA hydrolase (Fig. ​(Fig.1)1) (14). However, a mutant (PCT57) defective in malyl-CoA lyase (MclA1) (22), which contains no detectable malate synthase activity during growth on methanol, is able to grow on C₂ compounds (43).Open in a separate windowFIG. 1.Enzymes and genes involved in the ethylmalonyol-CoA pathway. The colors of gene names denote a change in gene expression from microarray results comparing wild-type cells grown on ethylamine to those grown on succinate: dark red, >3-fold increase; light red, 1.5- to 3-fold increase; black, no significant change (1.49-fold increase to 1.49-fold decrease); light green, 1.5- to 3-fold decrease; dark green, >3-fold decrease. Parentheses denote a predicted function not confirmed by the mutant phenotype. See Table ​Table11 for enzyme names.Clearly, the finding that glyoxylate is generated as a direct product of the EMC pathway presents a conundrum. Apparently acetyl-CoA is converted to glyoxylate via this pathway, but M. extorquens AM1 lacking malate synthase is able to grow on C₂ compounds. Another apparent conundrum involving the malyl-CoA lyase (mclA1) mutant is that the EMC pathway requires an enzyme that carries out ß-methylmalyl-CoA cleavage, a reaction that homologs of MclA1 are known to carry out (38). The MclA1 enzyme has been purified from M. extorquens AM1 and shown to have activity with glyoxylate and propionyl-CoA (27), which would produce ß-methylmalyl-CoA. These results have led to the suggestion that MclA homologs actually are malyl-CoA/ß-methylmalyl-CoA lyases (38). Since the mclA1 mutant does not contain detectable malyl-CoA lyase activity, and by inference has correspondingly low ß-methylmalyl-CoA lyase activity, it was not clear how M. extorquens AM1 could convert acetyl-CoA to propionyl-CoA and glyoxylate via the EMC pathway in the mclA1 mutant.The purpose of this study was to solve these conundrums and determine how mutants of M. extorquens AM1 grow on C₂ compounds in the absence of malyl-CoA/ß-methylmalyl-CoA lyase or malate synthase activity. Our results show (i) that the known homolog of MclA1 (MclA2) appears to be capable of supporting both ß-methylmalyl-CoA cleavage and condensation between glyoxylate and acetyl-CoA in the mclA1 mutant, and (ii) that an alternative route for glyoxylate consumption occurs in this bacterium, in which it is converted to intermediates of central metabolism via a part of the serine cycle coupled with the glycine cleavage system.     

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Background

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared.

Methodology/Principal Findings

The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).

Conclusion/Significance

These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.     

Identification of a fourth formate dehydrogenase in Methylobacterium extorquens AM1 and confirmation of the essential role of formate oxidation in methylotrophy

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.     

Thioesterases for ethylmalonyl–CoA pathway derived dicarboxylic acid production in <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

The ethylmalonyl–coenzyme A pathway (EMCP) is a recently discovered pathway present in diverse α-proteobacteria such as the well studied methylotroph Methylobacterium extorquens AM1. Its glyoxylate regeneration function is obligatory during growth on C1 carbon sources like methanol. The EMCP contains special CoA esters, of which dicarboxylic acid derivatives are of high interest as building blocks for chemical industry. The possible production of dicarboxylic acids out of the alternative, non-food competing C-source methanol could lead to sustainable and economic processes. In this work we present a testing of functional thioesterases being active towards the EMCP CoA esters including in vitro enzymatic assays and in vivo acid production. Five thioesterases including TesB from Escherichia coli and M. extorquens, YciA from E. coli, Bch from Bacillus subtilis and Acot4 from Mus musculus showed activity towards EMCP CoA esters in vitro at which YciA was most active. Expressing yciA in M. extorquens AM1 led to release of 70 mg/l mesaconic and 60 mg/l methylsuccinic acid into culture supernatant during exponential growth phase. Our data demonstrates the biotechnological applicability of the thioesterase YciA and the possibility of EMCP dicarboxylic acid production from methanol using M. extorquens AM1.     

Microbial growth on oxalate by a route not involving glyoxylate carboligase

1. The metabolism of oxalate by the pink-pigmented organisms, Pseudomonas AM1, Pseudomonas AM2, Protaminobacter ruber and Pseudomonas extorquens has been compared with that of the non-pigmented Pseudomonas oxalaticus. 2. During growth on oxalate, all the organisms contain oxalyl-CoA decarboxylase, formate dehydrogenase and oxalyl-CoA reductase. This is consistent with oxidation of oxalate to carbon dioxide taking place via oxalyl-CoA, formyl-CoA and formate as intermediates, and also reduction of oxalate to glyoxylate taking place via oxalyl-CoA. 3. The pink-pigmented organisms, when grown on oxalate, contain l-serine–glyoxylate aminotransferase and hydroxypyruvate reductase but do not contain glyoxylate carboligase. The converse of this obtains in oxalate-grown Ps. oxalaticus. This indicates that, in contrast with Ps. oxalaticus, synthesis of C₃ compounds from oxalate by the pink-pigmented organisms occurs by a variant of the `serine pathway' used by Pseudomonas AM1 during growth on C₁ compounds. 4. Evidence in favour of this scheme is provided by the finding that a mutant of Pseudomonas AM1 that lacks hydroxypyruvate reductase is not able to grow on oxalate.     

Genome Information of Methylobacterium oryzae,a Plant-Probiotic Methylotroph in the Phyllosphere

Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20^T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B₁₂ biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated.     

Cloning and characterization of indolepyruvate decarboxylase from <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

For the first time for methylotrophic bacteria an enzyme of phytohormone indole-3-acetic acid (IAA) biosynthesis, indole-3-pyruvate decarboxylase (EC 4.1.1.74), has been found. An open reading frame (ORF) was identified in the genome of facultative methylotroph Methylobacterium extorquens AM1 using BLAST. This ORF encodes thiamine diphosphate-dependent 2-keto acid decarboxylase and has similarity with indole-3-pyruvate decarboxylases, which are key enzymes of IAA biosynthesis. The ORF of the gene, named ipdC, was cloned into overexpression vector pET-22b(+). Recombinant enzyme IpdC was purified from Escherichia coli BL21(DE3) and characterized. The enzyme showed the highest k _cat value for benzoylformate, albeit the indolepyruvate was decarboxylated with the highest catalytic efficiency (k _cat/K _m). The molecular mass of the holoenzyme determined using gel-permeation chromatography corresponds to a 245-kDa homotetramer. An ipdC-knockout mutant of M. extorquens grown in the presence of tryptophan had decreased IAA level (46% of wild type strain). Complementation of the mutation resulted in 6.3-fold increase of IAA concentration in the culture medium compared to that of the mutant strain. Thus involvement of IpdC in IAA biosynthesis in M. extorquens was shown.     

Measurement of Respiration Rates of Methylobacterium extorquens AM1 Cultures by Use of a Phosphorescence-Based Sensor

Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.     

A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1

In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca²⁺-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La³⁺-dependent. Despite the absence of Ca²⁺ in the medium strain AM1 was able to grow on methanol in the presence of La³⁺. Addition of La³⁺ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La³⁺, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La³⁺ as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca²⁺, but was able to grow after supplementation with La³⁺. Taken together, these results show that XoxF1 participates in methanol metabolism as a La³⁺-dependent MDH in strain AM1.     

Methanol Assimilation in Methylobacterium extorquens AM1: Demonstration of All Enzymes and Their Regulation

Background

Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C₂) or succinate (C₄) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.

Methodology/Principal Findings

We present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C₁, C₂ or C₄ compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C₁ carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.

Conclusion/Significance

This study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.     

Metabolite Profiling Uncovers Plasmid-Induced Cobalt Limitation under Methylotrophic Growth Conditions

Background

The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases.

Methodology/Principal Findings

We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B₁₂-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import.

Conclusions/Significance

This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress.     

Multicopy Integration and Expression of Heterologous Genes in Methylobacterium extorquens ATCC 55366

High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (P_mxaF) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [β-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.