Improvement of the purification method for retaining the activity of the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The purification method of particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was improved, and purified pMMO retained its activity with duroquinol as a reductant. n-Dodecyl-,d-maltoside was used for the solubilization of pMMO and Brij 58 was used for the purification for anion exchange chromatography. Compared to the original pMMO activity in the membrane fraction, 88% of the activity was now retained in the purified material. The purified pMMO monomer (94 kDa) contained only two copper atoms and did not contain iron. Both copper ions showed only a typical type II copper EPR signal with a superhyperfine structure at the g region, indicating that the type II copper ions play an important role as the active site of methane hydroxylation in pMMO.     

Co-metabolic biodegradation of trichloroethylene by Methylosinus trichosporium is stimulated by low concentrations methane or methanol

Co-metabolic biodegradation of trichloroethylene by Methylosinus trichosporium OB3b was stimulated by low concentrations of methane (up to 70 M) or methanol (up to 0.4 mM) but inhibited at higher concentrations of them. A kinetic equation describing the dual effects of methane or methanol is proposed and the relevant kinetic constants have been determined.     

Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath

Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O₂, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hydroxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated K _mvalue for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.Abbreviations PMS Phenazine methosulphate - NADH nicotinamide adenine dinucleotide, reduced form - K _m Michaelis constant - NO ₂ ^- nitrite - NH₂OH hydroxylamine     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

The methane monooxygenase gene cluster of Methylosinus trichosporium: cloning and sequencing of the mmoC gene

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. The soluble MMO enzyme complex from Methylosinus trichosporium also oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study we have used heterologous DNA probes from the type X methanotroph Methylococcus capsulatus (Bath) to isolate mmo genes from the type II methanotroph M. trichosporium. We report here that the gene encoding the reductase component, Protein C of MMO, lies adjacent to the genes encoding the other components of soluble MMO in M. trichosporium but is separated by an open reading frame of unknown function, orfY. The complete nucleotide sequence of these genes is presented. Sequence analysis of mmoC indicates that the N-terminus of Protein C has significant homology with 2Fe2S ferredoxins from a wide range of organisms.Abbreviations MMO methane monooxygenase     

Effect of copper on membrane lipids and on methane monooxygenase activity of Methylococcus capsulatus (Bath)

The effect of copper supplementation on growth, methane monooxygenase activity and lipid composition of Methylococcus capsulatus (Bath) was studied. Copper increased biomass yield, methane monooxygenase activity and phospholipid content from 7.7 to 10.2% of dry weight. Cells from copper-deficient and copper supplemented cultures contained the same major fatty acids but in the presence of copper only the contents of C16:0 and the three C16:1 isomers were increased while the contents of C14:0 and cyclic C17:0 remained unchanged. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol and cardiolipin were analysed amongst the polar lipids. PE was the main component (about 60 mol-%) but the most notable copper-induced increment occurred in the proportion of PC, from about 10 to 16 mol-%. Concomitantly with this increment the fatty acids of PC were changed so that the mol-% of C16: 1 isomers were increased at the expense of other acids. Similar trends were seen also in the fatty acid compositions of other polar lipid fractions. It is therefore concluded that phosphatidylcholine would be one of the key factors when the role of membranous lipids in methane monooxygenase activity is to be considered.     

Optimization of methanol biosynthesis from methane using Methylosinus trichosporium OB3b

Methylosinus trichosporium OB3b oxidized methane to methanol in the presence of a high concentration of Cu2+. Further oxidation of methanol to formaldehyde was prevented by adding 200 mM NaCl which acted as a methanol dehydrogenase H inhibitor. The bacterium, 0.6 mg dry cell ml(-1), in methane/air (1:4, v/v) at 25 degrees C in 12.9 mM phosphate buffer (pH 7) containing 20 mM sodium formate and 200 mM NaCl accumulated 7.7 mM methanol over 36 h.     

Ammonia switch-off of nitrogenase from Rhodobacter sphaeroides and Methylosinus trichosporium: no evidence for Fe protein modification

In vivo switch-off of nitrogenase activity by NH ₄ ⁺ is a reversible process in Rhodobacter sphaeroides and Methylosinus trichosporium OB3b. The same pattern of switch-off in Rhodospirillum rubrum is explained by ADP-ribosylation of one of the Fe protein subunits, however, no evidence of covalent modification could be found in the subunits from either R. sphaeroides or M. trichosporium. Fe protein subunits from these organisms showed no variant behaviour on SDS-PAGE, nor were they ³²P-labeled following switch-off. These observations suggest either that the attachment of the modifying group to the Fe protein in these organisms is quite labile and does not survive in vitro manipulation, or that the mechanism of switch-off is different than that seen in Rhodospirillum.     

Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting β-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP () predicted 43 β-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8–3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins (). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.     

Effect of metal-binding agents and other compounds on methane oxidation by two strains of Methylococcus capsulatus

Inhibition studies of methane mono-oxygenase activity in whole cell suspensions of Methylococcus capsulatus (Texas) and M. capsulatus (Bath) were performed and the results compared. The inhibition pattern for M. capsulatus (Bath) was not only substantially different from the pattern obtained with M. capsulatus (Texas) but also very limited in the number of potent inhibitors specific for methane oxidation. To confirm the whole cell results of M. capsulatus (Bath) similar experiments were done using cell-free extracts. It was found that only acetylene (100% inhibition) and 8-hydroxyquinoline (71%) significantly inhibited methane oxidation, verifying the restricted inhibition pattern found with the whole cell suspensions. Eight acetylenic compounds were tested for specific inhibition of methane oxidation by whole cells and cell-free extracts of M. capsulatus (Bath). Only two compounds (acetylene and propyne) gave 100% inhibition in both cases with three other compounds (but-1-yne, but-2-yne and propyn-1-ol) giving weaker inhibitions. The inhibition pattern of methane oxidation by whole cell suspensions and cell-free extracts of M. capsulatus (Bath) is discussed and reasons for the prominent results are suggested.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N₂ and H₂O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

High-rate conversion of methane to methanol by Methylosinus trichosporium OB3b

Methanol was produced from methane with a high conversion rate using a high cell density process with Methylosinus trichosporium OB3b in the presence of a high concentration of phosphate buffer. More than 1.1 g/L methanol accumulated in the reaction media under optimized reaction conditions (17 g dry cell/L, 400 mmol/L phosphate, and 10 mmol/L MgCl₂) in the presence of 20 mmol/L sodium formate. The conversion rate of methane was over 60%. About 0.95 g/L methanol was produced when the biotransformation was carried out in a membrane aerated reactor into which methane and oxygen were introduced via two separate dense silicone tubing. Our results provide an efficient method and a promising process for high-rate conversion of methane to methanol.     

Effects of oxygen and nitrogen conditions on the transformation kinetics of 1,2-dichloroethenes by Methylosinus trichosporium OB3b and its sMMO C mutant

Transformation kinetics of trans- andcis-dichloroethylenes (DCE) by Methylosinus trichosporium OB3b wild type (WT)and PP319, a mutant that expresses soluble methane monooxygenase at copper levels upto 12 M Cu (sMMO^C), were determined to assess theeffects of O₂level and N₂-fixation on degradationcapabilities. Two issues were examined: (1) the influence of O₂level and nitrogen-limitation on DCE degradationkinetics and toxicity in both organisms, and (2) the relative utility of PP319 forcontaminant degradation in bioreactors. When both organisms were grown underhigh O₂conditions (80% saturation in air), maximum transformation rates(V_max) and apparent first-order rate constants(V_max/K_M) were lower compared with organisms grown under low O₂conditions (10% saturation in air)regardless of nitrogen level. Further, V_max values were near zero innitrogen-limited WT cultures when O₂was high (as expected), whereas PP319 retainedmoderate V_max levels even at high O₂levels. In general, elevatedO₂conditions reduced DCE degradation rates in OB3b, although the negative effectsof O₂were less in PP319 than in the WT. Given that PP319 retained moderate DCEdegradation rates under most O₂and copper conditions, the mutant appears to havesome utility for biodegradation applications.     

Immunocytochemical localization of glutamine synthetase in Rhodobacter capsulatus E1F1 and Rhodopseudomonas acidophila

Intracellular localization of glutamine synthetase has been studied by immunochemical techniques with cryosections and London Resin sections of Rhodobacter capsulatus E1F1 and Rhodopseudomonas acidophila. For immunostaining, sections were sequentially incubated with monospecific anti-glutamine synthetase antibodies (R. capsulatus) and gold labelled goat anti-rabbit antibodies. Gold label was present in the cytoplasm but not in the cell walls. The antigen is not associated with the cell membrane or with photosynthetic vesicle whether these are round and randomly distributed (R. capsulatus) or flattened and organized in well defined stacks (R. acidophila). Our results also indicate that glutamine synthetase is absent from the central, nucleoid part of the cell. The enzyme is present in dense cytoplasmic patches, which appear to be RNA-ribosome-containing areas.Abbreviations GS glutamine synthetase - LR London Resin White     

Diazotrophic mixed culture ofAzotobacter vinelandii andRhodobacter capsulatus

The question, whetherAzotobacter vinelandii can provide fixed N for the growth of other organisms, was studied with mixed cultures ofA. vinelandii andRhodobacter capsulatus, grown with aeration in the light. N₂-fixation byR. capsulatus was prevented by growing the cultures on either mannitol, glycerol or ethanol, which cannot be used by this organism. In the course of growth with mannitol, cell numbers of both organisms increased largely in parallel and attained a maximal ratio of about oneA. vinelandii per tenR. capsulatus. Prolonged growth of mixed cultures with mannitol did not lead to an adaptation ofR. capsulatus to this compound. After growth on either one of the three alcohols, mixed cultures exhibited almost twice as high protein levels as pure cultures ofA. vinelandii. Up to 80% of the protein of mixed cultures was incorporated intoR. capsulatus. The results suggest thatA. vinelandii provided an organic N-source for the growth ofR. capsulatus.     

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.Abbreviations A₆₀₀ absorbance due to cell density measured at 600 nm - HPLC high pressure liquid chromatography - NADH reduced nicotinamide adenine dinucleotide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - sMMO soluble methane monooxygenase - TCE trichloroethylene