Search

This study explores methane utilization by the methanotrophic microorganism Methylococcus capsulatus (Bath) for biomass production, presenting a promising approach to mitigate methane emissions and foster the development sustainable biomaterials. Traditional screening methods for gas cultivations involve either serum flasks without online monitoring or costly, low-throughput fermenters. To address these limitations, the Respiration Activity MOnitoring System was augmented with methane sensors for real-time methane transfer rate (MTR) monitoring in shake flasks. Utilizing online monitoring of the MTR in shake flasks results in enhanced throughput and cost-effectiveness for cultivating M. capsulatus. Simultaneous monitoring of transfer rates for oxygen, methane, and carbon dioxide was conducted in up to eight shake flasks, ensuring the success of the cultivation process. Alterations in methane-to-oxygen transfer rate ratios and carbon fixation rates reveal the impact of transfer limitations on microbial growth. Detection of gas transfer limitations, exploration of process parameter influences, and investigations of medium components were enabled by the introduced method. Optimal nitrogen concentrations could be determined to ensure optimal growth. This streamlined approach accelerates the screening process, offering efficient investigations into metabolic effects, limitations, and parameter influences in gas fermentations without the need for elaborate offline sampling, significantly reducing costs and enhanced reproducibility., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Biotechnology continues to drive innovation in the production of pharmaceuticals, biofuels, and other valuable compounds, leveraging the power of microbial systems for enhanced yield and sustainability. Genome-scale metabolic (GSM) modeling has become an essential approach in this field, which enables a guide for targeting genetic modifications and the optimization of metabolic pathways for various industrial applications. While single-species GSM models have traditionally been employed to optimize strains like Escherichia coli and Lactococcus lactis , the integration of these models into community-based approaches is gaining momentum. Herein, we present a pipeline for community metabolic modeling with a user-friendly GUI, applying it to analyze interactions between Methylococcus capsulatus , a biotechnologically important methanotroph, and Escherichia coli W3110 under oxygen- and nitrogen-limited conditions. We constructed models with unmodified and homoserine-producing E. coli strains using the pipeline implemented in the original BioUML platform. The E. coli strain primarily utilized acetate from M. capsulatus under oxygen limitation. However, homoserine produced by E. coli significantly reduced acetate secretion and the community growth rate. This homoserine was taken up by M. capsulatus , converted to threonine, and further exchanged as amino acids. In nitrogen-limited modeling conditions, nitrate and ammonium exchanges supported the nitrogen needs, while carbon metabolism shifted to fumarate and malate, enhancing E. coli TCA cycle activity in both cases, with and without modifications. The presence of homoserine altered cross-feeding dynamics, boosting amino acid exchanges and increasing pyruvate availability for M. capsulatus . These findings suggest that homoserine production by E. coli optimizes resource use and has potential for enhancing microbial consortia productivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH 4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus , can also use CO 2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH 4 and CO 2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO 2 -dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO 2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO 2 evolution from CH 4 -consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH 4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH 4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH 4 into CO 2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus , can also use CO 2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO 2 . We developed an engineered strain with improved CO 2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH 4 conversion bioprocesses., Competing Interests: Some aspects of this work are included as part of a U.S. patent application (PCT/US2024/034678).

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C 1 assimilation pathways in the gammaproteobacterial methanotrophs., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The effects of soluble and insoluble lanthanides on gene expression in Methylococcus capsulatus Bath were investigated. Genes for lanthanide-containing methanol dehydrogenases (XoxF-MDHs) and their calcium-containing counterparts (MxaFI-MDHs) were up- and down-regulated, respectively, by supplementation with soluble lanthanide chlorides, indicating that M. capsulatus has the "lanthanide switch" observed in other methanotrophs. Insoluble lanthanide oxides also induced the lanthanide switch and were dissolved by the spent medium of M. capsulatus, suggesting the presence of lanthanide-chelating compounds. A transcriptome ana-lysis indicated that a gene cluster for the synthesis of an enterobactin-like metal chelator contributed to the dissolution of insoluble lanthanides.

Particulate methane monooxygenase (pMMO) is a multi-subunit membrane metalloenzyme used by methanotrophic bacteria to convert methane to methanol. A major hurdle to studying pMMO is the lack of a recombinant expression system, precluding investigation of individual residues by mutagenesis and hampering a complete understanding of its mechanism. Here, we developed an Escherichia coli lysate-based cell-free protein synthesis (CFPS) system that can be used to express pMMO in vitro in the presence of nanodiscs. We used a SUMO fusion construct to generate the native PmoB subunit and showed that the SUMO protease (Ulp1) cleaves the protein in the reaction mixture. Using an affinity tag to isolate the complete pMMO complex, we demonstrated that the complex forms without the need for exogenous translocon machinery or chaperones, confirmed by negative stain electron microscopy. This work demonstrates the potential for using CFPS to express multi-subunit membrane-bound metalloenzymes directly into lipid bilayers.

Methanotrophs are bacteria capable on growing on methane as their sole carbon source. They may provide a promising route for upgrading natural gas into more valuable fuels and chemicals. However, natural gas may contain significant quantities of hydrogen sulfide. Little is known about how hydrogen sulfide affects the growth and physiology of methanotrophs aside from a few studies showing that it is inhibitory. This study investigated how hydrogen sulfide affects the growth and physiology of the model methanotroph, Methylococcus capsulatus Bath. Growth studies demonstrated that hydrogen sulfide inhibits the growth of M. capsulatus Bath when the concentration exceeds 0.5% (v/v). To better understand how hydrogen sulfide is inhibiting the growth of M. capsulatus Bath, transcription and metabolite concentrations were profiled using RNA sequencing and gas chromatography-mass spectrometry, respectively. Our analysis of the differentially expressed genes and changes in metabolite concentrations suggests that hydrogen sulfide inhibits cellular respiration. The cells respond to sulfide stress in part by increasing the rate of sulfide oxidation and by increasing the expression of sulfide quinone reductase and a putative persulfide dioxygenase. In addition, they reduce the expression of the native calcium-dependent methanol dehydrogenase and increase the expression of XoxF, a lanthanide-dependent methanol dehydrogenase. While the reason of this switch in unknown, XoxF has previously been shown to be induced by lanthanides or nitric oxide in methanotrophs. Collectively, these results further our understanding of how methanotrophs respond to sulfide stress and may aid in the engineering of strains resistant to hydrogen sulfide. KEY POINTS: • Hydrogen sulfide inhibits growth of Methylococcus capsulatus Bath • Sulfide stress inhibits cellular respiration • Sulfide stress induces XoxF, a lanthanide-dependent methanol dehydrogenase., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This work investigated the structural and electronic properties of the copper mononuclear site of the PmoB part of the pMMO enzyme at the molecular level. We propose that the Cu B catalytic site in the soluble portion of pMMO at room temperature and under physiological conditions is a mononuclear copper complex in a distorted octahedral arrangement with the residues His 33 , His 137 , and His 139 on the equatorial base and two water molecules on the axial axis. Our view was based on the molecular dynamics results and DFT calculations of the electronic paramagnetic resonance parameters and comparisons with experimental EPR data. This new proposed model for the Cu B site brings additional support concerning the recent experimental evidence, which pointed out that a saturated coordination sphere of the copper ion in the Cu B center is an essential factor that makes it less efficient than the Cu C site in the methane oxidation. Therefore, according to the Cu B site model proposed here, an additional step involving a displacement of at least one water molecule of the copper coordination sphere by the O 2 molecule prior to its activation must be necessary. This scenario is less likely to occur in the Cu C center once this one is buried in the alpha-helices, which are part of the pMMO structure bound to the membrane wall, and consequently located in a less solvent-exposed region. In addition, we also present a simple and efficient sequential S-MD/CPKS protocol to compute EPR parameters that can, in principle, be expanded for the study of other copper-containing proteins.

This study demonstrates the metabolic alteration of Methylococcus capsulatus (Bath), a representative bacterium among methanotrophs, in microbial gas-phase reactions. For comparative metabolome analysis, a bioreactor was designed to be capable of supplying gaseous substrates and liquid nutrients continuously. Methane degradation by M. capsulatus (Bath) was more efficient in a gas-phase reaction operated in the bioreactor than in an aqueous phase reaction operated in a batch reactor. Metabolome analysis revealed remarkable alterations in the metabolism of cells in the gas-phase reaction; in particular, pyruvate, 2-ketoglutarate, some amino acids, xanthine, and hypoxanthine were accumulated, whereas 2,6-diaminopimelate was decreased. Based on the results of metabolome analysis, cells in the gas-phase reaction seemed to alter their metabolism to reduce the excess ATP and NADH generated upon increased availability of methane and oxygen. Our findings will facilitate the development of efficient processes for methane-based bioproduction with low energy consumption., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Despite the wide-ranging proscription of hexavalent chromium, chromium(VI) remains among the major polluting heavy metals worldwide. Aerobic methane-oxidizing bacteria are widespread environmental microorganisms that can perform diverse reactions using methane as the feedstock. The methanotroph Methylococcus capsulatus Bath, like many other microorganisms, detoxifies chromium(VI) by reduction to chromium(III). Here, the interaction of chromium species with M. capsulatus Bath was examined in detail by using a range of techniques. Cell fractionation and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) indicated that externally provided chromium(VI) underwent reduction and was then taken up into the cytoplasmic and membranous fractions of the cells. This was confirmed by X-ray photoelectron spectroscopy (XPS) of intact cultures that indicated negligible chromium on the surfaces of or outside the cells. Distribution of chromium and other elements within intact and sectioned cells, as observed via transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS), was consistent with the cytoplasm/membrane location of the chromium(III), possibly as chromium phosphate. The cells could also take up chromium(III) directly from the medium in a metabolism-dependent fashion and accumulate it. These results indicate a novel pattern of interaction with chromium species distinct from that observed previously with other microorganisms. They also suggest that M. capsulatus and similar methanotrophs may contribute directly to chromium(VI) reduction and accumulation in mixed communities of microorganisms that are able to perform methane-driven remediation of chromium(VI). IMPORTANCE M. capsulatus Bath is a well-characterized aerobic methane-oxidizing bacterium that has become a model system for biotechnological development of methanotrophs to perform useful reactions for environmental cleanup and for making valuable chemicals and biological products using methane gas. Interest in such technology has increased recently owing to increasing availability of low-cost methane from fossil and biological sources. Here, it is demonstrated that this versatile methanotroph can reduce the toxic contaminating heavy metal chromium(VI) to the less toxic form chromium(III) while accumulating the chromium(III) within the cells. This is expected to diminish the bioavailability of the chromium and make it less likely to be reoxidized to chromium(VI). Thus, M. capsulatus has the capacity to perform methane-driven remediation of chromium-contaminated water and other materials and to accumulate the chromium in the low-toxicity chromium(III) form within the cells., (Copyright © 2021 Enbaia et al.)

The methanotrophic bacterium Methylococcus capsulatus is capable of assimilating methane and oxygen into protein-rich biomass, however, the diverse metabolism of the microorganism also allows for several undesired cometabolic side-reactions to occur. In this study, the ammonia cometabolism in Methylococcus capsulatus is investigated using pulse experiments. Surprisingly Methylococcus capsulatus oxidizes ammonia to nitrate through a yet unknown mechanism and fixes molecular nitrogen even at a high dissolved oxygen tension. The observed phenomena can be modeled using 14 ordinary differential equations and 18 kinetic parameters, of which 6 were revealed by Morris screening to be identifiable from the experimental data. Monte Carlo simulations showed that the model was robust and accurate even with uncertainty in the parameter values as confirmed by statistical error analysis., (© 2019 Wiley Periodicals, Inc.)

A wide range of microorganisms have been shown to transform selenium-containing oxyanions to reduced forms of the element, particularly selenium-containing nanoparticles. Such reactions are promising for the detoxification of environmental contamination and the production of valuable selenium-containing products, such as nanoparticles for application in biotechnology. It has previously been shown that aerobic methane-oxidizing bacteria, including Methylococcus capsulatus (Bath), are able to perform the methane-driven conversion of selenite (SeO 3 2- ) to selenium-containing nanoparticles and methylated selenium species. Here, the biotransformation of selenite by Mc . capsulatus (Bath) has been studied in detail via a range of imaging, chromatographic, and spectroscopic techniques. The results indicate that the nanoparticles are produced extracellularly and have a composition distinct from that of nanoparticles previously observed from other organisms. The spectroscopic data from the methanotroph-derived nanoparticles are best accounted for by a bulk structure composed primarily of octameric rings in the form Se 8 - x S x with an outer coat of cell-derived biomacromolecules. Among a range of volatile methylated selenium and selenium-sulfur species detected, methyl selenol (CH 3 SeH) was found only when selenite was the starting material, although selenium nanoparticles (both biogenic and chemically produced) could be transformed into other methylated selenium species. This result is consistent with methyl selenol being an intermediate in the methanotroph-mediated biotransformation of selenium to all the methylated and particulate products observed. IMPORTANCE Aerobic methane-oxidizing bacteria are ubiquitous in the environment. Two well-characterized strains, Mc . capsulatus (Bath) and Methylosinus trichosporium OB3b, representing gamma- and alphaproteobacterial methanotrophs, respectively, can convert selenite, an environmental pollutant, to volatile selenium compounds and selenium-containing particulates. Both conversions can be harnessed for the bioremediation of selenium pollution using biological or fossil methane as the feedstock, and these organisms could be used to produce selenium-containing particles for food and biotechnological applications. Using an extensive suite of techniques, we identified precursors of selenium nanoparticle formation and also found that these nanoparticles are made up of eight-membered mixed selenium and sulfur rings., (Copyright © 2019 Eswayah et al.)

Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS* , by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS * on an expression plasmid showed sensitivity to 10 mM p -chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS * as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS * marker in counterselection, we attempted to obtain an unmarked mutant of xoxF , a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS * is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath). IMPORTANCE Methanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such as Escherichia coli Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS *, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy., (Copyright © 2018 American Society for Microbiology.)

Sterols are essential eukaryotic lipids that are required for a variety of physiological roles. The diagenetic products of sterol lipids, sterane hydrocarbons, are preserved in ancient sedimentary rocks and are utilized as geological biomarkers, indicating the presence of both eukaryotes and oxic environments throughout Earth's history. However, a few bacterial species are also known to produce sterols, bringing into question the significance of bacterial sterol synthesis for our interpretation of sterane biomarkers. Recent studies suggest that bacterial sterol synthesis may be distinct from what is observed in eukaryotes. In particular, phylogenomic analyses of sterol-producing bacteria have failed to identify homologs of several key eukaryotic sterol synthesis enzymes, most notably those required for demethylation at the C-4 position. In this study, we identified two genes of previously unknown function in the aerobic methanotrophic γ-Proteobacterium Methylococcus capsulatus that encode sterol demethylase proteins (Sdm). We show that a Rieske-type oxygenase (SdmA) and an NAD(P)-dependent reductase (SdmB) are responsible for converting 4,4-dimethylsterols to 4α-methylsterols. Identification of intermediate products synthesized during heterologous expression of SdmA-SdmB along with 13 C-labeling studies support a sterol C-4 demethylation mechanism distinct from that of eukaryotes. SdmA-SdmB homologs were identified in several other sterol-producing bacterial genomes but not in any eukaryotic genomes, indicating that these proteins are unrelated to the eukaryotic C-4 sterol demethylase enzymes. These findings reveal a separate pathway for sterol synthesis exclusive to bacteria and show that demethylation of sterols evolved at least twice-once in bacteria and once in eukaryotes., Competing Interests: The authors declare no conflict of interest.

Future long-term manned space missions will require effective recycling of water and nutrients as part of a life support system. Biological waste treatment is less energy intensive than physicochemical treatment methods, yet anaerobic methanogenic waste treatment has been largely avoided due to slow treatment rates and safety issues concerning methane production. However, methane is generated during atmosphere regeneration on the ISS. Here we propose waste treatment via anaerobic digestion followed by methanotrophic growth of Methylococcus capsulatus to produce a protein- and lipid-rich biomass that can be directly consumed, or used to produce other high-protein food sources such as fish. To achieve more rapid methanogenic waste treatment, we built and tested a fixed-film, flow-through, anaerobic reactor to treat an ersatz wastewater. During steady-state operation, the reactor achieved a 97% chemical oxygen demand (COD) removal rate with an organic loading rate of 1740 g d -1  m -3 and a hydraulic retention time of 12.25 d. The reactor was also tested on three occasions by feeding ca. 500 g COD in less than 12 h, representing 50x the daily feeding rate, with COD removal rates ranging from 56-70%, demonstrating the ability of the reactor to respond to overfeeding events. While investigating the storage of treated reactor effluent at a pH of 12, we isolated a strain of Halomonas desiderata capable of acetate degradation under high pH conditions. We then tested the nutritional content of the alkaliphilic Halomonas desiderata strain, as well as the thermophile Thermus aquaticus, as supplemental protein and lipid sources that grow in conditions that should preclude pathogens. The M. capsulatus biomass consisted of 52% protein and 36% lipids, the H. desiderata biomass consisted of 15% protein and 7% lipids, and the Thermus aquaticus biomass consisted of 61% protein and 16% lipids. This work demonstrates the feasibility of rapid waste treatment in a compact reactor design, and proposes recycling of nutrients back into foodstuffs via heterotrophic (including methanotrophic, acetotrophic, and thermophilic) microbial growth., (Copyright © 2017. Published by Elsevier Ltd.)

Methylmercury (CH 3 Hg + ) is a potent neurotoxin produced by certain anaerobic microorganisms in natural environments. Although numerous studies have characterized the basis of mercury (Hg) methylation, no studies have examined CH 3 Hg + degradation by methanotrophs, despite their ubiquitous presence in the environment. We report that some methanotrophs, such as Methylosinus trichosporium OB3b, can take up and degrade CH 3 Hg + rapidly, whereas others, such as Methylococcus capsulatus Bath, can take up but not degrade CH 3 Hg + . Demethylation by M. trichosporium OB3b increases with increasing CH 3 Hg + concentrations but was abolished in mutants deficient in the synthesis of methanobactin, a metal-binding compound used by some methanotrophs, such as M. trichosporium OB3b. Furthermore, addition of methanol (>5 mM) as a competing one-carbon (C1) substrate inhibits demethylation, suggesting that CH 3 Hg + degradation by methanotrophs may involve an initial bonding of CH 3 Hg + by methanobactin followed by cleavage of the C-Hg bond in CH 3 Hg + by the methanol dehydrogenase. This new demethylation pathway by methanotrophs indicates possible broader involvement of C1-metabolizing aerobes in the degradation and cycling of toxic CH 3 Hg + in the environment.

Rationale: Traditional investigation of bacteriohopanepolyols (BHPs) has relied on derivatisation by acetylation prior to gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/MS (LC/MS) analysis. Here, modern chromatographic techniques (ultrahigh-performance liquid chromatography (UPLC)) and new column chemistries were tested to develop a method for BHP analysis without the need for derivatisation., Methods: Bacterial culture and sedimentary lipid extracts were analysed using a Waters Acquity Xevo TQ-S triple quadrupole mass spectrometer in positive ion atmospheric pressure chemical ionisation (APCI) mode. Waters BEH C18 and ACE Excel C18 were the central columns evaluated using a binary solvent gradient with 0.1% formic acid in the polar solvent phase in order to optimise performance and selectivity., Results: Non-amine BHPs and adenosylhopane showed similar performance on each C18 column; however, BHP-containing terminal amines were only identified eluting from the ultra-inert ACE Excel C18 column. APCI-MS/MS product ion scans revealed significant differences in fragmentation pathways from previous methods for acetylated compounds. The product ions used for targeted multiple reaction monitoring (MRM) are summarised., Conclusions: UPLC/MS/MS analysis using an ACE Excel C18 column produced superior separation for amine-containing BHPs and reduced run times from 60 to 9 min compared with previous methods. Unexpected variations in fragmentation pathways between structural subgroups must be taken into account when optimising MRM transitions for future quantitative studies. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The genome of Methylococcus capsulatus (bath) encodes a protein R-est6 that is annotated as a lipase family 3 protein. The phylogenetic and the sequence analyses linked this protein to the family 6 carboxylesterase. The gene encoding R-est6 was cloned and overexpressed in Escherichia coli and the recombinant 6x-His tagged protein was purified by Ni-NTA affinity chromatography. The buffers used in the purification were modified by adding 1% glycerol instead of the salt to prevent the protein aggregation. Far UV-CD spectrum and gel filtration chromatography of the purified R-est6 confirmed that the protein was well folded like a typical α/β hydrolase and had the quaternary structure of a tetramer, in addition to a compact monomer. The optimum pH was in the range of 7.0-9.0 and the optimum temperature was at 55 °C for the hydrolysis of pNP-butyrate. As expected, being a member of the family 6 carboxylesterase, R-est6 hydrolyzed triglycerides, pNP esters of the small and the medium fatty acid chain esters and an aryl ester-phenyl acetate. However, R-est6 was also found to hydrolyze the long-chain fatty acid ester which had never been reported for the family 6 carboxylesterase. Additionally, R-est6 was stable and active in the different water-miscible organic solvents. Therefore, the broad substrate range and the structural stability of R-est6 would be advantageous for its application in industrial processes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The bacteriohemerythrin (McHr) from Methylococcus capsulatus (Bath) is an oxygen carrier that serves as a transporter to deliver O2 from the cytosol of the bacterial cell body to the particulate methane monooxygenase residing in the intracytoplasmic membranes for methane oxidation. Here we report X-ray protein crystal structures of the recombinant wild type (WT) McHr and its L114A, L114Y and L114F mutants. The structure of the WT reveals a possible water tunnel in the McHr that might be linked to its faster autoxidation relative to hemerythrin in marine invertebrates. With Leu114 positioned at the end of this putative water tunnel, the hydrophobic side chain of this residue seems to play a prominent role in controlling the access of the water molecule required for autoxidation. This hypothesis is examined by comparing the autoxidation rates of the WT McHr with those of the L114A, L114Y and L114F mutants. The biochemical data are correlated with structural insights derived from the analysis of the putative water tunnels in the various McHr proteins provided by the X-ray structures., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Gram-negative methanotroph Methylococcus capsulatus (Bath) was recently demonstrated to abrogate inflammation in a murine model of inflammatory bowel disease, suggesting interactions with cells involved in maintaining mucosal homeostasis and emphasizing the importance of understanding the many properties of M. capsulatus. Secreted proteins determine how bacteria may interact with their environment, and a comprehensive knowledge of such proteins is therefore vital to understand bacterial physiology and behavior. The aim of this study was to systematically analyze protein secretion in M. capsulatus (Bath) by identifying the secretion systems present and the respective secreted substrates. Computational analysis revealed that in addition to previously recognized type II secretion systems and a type VII secretion system, a type Vb (two-partner) secretion system and putative type I secretion systems are present in M. capsulatus (Bath). In silico analysis suggests that the diverse secretion systems in M.capsulatus transport proteins likely to be involved in adhesion, colonization, nutrient acquisition and homeostasis maintenance. Results of the computational analysis was verified and extended by an experimental approach showing that in addition an uncharacterized protein and putative moonlighting proteins are released to the medium during exponential growth of M. capsulatus (Bath).

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.

Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A(||) =20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.

The mercury (II) ion is toxic and is usually detoxified in Bacteria by reduction to elemental mercury, which is less toxic. This is catalysed by an NAD(P)H-dependent mercuric reductase (EC 1.16.1.1). Here, we present strong evidence that Methylococcus capsulatus (Bath) - a methanotrophic member of the Gammaproteobacteria - uses this enzyme to detoxify mercury. In radiorespirometry studies, it was found that cells exposed to mercury dissimilated 100% of [(14) C]-methane provided to generate reducing equivalents to fuel mercury (II) reduction, rather than the mix of assimilation and dissimilation found in control incubations. The detoxification system is constitutively expressed with a specific activity of 352 (±18) nmol NADH oxidized min(-1) (mg protein)(-1) . Putative mercuric reductase genes were predicted in the M. capsulatus (Bath) genome and found in mRNA microarray studies. The MerA-derived polypeptide showed high identity (> 80%) with MerA sequences from the Betaproteobacteria., (2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Proteins on the cellular surface of a bacterium, its surfaceome, are part of the interface between the bacterium and its environment, and are essential for the cells response to its habitat. Methylococcus capsulatus Bath is one of the most extensively studied methane-oxidizers and is considered as a model-methanotroph. The composition of proteins of the surfaceome of M. capsulatus Bath varies with the availability of copper and changes significantly upon only minor changes of copper concentration in the sub-μM concentration range. Proteins that respond to the changes in copper availability include the assumed copper acquisition protein MopE, c-type heme proteins (SACCP, cytochrome c(553o) proteins) and several proteins of unknown function. The most intriguing observation is that multi-heme c-type cytochromes are major constituents of the M. capsulatus Bath surfaceome. This is not commonly observed in bacteria, but is a feature shared with the dissimilatory metal-reducing bacteria. Their presence on the M. capsulatus Bath cellular surface may be linked to the cells ability to efficiently adapt to changing growth conditions and environmental challenges. However, their possible role(s) in methane oxidation, nitrogen metabolism, copper acquisition, redox-reactions and/or electron transport remain(s) at present an open question. This review will discuss the possible significance of these findings., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

A small soluble cytochrome c-554 purified from Methylosinus trichosporium OB3b has been purified and analyzed by amino acid sequencing, mass spectrometry, visible, CD and EPR spectroscopies. It is found to be a mono heme protein with a characteristic cytochrome c fold, thus fitting into the class of cytochrome c(2), which is the bacterial homologue of mitochondrial cytochrome c. The heme iron has a Histidine/Methionine axial ligation and exhibits a highly anisotropic/axial low spin (HALS) EPR signal, with a g(max) at 3.40, and ligand field parameters V/ξ = 0.99, Δ/ξ = 4.57. This gives the rhombicity V/Δ = 0.22. The structural basis for this HALS EPR signal in Histidine/Methionine ligated hemes is not resolved. The ligand field parameters observed for cytochrome c-554 fits the observed pattern for other cytochromes with similar ligation and EPR behaviour.

Particulate methane monooxygenase (pMMO) is a multisubunit metalloenzyme complex used by methanotrophic bacteria to oxidize methane in the first step of carbon assimilation and energy production. In this chapter, we detail methods to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. We also describe protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. These approaches were used to address fundamental questions concerning the metal content and location of the pMMO active site., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We detected flavins in the growth medium of the methanotrophic bacterium Methylocystis species strain M. Flavin secretion correlates with growth stage and increases under iron starvation conditions. Two other methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), secrete flavins, suggesting that flavin secretion may be common to many methanotrophic bacteria.

1. The effects of increasing concentrations (0, 40, 80 or 120 g/kg) of bacterial protein meal (BPM) and bacterial protein autolysate (BPA) grown on natural gas on growth performance and carcase quality in broiler chickens were examined. 2. Adding BPM to diets reduced feed intake and improved gain: feed from 0 to 21 d and overall to 35 d, but did not significantly affect weight gain compared to the soybean meal based control diet. 3. Increasing concentrations of BPA significantly reduced growth rate, feed intake, gain: feed, carcase weight and dressing percentage, but significantly increased carcase dry matter, fat and energy content. 4. Adding BPM to diets had no effect on viscosity of diets and jejunal digesta, and minor effects on litter quality, whereas BPA increased the viscosity of diets and jejunal digesta, improved litter quality at 21 d, but decreased litter quality at 32 d. 5. To conclude, broiler chickens performed better on a BPM product with intact proteins than on an autolysate with ruptured cell walls and a high content of free amino acids and low molecular-weight peptides.

The physiology of the response in the methanotrophic bacterium Methylococcus capsulatus Bath towards thermal and solvent stress was studied. A systematic investigation of the toxic effects of organic compounds (chlorinated phenols and alkanols) on the growth of this bacterium was carried out. The sensitivity to the tested alkanols correlated with their chain length and hydrophobicity; methanol was shown to be an exception to which the cells showed a very high tolerance. This can be explained by the adaptation of these bacteria to growth on C1 compounds. On the other hand, M. capsulatus Bath was very sensitive towards the tested chlorinated phenols. The high toxic effect of phenolic compounds on methanotrophic bacteria might be explained by the occurrence of toxic reactive oxygen species. In addition, a physiological proof of the presence of cis-trans isomerization as a membrane-adaptive response mechanism in M. capsulatus was provided. This is the first report on physiological evidence for the presence of the unique postsynthetic membrane-adaptive response mechanism of the cis-trans isomerization of unsaturated fatty acids in a bacterium that does not belong to the genera Pseudomonas and Vibrio where this mechanism was already reported and described extensively.

Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates H(peroxo) and Q. The first intermediate, P*, is a precursor to H(peroxo) and was identified when the reaction of reduced MMOH and MMOB with O(2) was carried out in the presence of >or=540 microM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H(peroxo), with epsilon(420) = 3500 M(-1) cm(-1) and epsilon(720) = 1250 M(-1) cm(-1). These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/H(peroxo) formation and H(peroxo) decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D(2)O. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to H(peroxo) and H(peroxo) to Q conversion.

Methylococcus capsulatus strain Bath, a methane-oxidizing bacterium, and ammonia-oxidizing bacteria (AOB) carry out the first step of nitrification, the oxidation of ammonia to nitrite, through the intermediate hydroxylamine. AOB use hydroxylamine oxidoreductase (HAO) to produce nitrite. M. capsulatus Bath was thought to oxidize hydroxylamine with cytochrome P460 (cytL), until the recent discovery of an hao gene in its genome. We used quantitative PCR analyses of cDNA from M. capsulatus Bath incubated with CH(4) or CH(4) plus 5 mM (NH(4))(2)SO(4) to determine whether cytL and hao transcript levels change in response to ammonia. While mRNA levels for cytL were not affected by ammonia, hao mRNA levels increased by 14.5- and 31-fold in duplicate samples when a promoter proximal region of the transcript was analyzed, and by sixfold when a region at the distal end of the transcript was analyzed. A conserved open reading frame, orf2, located 3' of hao in all known AOB genomes and in M. capsulatus Bath, was cotranscribed with hao and showed increased mRNA levels in the presence of ammonia. These data led to designating this gene pair as haoAB, with the role of haoB still undefined. We also determined mRNA levels for additional genes that encode proteins involved in N-oxide detoxification: cytochrome c'-beta (CytS) and nitric oxide (NO) reductase (NorCB). Whereas cytS mRNA levels increased in duplicate samples by 28.5- and 40-fold in response to ammonia, the cotranscribed norC-norB mRNA did not increase. Our results strongly suggest that M. capsulatus Bath possesses a functional, ammonia-responsive HAO involved in nitrification.

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.

A putative operon containing homologues of essential eukaryotic sterol biosynthetic enzymes, squalene monooxygenase and oxidosqualene cyclase, has been identified in the genome of the prokaryote Methylococcus capsulatus. Expression of the squalene monooxygenase yielded a protein associated with the membrane fraction, while expression of oxidosqualene cyclase yielded a soluble protein, contrasting with the eukaryotic enzyme forms. Activity studies with purified squalene monooxygenase revealed a catalytic activity in epoxidation of 0.35 nmol oxidosqualene produced/min/nmol squalene monooxygenase, while oxidosqualene cyclase catalytic activity revealed cyclization of oxidosqualene to lanosterol with 0.6 nmol lanosterol produced/min/nmol oxidosqualene cyclase and no other products observed. The presence of prokaryotic sterol biosynthesis is still regarded as rare, and these are the first representatives of such prokaryotic enzymes to be studied, providing new insight into the evolution of sterol biosynthesis in general.

Cytochromes-P460 of Nitrosomonas europaea and Methylococcus capsulatus (Bath), and the cytochrome c' of M. capsulatus, believed to be involved in binding or transformation of N-oxides, are shown to represent an evolutionarily related new family of monoheme, approximately 17kDa, cytochromes c found in the genomes of diverse Proteobacteria. All members of this family have a predicted secondary structure predominantly of beta-sheets in contrast to the predominantly alpha-helical cytochromes c' found in photoheterotrophic and denitrifying Proteobacteria.

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

The effects of high dietary levels of phosphatidylethanolamine (PE) on plasma concentrations of homocysteine (tHcy) have not previously been studied. Eighteen mink (Mustela vison) studied were fed one of three diets during a 25 d period in a parallel-group design. The compared diets had 0, 17 and 67 % extracted lipids from natural gas-utilising bacteria (LNGB), which were rich in PE. The group with 0 % LNGB was fed a diet of 100 % soyabean oil (SB diet). Phospholipids are the main lipid components in LNGB and Methylococcus capsulatus is the main bacteria (90 %). The fasting plasma concentration of tHcy was significantly higher when the mink consumed the diet with 67 % LNGB than when they consumed the SB diet (P=0.039). A significantly lower glutathione peroxidase activity was observed in mink consuming the 17 % LNGB diet or the 67 % LNGB diet than was observed in mink fed the SB diet. The lack of significant differences in the level of plasma PE due to the diets indicates that most of the PE from the 67 % LNGB diet was converted to phosphatidylcholine (PC) in the liver. It has previously been hypothesised that phosphatidylethanolamine N-methyltransferase is an important source of tHcy. The present results indicate that plasma tHcy is at least partly regulated by phospholipid methylation from PE to PC. This methylation reaction is a regulator of physiological importance.

Completion of the genome sequence of Methylococcus capsulatus Bath is an important event in molecular microbiology, and an achievement for which the authors deserve congratulation. M. capsulatus, along with other methanotrophs, has been the subject of intense biochemical and molecular study because of its role in the global carbon cycle: the conversion of biogenic methane to carbon dioxide. The methane monooxygenase enzymes that are central to this process also have high biotechnological potential. Analysis of the genome sequence will potentially accelerate elucidation of the regulation of methane-dependent metabolism in obligate methanotrophs, and help explain the cause of obligate methanotrophy, the phenomenon making most methanotrophs unable to grow on any substrates other than methane and a very small number of other one-carbon compounds.

A three-component soluble methane monooxygenase (sMMO) enzyme system catalyzes the hydroxylation of methane to methanol at a carboxylate-bridged diiron center housed in the alpha-subunit of the hydroxylase (MMOH). Catalysis is facilitated by the presence of a regulatory protein (MMOB) and inhibited by MMOD, a protein of unknown function encoded in the sMMO operon. Both MMOB and MMOD are presumed to bind to the same region of the MMOH alpha-subunit. A colorimetric method for monitoring removal of Fe(II) from MMOH was developed using 1,10-phenanthroline and yields apo MMOH with <0.1 Fe/homodimer. With the use of this method, it was possible to investigate the X-ray structure of the apoenzyme and to perform metal reconstitution studies. Using MMOH from Methylococccus capsulatus (Bath), the effects of MMOB and MMOD on metal binding were studied and structural perturbations relevant to the function of this enzyme were identified. X-ray crystal structures of the apo, Mn(II)-soaked, and Co(II)-grown MMOH, determined to 2.3 A or greater resolution, reveal that the presence of metal ions is essential for the proper folding of helices E, F, and H of the alpha-subunit. The active sites of Mn(II)-soaked and Co(II)-grown MMOH are similar to that of reduced, native MMOH with notable differences in the metal-metal distances and ligand coordination sphere that may reflect how this dinuclear metal center might change in the presence of MMOB. MMOB and MMOD decrease the rate of removal of Fe(II) from the enzyme by 22- and 16-fold, respectively. On the basis of previous studies, it is hypothesized that MMOB, and perhaps MMOD, function to block solvent access to the MMOH active site. Finally, ITC studies and the observed disorder in helices E, F, and H in the apo and Mn(II)-soaked structures suggest that these regions of MMOH are critical for MMOB and MMOD binding.

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them.

The process of colonization of hydrophilic (glass) and hydrophobic (polysterene) carriers by pure cultures of methanotrophs Methylocystis parvus UCM B-3490T, Methylococcus capsulatus UCM B-3030, as well as by their cultures mixed with Bacillus megaterium UCM B 5723T and Pseudomonas putida VKPM B-4188 under the conditions efficient for methanotrophic bacteria. M. parvus demonstrated the highest intensity of this process on the above carriers owing to high hydrophobic cell surface. Both methanotrophs colonized the glass surface more quickly with formation of microcolonies on carriers after 6 days of incubation in pure and mixed cultures with B. megaterium. The number of bacilli on these carriers quickly decreased. In the mixed cultures with P. putida the glass and polysterene colonization intensity decreased, while the amount of pseudomonas on carriers increased.

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.

Experiments on cryptically chiral ethanes have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of configuration at the carbon center attacked. This result would seem to rule out a radical mechanism for the hydroxylation chemistry, at least as mediated by this enzyme. The interpretation of subsequent experiments on n-propane, n-butane, and n-pentane has been complicated by hydroxylation at both the pro-R and pro-S secondary C-H bonds, where the hydroxylation takes place. It has been suggested that these results merely reflect presentation of both the pro-R and pro-S C-H bonds to the hot "oxygen atom" species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of configuration. In the present work, we have augmented these earlier studies with experiments on [2,2-2H2]butane and designed d,l form chiral dideuteriobutanes. Essentially equal amounts of (2R)-[3,3-2H2]butan-2-ol and (2R)-[2-2H1]butan-2-ol are produced upon hydroxylation of [2,2-2H2]butane. The chemistry is stereospecific with full retention of configuration at the secondary carbon oxidized. In the case of the various chiral deuterated butanes, the extent of configurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of configuration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet oxene insertion across the C-H bond similar to that previously noted for singlet carbene insertion (Kirmse, W., and Ozkir, I. S. (1992) J. Am. Chem. Soc. 114, 7590-7591). Finally, we discuss how even the oxene insertion mechanism, with "spin crossover" in the transition state, could lead to small amounts of radical rearrangement products, if and when such products are observed. A scheme is described that unifies the two extreme mechanistic limits, namely the concerted oxene insertion and the hydrogen abstraction radical rebound mechanism within the same over-arching framework.

The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the oxidation of methane to methanol. Some methanotrophs, including Methylococcus capsulatus (Bath), possess two distinct MMOs. The level of copper in the environment regulates the biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural genes of sMMO were previously identified as mmoXYBZ, mmoD and mmoC. Putative transcriptional start sites, containing a sigma(70)- and a sigma(N)-dependent motif, were identified in the 5' region of mmoX. The promoter region of mmoX was mapped using truncated 5' end regions fused to a promoterless green fluorescent protein gene. A 9.5 kb region, adjacent to the sMMO structural gene cluster, was analysed. Downstream (3') from the last gene of the operon, mmoC, four ORFs were found, mmoG, mmoQ, mmoS and mmoR. mmoG shows significant identity to the large subunit of the bacterial chaperonin gene, groEL. In the opposite orientation, two genes, mmoQ and mmoS, showed significant identity to two-component sensor-regulator system genes. Next to mmoS, a gene encoding a putative sigma(N)-dependent transcriptional activator, mmoR was identified. The mmoG and mmoR genes were mutated by marker-exchange mutagenesis and the effects of these mutations on the expression of sMMO was investigated. sMMO transcription was impaired in both mutants. These results indicate that mmoG and mmoR are essential for the expression of sMMO in Mc. capsulatus (Bath).

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress.

We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria. Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications. The trains were found to be regenerated when rerunning individual polypeptide spots. The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al. (Protein Eng. 1990, 4, 155-161). The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse.

The relative importance of individual microbial pathways in nitrous oxide (N(2)O) production is not well known. The intramolecular distribution of (15)N in N(2)O provides a basis for distinguishing biological pathways. Concentrated cell suspensions of Methylococcus capsulatus Bath and Nitrosomonas europaea were used to investigate the site preference of N(2)O by microbial processes during nitrification. The average site preference of N(2)O formed during hydroxylamine oxidation by M. capsulatus Bath (5.5 +/- 3.5 per thousand) and N. europaea (-2.3 +/- 1.9 per thousand) and nitrite reduction by N. europaea (-8.3 +/- 3.6 per thousand) differed significantly (ANOVA, f((2,35)) = 247.9, p = 0). These results demonstrate that the mechanisms for hydroxylamine oxidation are distinct in M. capsulatus Bath and N. europaea. The average delta(18)O-N(2)O values of N(2)O formed during hydroxylamine oxidation for M. capsulatus Bath (53.1 +/- 2.9 per thousand) and N. europaea (-23.4 +/- 7.2 per thousand) and nitrite reduction by N. europaea (4.6 +/- 1.4 per thousand) were significantly different (ANOVA, f((2,35)) = 279.98, p = 0). Although the nitrogen isotope value of the substrate, hydroxylamine, was similar in both cultures, the observed fractionation (delta(15)N) associated with N(2)O production via hydroxylamine oxidation by M. capsulatus Bath and N. europaea (-2.3 and 26.0 per thousand, respectively) provided evidence that differences in isotopic fractionation were associated with these two organisms. The site preferences in this study are the first measured values for isolated microbial processes. The differences in site preference are significant and indicate that isotopomers provide a basis for apportioning biological processes producing N(2)O., (Copyright 2003 John Wiley & Sons, Ltd.)