Methane oxidation by cell-free extracts of Methylococcus capsulatus

The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso-octane extraction, (3) (NH(4))(2)SO(4) fractionation in the presence of sodium cholate. The fraction precipitating between 25-35 percent (NH(4))(2)SO(4) gave a difference spectrum with 11-deoxycorticosterone (11-DOC) but not with 20alpha-hydroxycholesterol (20alpha-HOC). This fraction showed high 11beta-hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45-60 percent (NH(4))(2)SO(4) gave a difference spectrum with 20alpha-HOC but not with 11-DOC and exhibited high S.C.C. activity but low 11beta-hydroxylase activity. The absorption spectrum of the 45-60 percent fraction indicated a preponderance of high spin hemoprotein (lambda(max) 395 nm).     

Mutation of the Methane Oxidizing Bacterium, Methylococcus capsulatus

S ummary : Spontaneous mutants of Methylococcus capsulatus resistant to antibiotics, amino acid analogues and other compounds were obtained at frequencies similar to those found in other bacteria. Attempts to increase these frequencies with the mutagens N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroso-N-methyl urethane, ethyl methane-sulphonate and UV were unsuccessful. Using these mutagens, only one auxotrophic mutant was isolated from 11,082 colonies examined. The growth characteristics of this p -aminobenzoic acid requiring mutant are described.     

Inhibition by ammonia of methane utilization in Methylococcus capsulatus (Bath)

Summary The kinetics of methane uptake by Methylococcus capsulatus (Bath) and its inhibition by ammonia were studied by stopped-flow membrane-inlet mass spectrometry. Measurements were done on suspensions of cells grown in high- and low-copper media. With both types of cells the kinetics of methane uptake are hyperbolic when oxygen is in excess. The apparent K _m and K _max for methane uptake are both higher in low-copper cells than in high-copper cells. Ammonia is a simple competitive inhibitor of methane uptake in high-copper cells when the oxygen concentration is above a few M. The findings agree with the assumption that ammonia is a week alternative substrate for particulate methane monooxygenase. In low-copper cells the effect of ammonia is complicated and cannot be explained in terms of current assumptions on the mechanism of soluble methane monooxygenase. Our data indicate that ammonia inhibition is likely to be a more serious problem in connection with cultivation in low-copper medium than in high-copper medium. Offprint requests to: H. N. Carlsen     

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.     

Quantitative Aspects of Growth of the Methane Oxidizing Bacterium Methylococcus capsulatus on Methane in Shake Flask and Continuous Chemostat Culture

S ummary : Experiments were directed towards the production of high biomass concentrations in cultures of Methylococcus capsulatus. In shake flasks the effects of ammonium ion, phosphate ion and various trace metals on growth were studied. In the chemostat the effects on growth of methane limitation, oxygen limitation, aeration, dilution rate, pH value and temperature were studied and carbon balances were made in steady state conditions. Growth in the fermenter was stimulated by the use of Amberlite CG–120 ion exchange resin in the medium. The probability that Amberlite removed a growth, inhibitor is discussed.     

Inhibition of dimethyl ether and methane oxidation in Methylococcus capsulatus and Methylosinus trichosporium.

Metal-chelating or -binding agents inhibited the oxidation of dimethyl ether and methane, but not methanol, by cell suspensions of Methylococcus capsulatus and Methylosinus trichosporium. Evidence suggests that the involvement of metal-containing enzymatic systems in the initial step of oxidation of dimethyl ether and methane.     

The fine structure of Methylococcus capsulatus

The fine structure of Methylococcus capsulatus is described. Particular emphasis is focused on the intracytoplasmic membrane system which is organized as a stacked array of flattened saccules. Each saccule is limited by a 75 A unit membrane and lies in close apposition to adjacent saccules. Methylococcus capsulatus is an obligate methylotroph whose sole source of carbon and energy is methane (or methanol). In this study methane oxidation is demonstrated for the first time in a cell-free system. Work is in progress to determine the cellular organelles which constitute the particulate fraction responsible for methane oxidation. The possible role of the intracytoplasmic membranes in energy transfer is considered in relation to the functions of stacked membrane arrays in other animal, plant and bacterial systems.     

Oxidation of ultrafast radical clock substrate probes by the soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.     

Reduction of RNA and DNA in Methylococcus capsulatus by endogenous nucleases

 A method for reducing RNA and DNA in the bacterium Methylococcus capsulatus (Bath) has been developed. Endogenous RNase and DNase were activated by a 10 s heat shock at 90°C. Cells were then incubated at 60°C for 20 min to allow degradation of the nucleic acids. The optimum pH for the process was 7.0. The protein loss was less than 10% and occurred during the initial heat shock. No protein loss was found during incubation. The total dry-weight loss in connection with an 80% reduction of the nucleic acid content was 20%–25%, giving a final product with a raw protein content of approximately 75%. Reduction of both RNA and DNA was inhibited by CuSO₄ and ZnSO₄. DNA reduction was stimulated by other minerals. Optimal stimulation was found at 1 mM FeSO₄. Reduction of RNA was not increased by any of the minerals tested. Received: 29 June 1995/Received last revision: 2 October 1995/Accepted: 16 October 1995     

Effect of fermentation conditions on N2 fixation by Methylococcus capsulatus

Nitrogen fixation has been investigated during chemostat fermentations with a culture of Methylococcus capsulatus with natural gas. It is demonstrated that nitrogen fixation occurs under conditions when either nitrate or ammonia as nitrogen source is insufficient for the growth on fixed supply of methane and oxygen. The fixation occurs contrary to expectations within a wide range of dilution rates and with variation of concentration of liquid source of nitrogen. An O₂ optimum is determined for the nitrogenase system of the culture in an assay. During fermentation a complete abolishment of nitrogenase reaction is attained at 15% air saturation (dissolved oxygen). Conditions for N₂ fixation is unaltered with change of pH from 6.8 to 5.7.     

Oxidation of C1 compounds by particulate fractions from Methylococcus capsulatus: properties of methanol oxidase and methanol dehydrogenase.

Methanol (and formaldehyde) oxidizing activities in crude extracts of Methylococcus capsulatus are associated mainly with particulate fractions sedimenting between 3,000 and 40,000 X g. Most of the phenazine methosulfate (PMS)-dependent methanol (and formaldehyde) dehydrogenase activity observed resides in the soluble fraction but represents only 40% of the total (PMS dependent plus independent) activity. Both PMS-dependent methanol dehydrogenase activity and PMS-independent methanol oxidase activity are found in particulate fractions, and the PMS-dependent dehydrogenase is easily solubilized by treatment with certain phospholipases or detergents. The properties of the PMS-dependent dehydrogenase activities in the soluble fraction and that solubilized from the particles suggested that they may be identical proteins. Their pH optima, temperature dependence, thermolabilities, and sensitivities to the presence of specific antisera were indistinguishable. Homogeneous preparations of the enzyme proteins obtained from the soluble fractions of extracts and the particulate fractions solubilized by detergents had similar: (i) electrophoretic mobilities in native and denatured states (subunit size in sodium dodecyl sulfate 62,000 daltons); (ii) molecular radii under native conditions, (iii) visible absorption spectra, lambdamax 350 nm, (iv) kinetic constants for methanol and formaldehyde; (v) substrate specificity; and (vi) immunological characteristics--antisera to each enzyme preparation showed precipitin lines of identity to either of the enzymes. It is suggested that the major site of methanol and formaldehyde oxidation in M. capsulatus occurs on the intracytoplasmic membranes in vivo and is coupled to oxygen reduction.     

Differential Inhibition by Allylsulfide of Nitrification and Methane Oxidation in Freshwater Sediment

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.     

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 - NO₂^- nitrite - NH₂OH hydroxylamine     

Outer membrane proteins of Methylococcus capsulatus (Bath)

Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of β-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl. Received: 20 December 1996 / Accepted: 11 April 1997     

Spatial Distribution and Inhibition by Ammonium of Methane Oxidation in Intertidal Freshwater Marshes

In two intertidal marshes, the vertical distribution in the sediment and inhibition by ammonium of methane oxidation were investigated by slurry incubation experiments. The two sites differ in their dominant vegetation type, i.e., reed and bulrush, and in their heights above sea level. The reed site was elevated with respect to the bulrush site, resulting in a lower frequency and duration of flooding and, consequently, a higher potential for methane oxidation. Methane oxidation decreased with depth in the bulrush and reed slurries, although methane oxidation associated with root material from the bulrush plants increased with depth. Reed root material had a limited capacity for methane oxidation and showed no significant increase with depth. Inhibition of methane oxidation by ammonium was observed in all samples and depended on methane and ammonium concentrations. Increasing ammonium concentrations resulted in greater inhibition, and increasing methane concentrations resulted in less. Ammonium concentrations had to exceed methane concentrations by at least 30-fold to become effective for inhibition. This ratio was found only in the surface layer of the sediment. Hence, the ecological relevance for ammonium inhibition of methane oxidation in intertidal marshes is rather limited and is restricted to the surface layer. Nitrate production was restricted to the 0- to 5-cm-depth slurries.     

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.