Phosphoribosylpyrophosphate synthetase as a metabolic valve advances Methylobacterium/Methylorubrum phyllosphere colonization and plant growth.

The proficiency of phyllosphere microbiomes in efficiently utilizing plant-provided nutrients is pivotal for their successful colonization of plants. The methylotrophic capabilities of Methylobacterium/Methylorubrum play a crucial role in this process. However, the precise mechanisms facilitating efficient colonization remain elusive. In the present study, we investigate the significance of methanol assimilation in shaping the success of mutualistic relationships between methylotrophs and plants. A set of strains originating from Methylorubrum extorquens AM1 are subjected to evolutionary pressures to thrive under low methanol conditions. A mutation in the phosphoribosylpyrophosphate synthetase gene is identified, which converts it into a metabolic valve. This valve redirects limited C1-carbon resources towards the synthesis of biomass by up-regulating a non-essential phosphoketolase pathway. These newly acquired bacterial traits demonstrate superior colonization capabilities, even at low abundance, leading to increased growth of inoculated plants. This function is prevalent in Methylobacterium/Methylorubrum strains. In summary, our findings offer insights that could guide the selection of Methylobacterium/Methylorubrum strains for advantageous agricultural applications.

Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism.

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Enhanced catabolism of glycine betaine and derivatives provides improved osmotic stress protection in Methylorubrum extorquens PA1.

Integration of metabolites into the overall metabolic network of a cell requires careful coordination dependent upon the ultimate usage of the metabolite. Different stoichiometric needs, and thus pathway fluxes, must exist for compounds destined for diverse uses, such as carbon sources, nitrogen sources, or stress-protective agents. Herein, we expand upon our previous work that highlighted the nature of glycine betaine (GB) metabolism in Methylobacteria to examine the utilization of GB-derivative compounds dimethylglycine (DMG) and sarcosine into Methylorubrum extorquens in different metabolic capacities, including as sole nitrogen and/or carbon sources. We isolated gain-of-function mutations that allowed M. extorquens PA1 to utilize dimethylglycine as a carbon source and dimethylglycine and sarcosine as nitrogen source. Characterization of mutants demonstrated selection for variants of the AraC-like regulator Mext_3735 that confer constitutive expression of the GB metabolic gene cluster, allowing direct utilization of the downstream GB derivatives. Finally, among the distinct isolates examined, we found that catabolism of the osmoprotectant used for selection (GB or dimethylglycine) enhanced osmotic stress resistance provided in the presence of that particular osmolyte. Thus, access to the carbon and nitrogen and osmoprotective effects of GB and DMG are made readily accessible through adaptive mutations. In M. extorquens PA1, the limitations to exploiting this group of compounds appear to exist predominantly at the levels of gene regulation and functional activity, rather than being constrained by transport or toxicity.IMPORTANCEOsmotic stress is a common challenge for bacteria colonizing the phyllosphere, where glycine betaine (GB) can be found as a prevalent osmoprotectant. Though Methylorubrum extorquens PA1 cannot use GB or its demethylation products, dimethylglycine (DMG) and sarcosine, as a sole carbon source, utilization is highly selectable via single nucleotide changes for both GB and DMG growth. The innate inability to use these compounds is due to limited flux through steps in the pathway and regulatory constraints. Herein, the characterization of the transcriptional regulator, Mext_3735 (GbdR), expands our understanding of the various roles in which GB derivatives can be used in M. extorquens PA1. Interestingly, increased catabolism of GB and derivatives does not interfere with, but rather improves, the ability of cells to thrive under increased salt stress conditions, suggesting that metabolic flux improves stress tolerance rather than providing a distinct tension between uses.

Glycine betaine metabolism is enabled in Methylorubrum extorquens PA1 by alterations to dimethylglycine dehydrogenase.

Low nutrient availability is a key characteristic of the phyllosphere (the aerial surface of plants). Phyllospheric bacteria utilize a wide array of carbon sources generated by plant hosts. Glycine betaine (GB) is a plant-derived compound that can be metabolized by certain members of the phyllosphere microbiota. Metabolism of glycine betaine generates formaldehyde, an intermediate of methylotrophic metabolism, leading us to investigate how the ubiquitous plant colonizing bacterium Methylorubrum extorquens PA1 might metabolize GB encountered in its native environment. M. extorquens PA1 cannot utilize GB as a sole carbon source. Through suppressor mutation analysis, we show that M. extorquens PA1 encodes a conserved GB utilization pathway that can be activated by single point mutations conferring GB utilization as a carbon source. We identified the gene cluster encoding the GB catabolic enzymes and found that gene expression was induced in the presence of GB. We show that utilization of GB is conserved among representative Methylobacterium species and generates the one-carbon metabolism intermediate formaldehyde, which M. extorquens utilizes as a source of energy. Our results support a model where suppressor mutations in Mext_3745 or ftsH (Mext_4840) prevent the degradation of the dimethylglycine dehydrogenase subunit DgcB by the membrane integral protease FtsH, conferring the ability to utilize GB by either (i) restoring stable membrane topology of DgcB or (ii) decreasing FtsH protease activity, respectively. Both mutations alleviate the bottleneck at the second step of GB degradation catalyzed by DgcAB.IMPORTANCEOvercoming low nutrient availability is a challenge many bacteria encounter in the environment. Facultative methylotrophs are able to utilize one-carbon and multi-carbon compounds as carbon and energy sources. The utilization of plant-derived glycine betaine (GB) represents a possible source of multi-carbon and one-carbon substrates. The metabolism of glycine betaine produces formaldehyde and glycine, which may be used simultaneously by facultative methylotrophs. However, the genes required for the utilization of GB in the ubiquitous plant-associated bacterium Methylorubrum extorquens have yet to be identified or described. Our work identifies and validates the genes required for glycine betaine metabolism in M. extorquens and shows that it directly intersects with methylotrophic metabolism through the production of formaldehyde.

Expression of toxic genes in Methylorubrum extorquens with a tightly repressed, cumate-inducible promoter.

Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.

Compartment-related aspects of XoxF protein functionality in Methylorubrum extorquens DM4 analysed using its cytoplasmic targeting.

The impact of periplasmic localisation on the functioning of the XoxF protein was evaluated in the well-studied dichloromethane-utilising methylotroph Methylorubrum extorquens DM4, which harbors only one paralogue of the xoxF gene. It was found that the cytoplasmic targeting of XoxF by expression of the corresponding gene without the sequence encoding the N-terminal signal peptide does not impair the activation and lanthanide-dependent regulation of the MxaFI-methanol dehydrogenase genes. Analysis of the viability of ΔxoxF cells complemented with the full-length and truncated xoxF gene also showed that the expression of cytoplasmically targeted XoxF even increases the resistance to acids. These results contradict the proposed function of the XoxF protein as an extracytoplasmic signal sensor. At the same time, the observed dynamics of growth with methanol, as well as with dichloromethane of strains expressing cytoplasmic-targeted XoxF, indicate the probable enzymatic activity of lanthanide-dependent methanol dehydrogenase in this compartment. Herewith, the only available substrate for this enzyme in cells growing with dichloromethane was formaldehyde, which is produced during the primary metabolism of the mentioned halogenated toxicant directly in the cytosol. These findings suggest that the maturation of XoxF-methanol dehydrogenase may occur already in the cytoplasm, while the factors changing affinity of this enzyme for formaldehyde are apparently absent there. Together with the demonstrated functioning of an enhancer-like upstream activating sequence in the promoter region of the xoxF gene in M. extorquens DM4, the obtained information enriches our understanding of the regulation, synthesis and role of the XoxF protein.

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation.

Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO2-but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.

Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake.

The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids. KEY POINTS: • 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations. • Deletion of dctA2 leads to reduction of dicarboxylic acid uptake. • Transporter-deficient strains show improved production of citramalic acid.

Isolation and Characterization of Homologically Expressed Methanol Dehydrogenase from Methylorubrum extorquens AM1 for the Development of Bioelectrocatalytical Systems.

(Ca2+)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6ß6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9-10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (KM = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002-0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.

Development of Methylorubrum extorquens AM1 as a promising platform strain for enhanced violacein production from co-utilization of methanol and acetate.

Violacein, a blue-violet compound with a wide range of beneficial bioactivities, is an attractive product for microbial production. Currently, violacein production has been demonstrated in several sugar heterotrophs through metabolic engineering; however, the cost of production remains an obstacle for business ventures. To address this issue, the development of host strains that can utilize inexpensive alternative substrates to reduce production costs would enable the commercialization of violacein. In this study, we engineered a facultative methylotroph, Methylorubrum extorquens AM1, to develop a methanol-based platform for violacein production. By optimizing expression vectors as well as inducer concentrations, 11.7 mg/L violacein production was first demonstrated using methanol as the sole substrate. Considering that unidentified bottlenecks for violacein biosynthesis in the shikimate pathway of M. extorquens AM1 would be difficult to address using generic metabolic engineering approaches, random mutagenesis and site-directed mutagenesis were implemented, and a 2-fold improvement in violacein production was achieved. Finally, by co-utilization of methanol and acetate, a remarkable enhancement of violacein production to 118 mg/L was achieved. Our results establish a platform strain for violacein production from non-sugar feedstocks, which may contribute to the development of an economically efficient large-scale fermentation system for violacein production.

Structural insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1.

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.

Functional diversity of isoprenoid lipids in Methylobacterium extorquens PA1.

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1.

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde.

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens.

AIM: Genetic tools are a prerequisite for engineering cell factories for synthetic biology and biotechnology. Methylorubrum extorquens is an important platform for a future one-carbon (C1) bioeconomy, but its application is currently limited by the availability of genetic tools. Small regulatory RNA (sRNA) is an important regulatory factor in bacteria and has been applied for gene repression in several strains. This study aimed to construct a synthetic sRNA system based on the MicC scaffold and the chaperone Hfq to control gene expression in M. extorquens. METHODS AND RESULTS: Initially, the exogenous lacZ gene was transposed into the M. extorquens chromosome as a reporter, and corresponding ß-galactosidase was measured to assess the knockdown efficiency of lacZ. A synthetic sRNA containing a 24-nt antisense RNA targeting lacZ and an Escherichia coli MicC scaffold were constructed, and different Hfqs from E. coli, M. extorquens AM1 and PA1 were further identified. The results showed that the expression of endogenous hfqs from the chromosome in M. extorquens strains was inadequate, and only when it was overexpressed via the plasmid did the colonies show a colour change and a corresponding decrease in ß-galactosidase expression. More specifically, M. extorquens strains with overexpressing their own Hfq showed the best gene repression efficiency. Furthermore, this E. coli MicC scaffold and AM1 Hfq system were combined to knock down crtI gene expression in AM1, leading to an 86% decrease in carotenoid production (0·09 mg g-1 ) compared to that (0·65 mg g-1 ) in the wild-type strain. CONCLUSION: A functional synthetic sRNA system combined with E. coli MicC and endogenous Hfq was constructed in M. extorquens strains, which was able to interfere with the target crtI gene and reduce carotenoid production. SIGNIFICANCE AND IMPACT OF THE STUDY: The synthetic sRNA system reported in this study provides a genetic tool for the manipulation of M. extorquens. The present findings might be helpful for achieving high-throughput gene knockdown expression.

Transposon mutagenesis for methylotrophic bacteria using Methylorubrum extorquens AM1 as a model system.

Transposon mutagenesis utilizes transposable genetic elements that integrate into a recipient genome to generate random insertion mutations which are easily identified. This forward genetic approach has proven powerful in elucidating complex processes, such as various pathways in methylotrophy. In the past decade, many methylotrophic bacteria have been shown to possess alcohol dehydrogenase enzymes that use lanthanides (Lns) as cofactors. Using Methylorubrum extorquens AM1 as a model organism, we discuss the experimental designs, protocols, and results of three transposon mutagenesis studies to identify genes involved in different aspects of Ln-dependent methanol oxidation. These studies include a selection for transposon insertions that prevent toxic intracellular formaldehyde accumulation, a fluorescence-imaging screen to identify regulatory processes for a primary Ln-dependent methanol dehydrogenase, and a phenotypic screen for genes necessary for function of a Ln-dependent ethanol dehydrogenase. We anticipate that the methods described in this chapter can be applied to understand other metabolic systems in diverse bacteria.

Metabolomic analysis improves bioconversion of methanol to isobutanol in Methylorubrum extorquens AM1.

BACKGROUND: Methylorubrum extorquens AM1 can be engineered to convert methanol to value-added chemicals. Most of these chemicals derive from acetyl-CoA involved in the serine cycle. However, recent studies on methylotrophic metabolism have suggested that C3 pyruvate is a good potential precursor for broadening the types of synthesized products. METHODS AND RESULTS: In the present study, we found that isobutanol was a model chemical that could be generated from pyruvate through a 2-keto acid pathway. Initially, the engineered M. extorquens AM1 could only produce a trace amount of isobutanol at 0.62 mgL-1 after introducing the heterologous 2-ketoisovalerate decarboxylase and alcohol dehydrogenase. Furthermore, the metabolomic analysis revealed that insufficient carbon fluxes through 2-ketoisovalerate and pyruvate were the key limitation steps for efficient biosynthesis of isobutanol. Based on this analysis, the titer of isobutanol was improved by over 20-fold after overexpressing alsS gene encoding acetolactate synthase and deleting ldhA gene for lactate dehydrogenase. Moreover, substituting the cell chassis with the isobutanol-tolerant strain isolated from adaptive evolution of M. extorquens AM1 further increased the production of isobutanol by 1.7-fold, resulting in the final titer of 19 mgL-1 in flask cultivation. CONCLUSION: Our current findings provided promising insights into engineering methylotrophic cell factories capable of converting methanol to isobutanol or value-added chemicals using pyruvate as the precursor.

Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens.

Methanol is assimilated through the serine cycle to generate acetyl-CoA without carbon loss. However, a highly active serine cycle requires high consumption of reducing equivalents and ATP, thereby leading to the impaired efficiency of methanol conversion to reduced chemicals. In the present study, a genome-scale flux balance analysis (FBA) predicted that the introduction of the heterologous ribulose monophosphate (RuMP) cycle, a more energy-efficient pathway for methanol assimilation, could theoretically increase growth rate by 31.3% for the model alphaproteobacterial methylotroph Methylorubrum extorquens AM1. Based on this analysis, we constructed a novel synergistic assimilation pathway in vivo by incorporating the RuMP cycle into M. extroquens metabolism with the intrinsic serine cycle. We demonstrated that the operation of the synergistic pathway could increase cell growth rate by 16.5% and methanol consumption rate by 13.1%. This strategy rewired the central methylotrophic metabolism through adjusting core gene transcription, leading to a pool size increase of C2 to C5 central intermediates by 1.2- to 3.6-fold and an NADPH cofactor improvement by 1.3-fold. The titer of 3-hydroxypropionic acid (3-HP), a model product in the newly engineered chassis of M. extorquens AM1, was increased to 91.2 mg/L in shake-flask culture, representing a 3.1-fold increase compared with the control strain with only the serine cycle. The final titer of 3-HP was significantly improved to 0.857 g/L in the fed-batch bioreactor, which was more competitive compared with the other 3-HP producers using methane and CO2 as C1 sources. Collectively, our current study demonstrated that engineering the synergistic methanol assimilation pathway was a promising strategy to increase the carbon assimilation and the yields of reduced chemicals in diverse host strains for C1 microbial cell factories.

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1.

Lanthanide elements have been recently recognized as "new life metals" yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.

Biochemical properties and crystal structure of formate-tetrahydrofolate ligase from Methylobacterium extorquens CM4.

Methylobacterium extorquens is a methylotroph model organism that has the ability to assimilate formate using the tetrahydrofolate (THF) pathway. The formate-tetrahydrofolate ligase from M. extorquens (MeFtfL) is an enzyme involved in the THF pathway that catalyzes the conversion of formate, THF, and ATP into formyltetrahydrofolate and ADP. To investigate the biochemical properties of MeFtfL, we evaluated the metal usage and enzyme kinetics of the enzyme. MeFtfL uses the Mg ion for catalytic activity, but also has activity for Mn and Ca ions. The enzyme kinetics analysis revealed that Km value of farmate was much higher than THF and ATP, which shows that the ligation activity of MeFtfL is highly dependent on formation concentration. We also determined the crystal structure of MeFtfL at 2.8 Å resolution. MeFtfL functions as a tetramer, and each monomer consists of three domains. The structural superposition of MeFtfL with FtfL from Moorella thermoacetica allowed us to predict the substrate binding site of the enzyme.

Preference for particular lanthanide species and thermal stability of XoxFs in Methylorubrum extorquens strain AM1.

XoxF-type methanol dehydrogenase was recently found to be lanthanide-dependent, while its counterpart MxaF is Ca2+-dependent. The lanthanide (Ln) series consists of 15 different elements, all of which exist in nature, although at different relative abundances. XoxF from Methylorubrum extorquens strain AM1 has been shown to be induced by four light Ln species (La3+ to Nd3+). The preference of XoxFs for certain co-existing Ln species and the catalytic activity and stability of XoxF metallated with different Ln species have not been well investigated. In this study, we found that (i) strain AM1 cells preferentially utilize La3+ rather than Nd3+ for growth, (ii) XoxF purified from cells grown with a La3+ and Nd3+ mixture contained a larger proportion of La3+, and (iii) La3+-metallated XoxF has higher activity and thermal stability than Nd3+-metallated XoxF, although (iv) both enzymes showed unchanged surface charges. Thermal shift assay in particular revealed that metallation affects the temperature for subunit denaturation but not for subunit dissociation. We concluded that, although La3+ and Nd3+ have similar distributions in nature, XoxF could chose La3+ preferentially.