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.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Rank orders and signed interactions in evolutionary biology.

Rank orders have been studied in evolutionary biology for almost a hundred years. Constraints on the order in which mutations accumulate are known from cancer drug treatment, and order constraints for species invasions are important in ecology. However, current theory on rank orders in biology is somewhat fragmented. Here, we show how our previous work on inferring genetic interactions from comparative fitness data (Crona et al., 2017) is related to an influential approach to rank orders based on sign epistasis. Our approach depends on order perturbations that indicate interactions. We apply our results to malaria parasites and find that order perturbations beyond sign epistasis are prevalent in the antimalarial drug-resistance landscape. This finding agrees with the observation that reversed evolution back to the ancestral type is difficult. Another application concerns the adaptation of bacteria to a methanol environment.

Microbial phenotypic heterogeneity in response to a metabolic toxin: Continuous, dynamically shifting distribution of formaldehyde tolerance in Methylobacterium extorquens populations.

While microbiologists often make the simplifying assumption that genotype determines phenotype in a given environment, it is becoming increasingly apparent that phenotypic heterogeneity (in which one genotype generates multiple phenotypes simultaneously even in a uniform environment) is common in many microbial populations. The importance of phenotypic heterogeneity has been demonstrated in a number of model systems involving binary phenotypic states (e.g., growth/non-growth); however, less is known about systems involving phenotype distributions that are continuous across an environmental gradient, and how those distributions change when the environment changes. Here, we describe a novel instance of phenotypic diversity in tolerance to a metabolic toxin within wild-type populations of Methylobacterium extorquens, a ubiquitous phyllosphere methylotroph capable of growing on the methanol periodically released from plant leaves. The first intermediate in methanol metabolism is formaldehyde, a potent cellular toxin that is lethal in high concentrations. We have found that at moderate concentrations, formaldehyde tolerance in M. extorquens is heterogeneous, with a cell's minimum tolerance level ranging between 0 mM and 8 mM. Tolerant cells have a distinct gene expression profile from non-tolerant cells. This form of heterogeneity is continuous in terms of threshold (the formaldehyde concentration where growth ceases), yet binary in outcome (at a given formaldehyde concentration, cells either grow normally or die, with no intermediate phenotype), and it is not associated with any detectable genetic mutations. Moreover, tolerance distributions within the population are dynamic, changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell time-lapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.

Methylofuran is a prosthetic group of the formyltransferase/hydrolase complex and shuttles one-carbon units between two active sites.

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.