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 XoxFMeDH 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 M 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. IMPORTANCEThe 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. Methylotrophs have gained worldwide interest as platforms for the production of value-added chemicals from singlecarbon compounds, turning atmospheric pollutants like methane and methanol into green chemicals, including biofuels and biodegradable plastics (1-5). A key step in this process is the oxidation of methanol to formaldehyde, which is carried out by different enzymes, including methanol dehydrogenase (MeDH) and alcohol oxidase, depending on the specific methylotroph (6, 7). Recently, it was discovered that some types of MeDHs require rareearth elements, specifically lantha...
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؉ IMPORTANCEExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxFtype 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. Methylotrophy is the capability of organisms to metabolize reduced carbon compounds lacking carbon-carbon bonds as the sole source of carbon and energy (1). The genus Methylobacterium is comprised of aerobic facultative methylotrophs that can metabolize single-carbon compounds, such as methanol and methylamine, as well as multicarbon substrates like ethanol, acetate, ethylamine, pyruvate, and succinate (2, 3). Members of the genus Methylobacterium are wide-spread plant epiphytes (4, 5) that utilize their metabolic flexibility to gain an advantage in the phyllosphere, an oligotrophic environment with transient substrate availability (6, 7).Methanol dehydrogenase (MDH) is an essential enzyme for the methylotrophic metabolism of methanol and methane (8). In Gram-negative methylotrophic bacteria, MDHs are soluble, periplasmic proteins with pyrroloquinoline quinone (PQQ) as the prosthetic group (9, 10). The best studied PQQ-containing MDHs are ␣ 2  2 tetramers consisting of the MxaF and MxaI proteins (11-14) that contain calcium (Ca 2ϩ ) in the active site (15, 16). Studies have provided evidence for the physiological role of a second type of PQQ-dependent MDH, XoxF, which has ϳ50% amino acid identity to MxaF from MxaFI-type MDHs (17). Metagenomic and environmental proteomics studies have demonstrated that xoxF is more widespread than mxaF in environmental samples (18)(19)(20)(21). Phylogenetic analysis of putative PQQ-containing MDHs has shown that XoxF-type MDHs are genetically diverse with at least five distinct clades, and it has been suggested that MxaFI-type MDHs represent a minor fraction of these MDHs (8,22). It has been further proposed that MxaFI-type MDHs may be the result of a second evolutionary event, with an ancestral XoxF-type MDH prototype (22). Together, these suppositions suggest that XoxFtype MDHs may be the primary MDHs for meth...
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.
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-dichlorophenolindophenol (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. A direct link between the Ln elements and microbial metabolism has been firmly established with the discovery of PQQ-dependent alcohol dehydrogenases (ADHs), from methylotrophic bacteria, that contain a Ln atom in the active site 1-5. Thus far, Ln-PQQ ADHs can be grouped by their phylogeny and primary substrate as either XoxF-MDHs or ExaF-type ethanol dehydrogenases (EtDHs). MxaFI MDH has been considered the canonical primary catalyst for methanol oxidation in Gram-negative methylotrophs 6,7. The heterotetramer MxaFI contains PQQ that coordinates the calcium (Ca) ion 8-10. The discovery that Ln is incorporated into the active site of XoxF MDH in place of Ca, allowing catalytic function, has prompted the reexamination of methanol oxidation in methylotrophic bacteria 3,5,11-18. To date, only a few XoxF MDHs have been kinetically characterized 1,3,4,19,20. Phylogenetic analyses show there are at least five distinct families of XoxF MDHs 11,21 , and while it has been suggested that all XoxF MDHs may exhibit similar kinetic properties, reported data for these enzymes are currently inadequate to support such a broad characterization. In fact, recent studies have begun to identify differences in kinetic properties, cofactor usage, and pH optima of phylogenetically distinct XoxF enzymes 20,22,23. Lack of genes encoding the
Some methanol-using bacteria may depend on lanthanide elements for carbon capture and energy generation
Lanthanide chemistry has only been extensively studied for the last 2 decades, when it was recognized that these elements have unusual chemical characteristics including fluorescent and potent magnetic properties because of their unique 4f electrons.1,2 Chemists are rapidly and efficiently integrating lanthanides into numerous compounds and materials for sophisticated applications. In fact, lanthanides are often referred to as "the seeds of technology" because they are essential for many technological devices including smartphones, computers, solar cells, batteries, wind turbines, lasers, and optical glasses.3-6 However, the effect of lanthanides on biological systems has been understudied. Although displacement of Ca by lanthanides in tissues and enzymes has long been observed,7 only a few recent studies suggest a biological role for lanthanides based on their stimulatory properties toward some plants and bacteria.8,9 Also, it was not until 2011 that the first biochemical evidence for lanthanides as inherent metals in bacterial enzymes was published.10 This forum provides an overview of the classical and current aspects of lanthanide coordination chemistry employed in the development of technology along with the biological role of lanthanides in alcohol oxidation. The construction of lanthanide-organic frameworks will be described. Examples of how the luminescence field is rapidly evolving as more information about lanthanide-metal emissions is obtained will be highlighted, including biological imaging and telecommunications.11 Recent breakthroughs and observations from different exciting areas linked to the coordination chemistry of lanthanides that will be mentioned in this forum include the synthesis of (i) macrocyclic ligands, (ii) antenna molecules, (iii) coordination polymers, particularly nanoparticles, (iv) hybrid materials, and (v) lanthanide fuel cells. Further, the role of lanthanides in bacterial metabolism will be discussed, highlighting the discovery that lanthanides are cofactors in biology, particularly in the enzymatic oxidation of alcohols. Finally, new and developing chemical and biological lanthanide mining and recycling extraction processes will be introduced.
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.
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.Thiamin pyrophosphate (TPP) is an essential cofactor formed from 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) and thiazole monophosphate (THZ-P) moieties. In bacteria, HMP-PP and THZ-P are independently synthesized and combined by thiamin phosphate (TMP) synthase (ThiE, E.C. 2.5.1.3) yielding TMP prior its conversion to TPP by the TMP kinase (ThiL, E.C. 2.7.4.16) (1) Figure 1A. Synthesis of the THZ moiety has been reconstituted in vitro using components from Gram-positive (B. subtilis) and Gram-negative (E. coli, S. enterica) bacteria (2-7). In contrast, our understanding of the biochemical steps leading to the synthesis of HMP is limited. Genetic and biochemical studies have shown that HMP is derived from 5-aminoimidazole ribotide (AIR) (8)(9)(10)(11)(12). In vivo labeling studies showed that all carbon and nitrogen atoms present in HMP are derived from AIR, as illustrated in Figure 1B (8,9,13,14). These labeling data suggested that the conversion from AIR to HMP involves the breakage and reforming of multiple bonds. Despite this complex intramolecular rearrangement, only one gene product (ThiC) has been shown to be essential for this conversion in vivo.Formation of HMP was reported in cell-free extracts of an E. coli strain that overproduced ThiC (14). In the cited study, the ThiC-dependent conversion of AIR to the pyrimidine was measured by in situ conversion of the product to TMP, which was quantified by a thiochrome assay (5). The ThiC-dependent activity detected in the cell-free extract was low, and increased by the addition of dialyzed cell-free extract of a thiC mutant strain. These studies indicated that AIR, AdoMet, one of the following: NAD + , NADH, or NADPH, and an additional cellular component were required for optimal ThiC activity. Purification of ThiC resulted in loss of all activity, and no evidence for an [Fe-S] Strain DM7474 (E.coli; SG-13009 pREP4 pMD34) was used to produce ThiC-His 6 . When purified under anoxic conditions 12 mg of ThiC protein sample was obtained from 30g of cells. The ThiC protein sample (6 mg/ml) had a brown color with a UV-visible spectrum characteristic of Fe-S cluster proteins, including a shoulder at 410 nm (Figure 2A green). The signal at 410 nm decreased when the sample was exposed to oxyge...
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