Characterization of recombinant PPi-dependent 6-phosphofructokinases from <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b and <Emphasis Type="Italic">Methylobacterium nodulans</Emphasis> ORS 2060

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His₆-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.     

Immobilization of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b for methanol production

Due to the natural gas boom in North America, there is renewed interest in the production of other chemical products from methane. We investigated the feasibility of immobilizing the obligate methanotrophic bacterium Methylosinus trichosporium OB3b in alginate beads, and selectively inactivating methanol dehydrogenase (MDH) with cyclopropane to produce methanol. In batch cultures and in semi-continuous flow columns, the exposure of alginate-immobilized cells to cyclopropane or cyclopropanol resulted in the loss of the majority of MDH activity (> 80%), allowing methanol to accumulate to significant concentrations while retaining all of M. trichosporium OB3b’s methane monooxygenase capacity. Thereafter, the efficiency of methanol production fell due to recovery of most of the MDH activity; however, subsequent inhibition periods resulted in renewed methanol production efficiency, and immobilized cells retained methane-oxidizing activity for at least 14 days.     

Factors affecting competition between type I and type II methanotrophs in two-organism,continuous-flow reactors

Competition experiments were performed in a continuous-flow reactor using Methylosinus trichosporium OB3b, a type II methanotroph, and Methylomonas albus BG8, a type I methanotroph. The experiments were designed to establish conditions under which type II methanotrophs, which have significant cometabolic potential, prevail over type I strains. The primary determinants of species selection were dissolved methane, copper, and nitrate concentrations. Dissolved oxygen and methanol concentrations played secondary roles. M. trichosporium OB3b proved dominant under copper and nitratelimited conditions. The ratio of M. trichosporium to M. albus in the reactor increased ten-fold in less than 100 hours following the removal of copper from the reactor feed. Numbers of M. albus declined to levels that were below detection limits (<106/ml) under nitrogen-limited conditions. In the latter experiment, the competitive success of M. trichosporiumdepended on the maintenance of an ambient dissolved oxygen level below about 7.5 × 10^–5 M, or 30% of saturation with air. The ability of M. trichosporium to express soluble methane monooxygenase under copper limitation and nitrogenase under nitrate limitation was very significant. M. albus predominated under methane-limited conditions, especially when low levels of methanol were simultaneously added with methane to the reactor. The results imply that nitrogen limitation can be used to select for type II strains such as M. trichosporium OB3b. Offprint requests to: Pierre Servais     

Methylosinus trichosporium OB3b Mutants Having Constitutive Expression of Soluble Methane Monooxygenase in the Presence of High Levels of Copper

The methanotrophic bacterium Methylosinus trichosporium OB3b is unusually active in degrading recalcitrant haloalkanes such as trichloroethylene (TCE). The first and rate-limiting step in the degradation of TCE is catalyzed by a soluble methane monooxygenase (sMMO). This enzyme is not expressed when the cells are grown in the presence of copper at concentrations typically found in polluted groundwater. Under these conditions, M. trichosporium OB3b expresses a particulate form of the enzyme (pMMO), which has a narrow substrate specificity and does not degrade TCE at any significant rate. We have isolated M. trichosporium OB3b mutants that are deficient in pMMO and express sMMO constitutively in the presence of elevated concentrations of copper. One mutant (PP358) exhibited a TCE degradation rate which was almost twice as high as that of the wild-type strain grown under optimal conditions (without copper). All of the mutants lost the ability to express pMMO activity and to form stacked intracellular membranes characteristic of wild-type cells expressing pMMO.     

Spectral and thermodynamic properties of methanobactin from γ-proteobacterial methane oxidizing bacteria: A case for copper competition on a molecular level

Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.     

Detoxification of Mercury by Methanobactin from Methylosinus trichosporium OB3b

Many methanotrophs have been shown to synthesize methanobactin, a novel biogenic copper-chelating agent or chalkophore. Methanobactin binds copper via two heterocyclic rings with associated enethiol groups. The structure of methanobactin suggests that it can bind other metals, including mercury. Here we report that methanobactin from Methylosinus trichosporium OB3b does indeed bind mercury when added as HgCl₂ and, in doing so, reduced toxicity associated with Hg(II) for both Alphaproteobacteria methanotrophs, including M. trichosporium OB3b, M. trichosporium OB3b ΔmbnA (a mutant defective in methanobactin production), and Methylocystis sp. strain SB2, and a Gammaproteobacteria methanotroph, Methylomicrobium album BG8. Mercury binding by methanobactin was evident in both the presence and absence of copper, despite the fact that methanobactin had a much higher affinity for copper due to the rapid and irreversible binding of mercury by methanobactin. The formation of a gray precipitate suggested that Hg(II), after being bound by methanobactin, was reduced to Hg(0) but was not volatilized. Rather, mercury remained associated with methanobactin and was also found associated with methanotrophic biomass. It thus appears that although the mercury-methanobactin complex was cell associated, mercury was not removed from methanobactin. The amount of biomass-associated mercury in the presence of methanobactin from M. trichosporium OB3b was greatest for M. trichosporium wild-type strain OB3b and the ΔmbnA mutant and least for M. album BG8, suggesting that methanotrophs may have selective methanobactin uptake systems that may be based on TonB-dependent transporters but that such uptake systems exhibit a degree of infidelity.     

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     

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K _iE and K _iES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO. Received: 3 November 1998 / Accepted: 1 March 1999     

Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 ± 27 and 321 ± 16 nmol h^-1 mg of protein ^-1, respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 ± 260 and 995 ± 160 nmol h^-1 mg of protein^-1, respectively. The substrate affinity of 68-1 sMMO to naphthalene (K_m, 70 ± 4 μM) and TCE (K_m, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.     

Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl₂, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl₂, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl₂. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl₂. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.     

Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading Methylomonas sp. Strains and Detection of Methanotrophs during In Situ Bioremediation

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.     

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.     

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.     

Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

The tetrazolium salt sodium 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) was examined for use as a colorimetric indicator of viability in respiring bacteria. XTT was reduced to an orange, water-soluble formazan product by Methylosinus trichosporium OB3b, Pseudomonas putida, Escherichia coli, and Bacillus subtilis. Formazan production was proportional to live cell biomass, and XTT was reduced by all cultures in the absence of added electron-coupling agents. XTT reduction by M. trichosporium OB3b was linear over several hours and was stimulated by the presence of an exogenous substrate (methanol). Addition of cyanide to cultures incubated under oxic conditions gave an initial 10-fold increase in XTT reduction. Viability of bacteria incubated in the absence of exogenous carbon substrates was measured as XTT reduction and compared with viability estimates from plate counts. Results obtained with the two methods were generally comparable, but the XTT assay was superior when cell recovery on plates was low. Incubation of E. coli for 7 days in the absence of exogenous carbon substrates decreased viability by 90%, whereas the corresponding decreases for cultures of M. trichosporium OB3b, P. putida, and B. subtilis were less than 40%.     

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm^r mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm^r mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.     

Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O₂- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)₂ complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.     

Use of allylthiourea to produce soluble methane monooxygenase in the presence of copper

Methanotrophs expressing soluble methane monooxygenase (sMMO) may find use in a variety of industrial applications. However, sMMO expression is strongly inhibited by copper, and the growth rate may be limited by the aqueous solubility of methane. In this study, addition of allylthiourea decreased intracellular copper in Methylosinus trichosporium OB3b, allowing sMMO production at Cu/biomass ratios normally not permitting sMMO synthesis. The presence of about 1.5 μmoles intracellular Cu g⁻¹ dry biomass resulted in sMMO activity of about 250 μmoles 1-napthol formed per hour gram dry biomass whether this intracellular Cu concentration was achieved by Cu limitation or by allylthiourea addition. No loss of sMMO activity occurred when the growth substrate was switched from methane to methanol when allylthiourea had been added to growth medium containing copper. Addition of copper to medium that was almost copper-free increased the yield of dry biomass from methanol from 0.20 to 0.36 g g⁻¹, demonstrating that some copper was necessary for good growth. This study demonstrated a method by which sMMO can be produced by M. trichosporium OB3b while growing on methanol in copper-containing medium.