Lanthanide-dependent isolation of phyllosphere methylotrophs selects for a phylogenetically conserved but metabolically diverse community.

The influence of lanthanide biochemistry during methylotrophy demands a reassessment of how the composition and metabolic potential of methylotrophic phyllosphere communities are affected by the presence of these metals. To investigate this, methylotrophs were isolated from soybean leaves by selecting for bacteria capable of methanol oxidation with lanthanide cofactors. Of the 344 pink-pigmented facultative methylotroph isolates, none were obligately lanthanide-dependent. Phylogenetic analyses revealed that all strains were nearly identical to each other and to model strains from the extorquens clade of Methylobacterium, with rpoB providing higher resolution than 16s rRNA for strain-specific identification. Despite the low species diversity, the metabolic capabilities of the community diverged greatly. Strains encoding identical PQQ-dependent alcohol dehydrogenases displayed significantly different growth from each other on alcohols in the presence and absence of lanthanides. Several strains also lacked well-characterized lanthanide-associated genes thought to be important for phyllosphere colonization. Additionally, 3% of our isolates were capable of growth on sugars and 23% were capable of growth on aromatic acids, substantially expanding the range of multicarbon substrates utilized by members of the extorquens clade in the phyllosphere. Whole genome sequences of eleven novel strains are reported. Our findings suggest that the expansion of metabolic capabilities, as well as differential usage of lanthanides and their influence on metabolism among closely related strains, point to evolution of niche partitioning strategies to promote colonization of the phyllosphere.

A Novel Protein for the Bioremediation of Gadolinium Waste.

Several hundreds of tons of gadolinium-based contrast agents (GBCAs) are being dumped into the environment every year. Although macrocyclic GBCAs exhibit superior stability compared to their linear counterparts, we have found that the structural integrity of chelates are susceptible to ultraviolet light, regardless of configuration. In this study, we present a synthetic protein termed GLamouR that binds and reports gadolinium in an intensiometric manner. We then explore the extraction of gadolinium from GBCA-spiked artificial urine samples and investigate if the low picomolar concentrations reported in gadolinium-contaminated water sources pose a barrier for bioremediation. Based on promising results, we anticipate GLamouR can be used for detecting and mining REEs beyond gadolinium as well and hope to expand the biological toolbox for such applications.

A perspective on the role of lanthanides in biology: Discovery, open questions and possible applications.

Because of their use in high technologies like computers, smartphones and renewable energy applications, lanthanides (belonging to the group of rare earth elements) are essential for our daily lives. A range of applications in medicine and biochemical research made use of their photo-physical properties. The discovery of a biological role for lanthanides has boosted research in this new field. Several methanotrophs and methylotrophs are strictly dependent on the presence of lanthanides in the growth medium while others show a regulatory response. After the first demonstration of a lanthanide in the active site of the XoxF-type pyrroloquinoline quinone methanol dehydrogenases, follow-up studies showed the same for other pyrroloquinoline quinone-containing enzymes. In addition, research focused on the effect of lanthanides on regulation of gene expression and uptake mechanism into bacterial cells. This review briefly describes the discovery of the role of lanthanides in biology and focuses on open questions in biological lanthanide research and possible application of lanthanide-containing bacteria and enzymes in recovery of these special elements.

Methylothon: a Versatile Course-Based High School Research Experience in Microbiology and Bioinformatics with Pink Bacteria.

Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here, we present an overview of the module's learning goals, describe course resources (available for public use at http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

Comprehensive Phylogenomics of Methylobacterium Reveals Four Evolutionary Distinct Groups and Underappreciated Phyllosphere Diversity.

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum.

Hyperaccumulation of Gadolinium by Methylorubrum extorquens AM1 Reveals Impacts of Lanthanides on Cellular Processes Beyond Methylotrophy.

Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57-62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo-HLn, for "evolved for Heavy Lanthanides." A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Ln accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the "lanthasome." Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo-HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo-HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery.

Expression, purification and testing of lanthanide-dependent enzymes in Methylorubrum extorquens AM1.

With mounting evidence of the importance of lanthanide metals in biology and among diverse bacterial phyla, a platform for high-throughput microbial growth for expression and purification of lanthanide-dependent enzymes is increasingly important. Presented in this chapter is a stream-lined approach for growth of the model methylotrophic bacterium Methylorubrum extorquens AM1 for the expression of lanthanide-dependent enzymes. Growth is optimized for both high-throughput phenotypic characterization facilitating in vivo studies, as well as for scaled-up batch cultivation for enzyme purification allowing for in vitro enzymatic studies. Both approaches have been shown to be important to understanding the function and structure of these enzymes. Expression systems have been designed for production of enzymes with and without lanthanide metals, allowing for detection of lanthanide dependence. The protocol described herein is expected to accelerate the discovery of novel lanthanide-dependent enzymes and our understanding of the role of these metals in the greater biological world.

Lanthanide-dependent alcohol dehydrogenases require an essential aspartate residue for metal coordination and enzymatic function.

The lanthanide elements (Ln3+), those with atomic numbers 57-63 (excluding promethium, Pm3+), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca2+-PQQ cofactor. An important missing piece of the Ln3+ puzzle is defining what features distinguish enzymes that use Ln3+-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium Methylorubrum extorquens AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La3+ bound in the active-site region and coordinated by Asp320 Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp320 is needed for in vivo catalytic function, in vitro activity, and La3+ coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La3+, coordinated Ca2+ but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La3+ These results provide evidence that a Ln3+-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln3+ metabolism.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Contrasting in vitro and in vivo methanol oxidation activities of lanthanide-dependent alcohol dehydrogenases XoxF1 and ExaF from Methylobacterium extorquens AM1.

Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) from Methylobacterium extorquens AM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol- and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9-12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation of xox1 and downregulation of mxa genes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation of fdh4 formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in the fae mutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy.

Facile Arsenazo III-Based Assay for Monitoring Rare Earth Element Depletion from Cultivation Media for Methanotrophic and Methylotrophic Bacteria.

Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low µM (0.1 to 10 µM) range. We designed this assay to follow LaIII and EuIII depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as PrIII, or to monitor the depletion of LaIII from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS).IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria.

Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates.

Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La3+). ExaF was purified from cells grown with both calcium (Ca2+) and La3+ and with Ca2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La3+ over Ca2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca2+-plus-La3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca2+-only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La3+ is present, expanding the role of lanthanides to multicarbon metabolism. IMPORTANCE: ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth.

UNLABELLED: Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 µM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. IMPORTANCE: The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements.

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1.

UNLABELLED: During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH4MPT), an early intermediate during methanol metabolism, transiently accumulated 7- to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H4MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH4MPT did not accumulate methenyl-dH4MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH4MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH4MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH4MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde. IMPORTANCE: The identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional regulatory mechanism that was found to operate as an immediate response for acclimation during changes in substrate availability. Methenyl-dH4MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H4MPT dehydrogenase, MtdA, when Methylobacterium extorquens is challenged to switch from succinate to methanol growth. Bypassing this regulatory mechanism causes formaldehyde to accumulate. Fae, the enzyme catalyzing the conversion of formaldehyde to methylene-dH4MPT, was also identified as another potential regulatory target using this strategy. The results herein further our understanding of the complex regulatory network in methylotrophy and will allow us to improve metabolic engineering strategies of methylotrophs for the production of value-added products.

C1-Pathways in Methyloversatilis universalis FAM5: Genome Wide Gene Expression and Mutagenesis Studies.

Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. As expected, all genes for the N-methylglutamate pathway were induced during growth on methylamine. Among other functions responding to the aminated source of C1-carbon, are a heme-containing amine dehydrogenase (Qhp), a distant homologue of formaldehyde activating enzyme (Fae3), molybdenum-containing formate dehydrogenase, ferredoxin reductase, a set of homologues to urea/ammonium transporters and amino-acid permeases. Mutants lacking one of the functional subunits of the amine dehydrogenase (ΔqhpA) or Δfae3 showed no growth defect on C1-compounds. M. universalis FAM5 strains with a lesion in the H4-folate pathway were not able to use any C1-compound, methanol or methylamine. Genes essential for C1-assimilation (the serine cycle and glyoxylate shunt) and H4MTP-pathway for formaldehyde oxidation showed similar levels of expression on both C1-carbon sources. M. universalis FAM5 possesses three homologs of the formaldehyde activating enzyme, a key enzyme of the H4MTP-pathway. Strains lacking the canonical Fae (fae1) lost the ability to grow on both C1-compounds. However, upon incubation on methylamine the fae1-mutant produced revertants (Δfae1(R)), which regained the ability to grow on methylamine. Double and triple mutants (Δfae1(R)Δfae3, or Δfae1(R)Δfae2 or Δfae1(R)Δfae2Δfae3) constructed in the revertant strain background showed growth similar to the Δfae1(R) phenotype. The metabolic pathways for utilization of methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed based on these gene expression and phenotypic data.

Ethylmalonyl coenzyme A mutase operates as a metabolic control point in Methylobacterium extorquens AM1.

The metabolism of one- and two-carbon compounds by the methylotrophic bacterium Methylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-type M. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts for ecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressing ecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation.

Elucidation of the role of the methylene-tetrahydromethanopterin dehydrogenase MtdA in the tetrahydromethanopterin-dependent oxidation pathway in Methylobacterium extorquens AM1.

The methylotroph Methylobacterium extorquens AM1 oxidizes methanol and methylamine to formaldehyde and subsequently to formate, an intermediate that serves as the branch point between assimilation (formation of biomass) and dissimilation (oxidation to CO2). The oxidation of formaldehyde to formate is dephosphotetrahydromethanopterin (dH4MPT) dependent, while the assimilation of carbon into biomass is tetrahydrofolate (H4F) dependent. This bacterium contains two different enzymes, MtdA and MtdB, both of which are dehydrogenases able to use methylene-dH4MPT, an intermediate in the oxidation of formaldehyde to formate. Unique to MtdA is a second enzymatic activity with methylene-H4F. Since methylene-H4F is the entry point into the biomass pathways, MtdA plays a key role in assimilatory metabolism. However, its role in oxidative metabolism via the dH4MPT-dependent pathway and its apparent inability to replace MtdB in vivo on methanol growth are not understood. Here, we have shown that an mtdB mutant is able to grow on methylamine, providing a system to study the role of MtdA. We demonstrate that the absence of MtdB results in the accumulation of methenyl-dH4MPT. Methenyl-dH4MPT is shown to be a competitive inhibitor of the reduction of methenyl-H4F to methylene-H4F catalyzed by MtdA, with an estimated Ki of 10 µM. Thus, methenyl-dH4MPT accumulation inhibits H4F-dependent assimilation. Overexpression of mch in the mtdB mutant strain, predicted to reduce methenyl-dH4MPT accumulation, enhances growth on methylamine. Our model proposes that MtdA regulates carbon flux due to differences in its kinetic properties for methylene-dH4MPT and for methenyl-H4F during growth on single-carbon compounds.

Good codons, bad transcript: large reductions in gene expression and fitness arising from synonymous mutations in a key enzyme.

Biased codon usage in protein-coding genes is pervasive, whereby amino acids are largely encoded by a specific subset of possible codons. Within individual genes, codon bias is stronger at evolutionarily conserved residues, favoring codons recognized by abundant tRNAs. Although this observation suggests an overall pattern of selection for translation speed and/or accuracy, other work indicates that transcript structure or binding motifs drive codon usage. However, our understanding of codon bias evolution is constrained by limited experimental data on the fitness effects of altering codons in functional genes. To bridge this gap, we generated synonymous variants of a key enzyme-coding gene in Methylobacterium extorquens. We found that mutant gene expression, enzyme production, enzyme activity, and fitness were all significantly lower than wild-type. Surprisingly, encoding the gene using only rare codons decreased fitness by 40%, whereas an allele coded entirely by frequent codons decreased fitness by more than 90%. Increasing gene expression restored mutant fitness to varying degrees, demonstrating that the fitness disadvantage of synonymous mutants arose from a lack of beneficial protein rather than costs of protein production. Protein production was negatively correlated with the frequency of motifs with high affinity for the anti-Shine-Dalgarno sequence, suggesting ribosome pausing as the dominant cause of low mutant fitness. Together, our data support the idea that, although a particular set of codons are favored on average across a genome, in an individual gene selection can either act for or against codons depending on their local context.

The rhodanese domain of ThiI is both necessary and sufficient for synthesis of the thiazole moiety of thiamine in Salmonella enterica.

In Salmonella enterica, ThiI is a bifunctional enzyme required for the synthesis of both the 4-thiouridine modification in tRNA and the thiazole moiety of thiamine. In 4-thiouridine biosynthesis, ThiI adenylates the tRNA uridine and transfers sulfur from a persulfide formed on the protein. The role of ThiI in thiazole synthesis is not yet well understood. Mutational analysis described here found that ThiI residues required for 4-thiouridine synthesis were not involved in thiazole biosynthesis. The data further showed that the C-terminal rhodanese domain of ThiI was sufficient for thiazole synthesis in vivo. Together, these data support the conclusion that sulfur mobilization in thiazole synthesis is mechanistically distinct from that in 4-thiouridine synthesis and suggest that functional annotation of ThiI in genome sequences should be readdressed. Nutritional studies described here identified an additional cysteine-dependent mechanism for sulfur mobilization to thiazole that did not require ThiI, IscS, SufS, or glutathione. The latter mechanism may provide insights into the chemistry used for sulfur mobilization to thiazole in organisms that do not utilize ThiI.

Reaction of AdoMet with ThiC generates a backbone free radical.

ThiC is an [4Fe-4S] cluster protein that catalyzes the formation of 4-amino-5-hydroxymethyl-2-methylpyrimidine. EPR spectroscopic studies demonstrate that, upon interaction with AdoMet, active ThiC from Salmonella enterica generates a persistent free radical on the alpha-carbon of an amino acid residue. The EPR properties of the radical are consistent with any residue other than a Gly or Ala. Exposure to oxygen was accompanied by a fission of the radical-carrying polypeptide chain between the Gly436 and His437 residues in ThiC. Regardless of whether the backbone radical is part of the catalytic machinery, its presence provides evidence that ThiC employs free radical chemistry as expected for radical SAM enzymes.