Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene.

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.

Transcriptional Regulation of Methanol Dehydrogenases in the Methanotrophic Bacterium Methylococcus capsulatus Bath by Soluble and Insoluble Lanthanides.

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.

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

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 C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Cell-Free Protein Synthesis of Particulate Methane Monooxygenase into Nanodiscs.

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.

Systems analysis of the effect of hydrogen sulfide on the growth of Methylococcus capsulatus Bath.

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.

Refining details of the structural and electronic properties of the CuB site in pMMO enzyme through sequential molecular dynamics/CPKS-EPR calculations.

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 CuB 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 His33, His137, and His139 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 CuB site brings additional support concerning the recent experimental evidence, which pointed out that a saturated coordination sphere of the copper ion in the CuB center is an essential factor that makes it less efficient than the CuC site in the methane oxidation. Therefore, according to the CuB site model proposed here, an additional step involving a displacement of at least one water molecule of the copper coordination sphere by the O2 molecule prior to its activation must be necessary. This scenario is less likely to occur in the CuC 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.

Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction.

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.

Detoxification, Active Uptake, and Intracellular Accumulation of Chromium Species by a Methane-Oxidizing Bacterium.

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).IMPORTANCEM. 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.

Methyl Selenol as a Precursor in Selenite Reduction to Se/S Species by Methane-Oxidizing Bacteria.

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 (SeO32-) 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 Se8 -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 (CH3SeH) 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.

Dynamic investigation and modeling of the nitrogen cometabolism in Methylococcus capsulatus ( Bath).

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.

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene.

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.

C-4 sterol demethylation enzymes distinguish bacterial and eukaryotic sterol synthesis.

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 13C-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.

Coupling of anaerobic waste treatment to produce protein- and lipid-rich bacterial biomass.

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.

Methylmercury uptake and degradation by methanotrophs.

Methylmercury (CH3Hg+) 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 CH3Hg+ 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 CH3Hg+ rapidly, whereas others, such as Methylococcus capsulatus Bath, can take up but not degrade CH3Hg+. Demethylation by M. trichosporium OB3b increases with increasing CH3Hg+ 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 CH3Hg+ degradation by methanotrophs may involve an initial bonding of CH3Hg+ by methanobactin followed by cleavage of the C-Hg bond in CH3Hg+ 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 CH3Hg+ in the environment.

Analysis of non-derivatised bacteriohopanepolyols by ultrahigh-performance liquid chromatography/tandem mass spectrometry.

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.

Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath).

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/ß 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.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

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.

The bacteriohemerythrin from Methylococcus capsulatus (Bath): Crystal structures reveal that Leu114 regulates a water tunnel.

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.

Computational and experimental analysis of the secretome of Methylococcus capsulatus (Bath).

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).

Draft genome sequence of the methane-oxidizing bacterium Methylococcus capsulatus (Texas).

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.