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Genetic regulation by lanthanides in the Type I methanotroph Methylotuvimicrobium buryatense 5GB1C
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The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO2 and biomass, a value-added product and a cobenefit of methane removal. Typically, methanotrophs grow best at around 5,000 to 10,000 ppm methane, but methane in the atmosphere is 1.9 ppm. Air above emission sites such as landfills, anaerobic digestor effluents, rice paddy effluents, and oil and gas wells contains elevated methane in the 500 ppm range. If such sites are targeted for methane removal, technology harnessing aerobic methanotroph metabolism has the potential to become economically and environmentally viable. The first step in developing such methane removal technology is to identify methanotrophs with enhanced ability to grow and consume methane at 500 ppm and lower. We report here that some existing methanotrophic strains grow well at 500 ppm methane, and one of them, Methylotuvimicrobium buryatense 5GB1C, consumes such low methane at enhanced rates compared to previously published values. Analyses of bioreactor-based performance and RNAseq-based transcriptomics suggest that this ability to utilize low methane is based at least in part on extremely low non-growth-associated maintenance energy and on high methane specific affinity. This bacterium is a candidate to develop technology for methane removal at emission sites. If appropriately scaled, such technology has the potential to slow global warming by 2050.
Atmospheric methane removal (e.g. in situ methane oxidation to carbon dioxide) may be needed to offset continued methane release and limit the global warming contribution of this potent greenhouse gas. Because mitigating most anthropogenic emissions of methane is uncertain this century, and sudden methane releases from the Arctic or elsewhere cannot be excluded, technologies for methane removal or oxidation may be required. Carbon dioxide removal has an increasingly well-established research agenda and technological foundation. No similar framework exists for methane removal. We believe that a research agenda for negative methane emissions—‘removal' or atmospheric methane oxidation—is needed. We outline some considerations for such an agenda here, including a proposed Methane Removal Model Intercomparison Project (MR-MIP).
This article is part of a discussion meeting issue 'Rising methane: is warming feeding warming? (part 1)'.
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.
Lanthanides were previously thought to be biologically inert owing to their low solubility; however, they have recently been shown to strongly impact the metabolism of methylotrophic bacteria. Leading efforts in this emergent field have demonstrated far-reaching impacts of lanthanide metabolism in biology; from the identification of novel roles of enzymes and pathways dependent on lanthanide-chemistry to the control of transcriptional regulatory networks to the modification of microbial community interactions. Even further, the recent discovery of lanthanide-dependent enzymes associated with multi-carbon metabolism in both methylotrophs and non-methylotrophs alike suggests that lanthanide biochemistry may be more widespread than initially thought. Current efforts aim to understand how lanthanide chemistry and lanthanide-dependent enzymes affect numerous ecosystems and metabolic functions. These efforts will likely have a profound impact on biotechnological processes involving methylotrophic communities and the biologically mediated recovery of these critical metals from a variety of waste streams while redefining our understanding of a fundamental set of metals in biology.
Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.
Lanthanides (Ln3+), known as rare earth elements, have recently emerged as enzyme cofactors, contrary to prior assumption of their biological inertia. Several bacterial alcohol dehydrogenases have been characterized so far that depend on Ln3+ for activity and expression, belonging to the methanol dehydrogenase clade XoxF and the ethanol dehydrogenase clade ExaF/PedH. Here we compile an inventory of genes potentially encoding Ln3+-dependent enzymes, closely related to the previously characterized XoxF and ExaF/PedH enzymes. We demonstrate their wide distribution among some of the most numerically abundant and environmentally important taxa, such as the phylogenetically disparate rhizobial species and metabolically versatile bacteria inhabiting world’s oceans, suggesting that reliance on Ln3+-mediated biochemistry is much more widespread in the microbial world than previously assumed. Through protein expression and analysis, we here more than double the extant collection of the biochemically characterized Ln3+-dependent enzymes, demonstrating a range of catalytic properties and substrate and cofactor specificities. Many of these enzymes reveal propensity for oxidation of methanol. This observation, in combination with genome-based reconstruction of methylotrophy pathways for select species suggests a much wider occurrence of this metabolic capability among bacterial species, and thus further suggests the importance of methylated compounds as parts of the global carbon cycling.
Until recently, rare‐earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca²⁺‐dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La³⁺) is sensed both in a XoxF‐dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsis thaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 μg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La³⁺‐regulated proteins under defined artificial media conditions. Among these was a REE‐binding protein that is encoded next to a gene for a TonB‐dependent transporter. The latter was essential for REE‐dependent growth on methanol indicating chelator‐assisted uptake of REEs.
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Background: Rare Earth Elements (REEs) control methanol utilization in both methane- and methanol-utilizing microbes. It has been established that the addition of REEs leads to the transcriptional repression of MxaFI-MeDH [a two-subunit methanol dehydrogenase (MeDH), calcium-dependent] and the activation of XoxF-MeDH (a one-subunit MeDH, lanthanum-dependent). Both enzymes are pyrroquinoline quinone-dependent alcohol dehydrogenases and show significant homology; however, they display different kinetic properties and substrate specificities. This study investigates the impact of the MxaFI to XoxF switch on the behavior of metabolic networks at a global scale.
Results: In this study we investigated the steady-state growth of Methylomicrobium alcaliphilum 20ZR in media containing calcium (Ca) or lanthanum (La, a REE element). We found that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca. Three complementary global-omics approaches–RNA-seq transcriptomics, proteomics, and metabolomics–were applied to investigate the mechanisms of improved growth vs. carbon conversion. Cells grown with La showed the transcriptional activation of the xoxF gene, a homolog of the formaldehyde-activating enzyme (fae2), a putative transporter, genes for hemin-transport proteins, and nitrate reductase. In contrast, genes for mxaFI and associated cytochrome (mxaG) expression were downregulated. Proteomic profiling suggested additional adjustments of the metabolic network at the protein level, including carbon assimilation pathways, electron transport systems, and the tricarboxylic acid (TCA) cycle. Discord between gene expression and protein abundance changes points toward the possibility of post-transcriptional control of the related systems including key enzymes of the TCA cycle and a set of electron-transport carriers. Metabolomic data followed proteomics and showed the reduction of the ribulose-monophosphate (RuMP) pathway intermediates and the increase of the TCA cycle metabolites.
Conclusion: Cells exposed to REEs display higher rates of growth but have lower carbon conversion efficiency compared to cells supplemented with Ca. The most plausible explanation for these physiological changes is an increased conversion of methanol into formate by XoxF-MeDH, which further stimulates methane oxidation but limits both the supply of reducing power and flux of formaldehyde into the RuMP pathway.
A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in xoxG, a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using Methylomonas sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome c, based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level.
Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca²⁺-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La³⁺. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La3³⁺; this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides.
In aerobic methanotrophs, copper and cerium control the expression and activity of different forms of methane monooxygenase and methanol dehydrogenase, respectively. To exploit methanotrophy for the valorization of methane, it is crucial to determine if these metals exert more global control on gene expression in methanotrophs. Using RNA-Seq analysis we compared the transcriptome of Methylosinus trichosporium OB3b grown in the presence of varying amounts of copper and cerium. When copper was added in the absence of cerium, expression of genes encoding for both soluble and particulate methane monooxygenases varied as expected. Genes encoding for copper uptake, storage, and efflux also increased, indicating that methanotrophs must carefully control copper homeostasis. When cerium was added in the absence of copper, expression of genes encoding for alternative methanol dehydrogenases varied as expected, but few other genes were found to have differential expression. When cerium concentrations were varied in the presence of copper, few genes were found to be either up- or downregulated, indicating that copper over rules any regulation by cerium. When copper was increased in the presence of cerium, however, many genes were upregulated, most notably multiple steps of the central methane oxidation pathway, the serine cycle, and the ethylmalonyl-CoA pathway. Many genes were also downregulated, including those encoding for nitrogenase and hydrogenase. Collectively, these data suggest that copper plays a larger role in regulating gene expression in methanotrophs, but that significant changes occur when both copper and cerium are present.
The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca²⁺-dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La³⁺, Ce³⁺, Pr³⁺, Sm³⁺, or Nd³⁺. Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.
Many methylotrophs, microorganisms that consume carbon compounds lacking carbon?carbon bonds, use two different systems to oxidize methanol for energy production and biomass accumulation. The MxaFI methanol dehydrogenase (MDH) contains calcium in its active site, while the XoxF enzyme contains a lanthanide in its active site. The genes encoding the MDH enzymes are differentially regulated by the presence of lanthanides. In this study, we found that the histidine kinase MxaY controls the lanthanide-mediated switch in Methylomicrobium buryatense 5GB1C. MxaY controls the transcription of genes encoding MxaFI and XoxF at least partially by controlling the transcript levels of the orphan response regulator MxaB. We identify a constitutively active version of MxaY, and identify the mutated residue that may be involved in lanthanide sensing. Lastly, we find evidence to suggest that tight control of active MDH production is required for wild-type growth rates.
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 multi-carbon 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.
Importance:
Aerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph.
Methane is becoming a major candidate for a prominent carbon feedstock in the future and bioconversion of methane into valuable
products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate
our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one of
such enabling tools, but to date it has not been successful in a group of methanotrophs of interest for the production of
chemicals and fuels, the gamma-proteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques
with high transformation efficiency for three different species of type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with high carbon conversion efficiency and is well
suited for industrial use for the bioconversion of methane. Based on the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation
of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin
were validated in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development
in these strains.
Methanotrophs have multiple methane monooxygenases that are well-known to be regulated by copper, i.e., a "copper-switch". At low copper:biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDH), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. Here we show expression of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased respectively when grown in the presence of cerium but the absence of copper as compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower as compared to just the presence of copper, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression to significantly decrease and increase respectively, but the simultaneous addition of cerium had no discernable effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b, but not from pMMO.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Methane, as natural gas or biogas, is the least expensive source of carbon for (bio)chemical synthesis. Scalable biological upgrading of this simple alkane to chemicals and fuels can bring new sustainable solutions to a number of industries with large environmental footprints, such as natural gas/petroleum production, landfills, wastewater treatment, and livestock. Microbial biocatalysis with methane as a feedstock has been pursued off and on for almost a half century, with little enduring success. Today, biological engineering and systems biology provide new opportunities for metabolic system modulation and give new optimism to the concept of a methane-based bio-industry. Here we present an overview of the most recent advances pertaining to metabolic engineering of microbial methane utilization. Some ideas concerning metabolic improvements for production of acetyl-CoA and pyruvate, two main precursors for bioconversion, are presented. We also discuss main gaps in the current knowledge of aerobic methane utilization, which must be solved in order to release the full potential of methane-based biosystems.
Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Aerobic methanotrophs oxidize methane at ambient temperatures and pressures, and are therefore attractive systems for methane-based bioconversions. In this work we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a three hour doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small IncP-based broad host range vectors, and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Robust growth of the gammaproteobacterium Methylomicrobium buryatense strain 5G on methane makes it an attractive system for CH4-based biocatalysis. Here we present a draft genome sequence of the strain that will provide a valuable framework for metabolic
engineering of the core pathways for the production of valuable chemicals from methane.
In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde
in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins,
which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other.
It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate
that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM,
which are required for the expression of the methanol dehydrogenase genes.
Antibodies to methanol dehydrogenase purified from Methylobacterium sp. strain AM1 and Methylomonas sp. strain A4 were raised. The antibody preparations were used in indirect immunogold labeling studies. With this approach, methanol dehydrogenase was found to be preferentially localized to the periplasmic region of the methylotroph Methylobacterium sp. strain AM1 and to the intracytoplasmic membrane of the methanotroph Methylomonas sp. strain A4. Antibody cross-reactivity to other methylotrophic bacteria was detected.
Five strains of obligate methanotrophic bacteria (4G, 5G, 6G, 7G and 5B) isolated from bottom sediments of Southeastern Transbaikal soda lakes (pH 9.5-10.5) are taxonomically described. These bacteria are aerobic, Gram-negative monotrichous rods having tightly packed cup-shaped structures on the outer cell wall surface (S-layers) and Type I intracytoplasmic membranes. All the isolates possess particulate methane monooxygenase (pMMO) and one strain (5G) also contains soluble methane monooxygenase (sMMO). They assimilate methane and methanol via the ribulose monophosphate pathway (RuMP). The isolates are alkalitolerant or facultatively alkaliphilic, able to grow at pH 10.5-11.0 and optimally at pH 8.5-9.5. These organisms are obligately dependent on the presence of sodium ions in the growth medium and tolerate up to 0.9-1.4 M NaCl or 1 M NaHCO3. Although being mesophilic, all the isolates are resistant to heating (80 degrees C, 20 min), freezing and drying. Their cellular fatty acids profiles primarily consist of C(16:1). The major phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The main quinone is Q-8. The DNA G+C content ranges from 49.2-51.5 mol %. Comparative 16S rDNA sequencing showed that the newly isolated methanotrophs are related to membres of the Methylomicrobium genus. However, they differ from the known members of this genus by DNA-DNA relatedness. Based on pheno- and genotypic characteristics, we propose a new species of the genus Methylomicrobium Methylomicrobium buryatense sp. nov.
Lanthanides (Lns) have been shown recently to be essential cofactors in certain enzymes in methylotrophic bacteria. Here we identify in the model methylotroph, Methylobacterium extorquens, a highly selective Ln(III)-binding protein, which we name lanmodulin (LanM). LanM possesses four metal-binding EF-hand motifs, commonly associated with Ca(II) binding proteins. In contrast to other EF hand-containing proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concentrations of all Ln(III) (Ln = La-Lu, Y), whereas it only responds to Ca(II) at near-millimolar concentrations. Mutagenesis of conserved proline residues present in LanM’s EF hands, not encountered in Ca(II)-binding EF hands, to alanine pushes Ca(II) responsiveness into the micromolar concentration range while retaining picomolar Ln(III) affinity, suggesting that these unique proline residues play a key role in ensuring metal selectivity in vivo. Identification and characterization of LanM provides insights into how biology selectively recognizes low-abundance Ln(III) over higher-abundance Ca(II), pointing toward biotechnologies for detecting, sequestering, and separating these technologically important elements.
Significance
A great challenge in microbial ecology lies in determining the underlying mechanisms that drive interactions in complex natural communities. In this study we used bacterial isolates from Lake Washington sediment that are important for the utilization of the greenhouse gas methane. We show how simple cocultures can be used to identify mechanisms involved in cross-feeding in microbial communities; these mechanisms cannot be deduced from pure cultures alone. We demonstrate that the presence of one species alters gene expression and metabolism in another species such that the second species excretes a carbon and energy source to sustain the cross-fed species. This mode of cross-feeding based on partner-induced altered gene expression may have important implications for microbial interactions in the environment.
Recent advances in lanthanide chemistry include the synthesis of stable lanthanide complexes with tunable luminescent properties. Further, the recent characterization of a few lanthanide-dependent enzymes highlights lanthanides as inherent cofactors for several enzymes in bacterial metabolism and demands further characterization of metabolic pathways affected by these potent Lewis acids. As the demand for lanthanides rises, the development of new strategies for efficient, nonhazardous extraction and purification offers innovative advances in biometallurgy and inorganic chemistry.
The molecular structure and nucleotide sequence of Ce(3+)-induced methanol dehydrogenase (MDH) of Bradyrhizobium sp. MAFF211645 were investigated. The addition of 30 μM Ce(3+) to 1/10 nutrient broth containing 0.5% methanol remarkably increased MDH activity. Furthermore, La(3+) increased MDH activity, but other heavier rare earth and metal elements did not have the same effect. MDH increased by Ce(3+) was purified by sequential column chromatography, and the purified MDH migrated as a single band with an apparent molecular weight of 68 kDa on SDS-PAGE. The apparent molecular weight of native MDH was estimated to be 108,000 by gel chromatography. The MDH was comprised of two identical subunits. N-terminal 23-amino acid sequence, 1-NDELHKMAQNPKDWVMPAGDYAN-23, of the purified MDH exhibited 91.3% identity to that of the MDH large subunit-like protein encoded by mxaF' of Bradyrhizobium japonicum USDA110. Nucleotide sequencing of the MDH gene of strain MAFF211645 yielded a deduced amino acid sequence comprising 601 amino acid residues, an N-terminal signal peptide, and a mature MDH comprising 578 amino acid residues with a predicted molecular mass of 62,918 Da. Further analysis of the deduced amino acid sequence of mature MDH revealed that the functional amino acids in its active site, such as two adjacent Cys residues, and bacterial quinoprotein signatures 1 and 2 were conserved. These results indicate that Ce(3+)-induced MDH encoded by mxaF' may be involved in methanol metabolism in Bradyrhizobium sp. MAFF211645.
La(3+) and not Ca(2+) increases methanol dehydrogenase (MDH) activity in Methylobacterium radiotolerans NBRC15690. La(3+)- and Ca(2+)-MDH-like proteins were found to be homodimeric (α(2)) and heterotetrameric (α(2)β(2)), respectively. N-terminal amino acid sequences of these proteins revealed that La(3+)- and Ca(2+)-MDH-like proteins were encoded by xoxF and mxaFI, respectively.
Methanotrophs, cells that consume methane (CH(4)) as their sole source of carbon and energy, play key roles in the global carbon cycle, including controlling anthropogenic and natural emissions of CH(4), the second-most important greenhouse gas after carbon dioxide. These cells have also been widely used for bioremediation of chlorinated solvents, and help sustain diverse microbial communities as well as higher organisms through the conversion of CH(4) to complex organic compounds (e.g. in deep ocean and subterranean environments with substantial CH(4) fluxes). It has been well-known for over 30 years that copper (Cu) plays a key role in the physiology and activity of methanotrophs, but it is only recently that we have begun to understand how these cells collect Cu, the role Cu plays in CH(4) oxidation by the particulate CH(4) monooxygenase, the effect of Cu on the proteome, and how Cu affects the ability of methanotrophs to oxidize different substrates. Here we summarize the current state of knowledge of the phylogeny, environmental distribution, and potential applications of methanotrophs for regional and global issues, as well as the role of Cu in regulating gene expression and proteome in these cells, its effects on enzymatic and whole-cell activity, and the novel Cu uptake system used by methanotrophs.
Five genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains. These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood. In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xylE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF. The nucleotide sequence suggests that mxbD encodes a histidine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator. A xylE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate. Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression. The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes. xylE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation. These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation.
Investigation of lanthanide-dependent methylotrophy uncovers complementary roles for alcohol dehydrogenase enzymes
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