Search

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.

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 transfers. We showed that Methylobacterium contains four evolutionary 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 of 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), which early branching in Methylobacterium history was heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandon of Methylorubrum.

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 fromMethylorubrum extorquensAM1 andMethylacidiphilum fumariolicumSolV with WB, along with handling recommendations, is presented.Graphic abstractLanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

While lanthanide-dependent metabolism is widespread in nature and has been proven to drive one-carbon metabolism in bacteria, details about the machinery necessary to sense, sequester, and traffic lanthanides (Ln) remain unknown. This gap in knowledge is in part because nearly all bacterial growth studies with Ln to date have used soluble chloride salts, compounds that do not reflect the insoluble Ln sources common in the natural environment. Here, we describe the changes in the metabolic machinery of Methylorubrum extorquens AM1 in response to poorly soluble Nd2O3, including 4-fold increases in orphan pqqA genes and the Ln-dependent ADHs xoxF1 and exaF compared to growth with soluble NdCl3. We report the first description of a Ln-chelator biosynthetic gene cluster, encoded by META1p4129 through META1p4138 that we named the Lanthanide Chelation Cluster (LCC). The LCC encodes a TonB dependent receptor and NRPS biosynthetic enzymes and is predicted to produce a metal-chelating molecule. As some LCC protein sequences share similarity to biosynthetic enzymes producing the Fe-chelating siderophore aerobactin, the capacity of aerobactin for binding Ln was tested. It was found that while aerobactin can bind lanthanum (La), neodymium (Nd) and lutetium (Lu) at physiological pH, providing only exogenous aerobactin did not affect growth rate or yield. The LCC was highly upregulated when M. extorquens AM1 was grown using Nd2O3 and expression in trans enabled an increase of Nd bioaccumulation by over 50%. Expression of the LCC in trans did not affect iron bioaccumulation, providing further evidence that its product is a novel Ln-chelator. Finally, expression of the LCC in trans increased Nd, dysprosium (Dy), and praseodymium (Pr) bioaccumulation from the complex Ln source NdFeB magnet swarf by over 60%, opening new strategies for sustainable recovery of these critical Rare Earth Elements.

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

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 on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. 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.

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.

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.

The lanthanide elements (Ln(3+)), those with atomic numbers 57–63 (excluding promethium, Pm(3+)), 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 Ca(2+)-PQQ cofactor. An important missing piece of the Ln(3+) puzzle is defining what features distinguish enzymes that use Ln(3+)-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 La(3+) bound in the active-site region and coordinated by Asp(320). Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp(320) is needed for in vivo catalytic function, in vitro activity, and La(3+) coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La(3+), coordinated Ca(2+) 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 La(3+). These results provide evidence that a Ln(3+)-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 Ln(3+) metabolism.

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 (La 3+ ). ExaF was purified from cells grown with both calcium (Ca 2+ ) and La 3+ and with Ca 2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La 3+ over Ca 2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca 2+ -plus-La 3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca 2+ -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 La 3+ 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 (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.

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.

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 La III and Eu III 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 Pr III , or to monitor the depletion of La III 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.

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-dH 4 MPT), 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-H 4 MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene ( mch ) encoding the enzyme that consumes methenyl-dH 4 MPT did not accumulate methenyl-dH 4 MPT 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 dH 4 MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH 4 MPT 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-dH 4 MPT 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-dH 4 MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H 4 MPT 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-dH 4 MPT, 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.

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.

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 (Δfae1R), which regained the ability to grow on methylamine. Double and triple mutants (Δfae1RΔfae3, or Δfae1RΔfae2 or Δfae1RΔfae2Δfae3) constructed in the revertant strain background showed growth similar to the Δfae1R phenotype. The metabolic pathways for utilization of methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed based on these gene expression and phenotypic data.

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.

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 CO 2 ). The oxidation of formaldehyde to formate is dephosphotetrahydromethanopterin (dH 4 MPT) dependent, while the assimilation of carbon into biomass is tetrahydrofolate (H 4 F) dependent. This bacterium contains two different enzymes, MtdA and MtdB, both of which are dehydrogenases able to use methylene-dH 4 MPT, an intermediate in the oxidation of formaldehyde to formate. Unique to MtdA is a second enzymatic activity with methylene-H 4 F. Since methylene-H 4 F is the entry point into the biomass pathways, MtdA plays a key role in assimilatory metabolism. However, its role in oxidative metabolism via the dH 4 MPT-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-dH 4 MPT. Methenyl-dH 4 MPT is shown to be a competitive inhibitor of the reduction of methenyl-H 4 F to methylene-H 4 F catalyzed by MtdA, with an estimated K i of 10 μM. Thus, methenyl-dH 4 MPT accumulation inhibits H 4 F-dependent assimilation. Overexpression of mch in the mtdB mutant strain, predicted to reduce methenyl-dH 4 MPT accumulation, enhances growth on methylamine. Our model proposes that MtdA regulates carbon flux due to differences in its kinetic properties for methylene-dH 4 MPT and for methenyl-H 4 F during growth on single-carbon compounds.

Thiamin pyrophosphate is a required cofactor in all organisms. The biosynthesis of thiamin requires the independently synthesized 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) and 5-hydroxyethyl-4-methyl thiazole phosphate (THZ-P) moieties. In bacteria, the pyrimidine moiety is derived from 5-aminoimidazole ribotide (AIR) and ThiC is the only gene product known to be required for this conversion in vivo. We report here the purification and characterization of the ThiC protein from Salmonella enterica. The data showed this protein generated HMP when AIR, S-adenosyl methionine (AdoMet), and an appropriate reducing agent were present. It is further shown that ThiC carries an oxygen labile [Fe-S] cluster essential for this activity.