Effect of Copper Speciation on Whole-Cell Soluble Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

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.     

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     

Production of soluble methane monooxygenase during growth of Methylosinus trichosporium on methanol

Soluble methane monooxygenase (sMMO) can degrade many chlorinated and aromatic pollutants. It is produced by certain methanotrophs such as Methylosinus trichosporium when grown on methane under copper limitation but, due to its low aqueous solubility, methane cannot support dense biomass growth. Since it is water soluble, methanol may be a more attractive growth substrate, but it is widely believed that sMMO is not produced on methanol. In this study, when the growth-limiting substrate was switched from methane to methanol, in the presence of the particulate MMO inhibitor, allylthiourea, growth of M. trichosporium OB3b continued unabated and sMMO activity was completely retained. When allylthiourea was then removed, sMMO activity was maintained for an additional 24 generations, albeit at a slightly lower level due to the presence of 0.70 microM of Cu(2+) in the feed medium. While a biomass density of only 2 g l(-1) could be obtained on methane, 7.4 g l(-1) was achieved by feeding methanol exponentially, and 29 g l(-1) was obtained using a modified feeding strategy employing on-line carbon dioxide production measurement. It was concluded that methanol can be employed to produce large amounts of M. trichosporium biomass containing sMMO.     

Phenotypic characterization of copper-resistant mutants of Methylosinus trichosporium OB3b.

Cultures of Methylosinus trichosporium OB3b grown in the presence of very low concentrations of copper synthesize a soluble methane monooxygenase (sMMO) that efficiently catalyzes the oxidation of trichloroethylene and other organic pollutants. Recently, we isolated five M. trichosporium OB3b mutants that express sMMO activity when grown in the presence of elevated copper concentrations (P.A. Phelps, S. K. Agarwal, G. E. Speitel, Jr., and G. Georgiou, Appl. Environ. Microbiol. 58:3701-3708, 1992). Here we show that, in contrast to the results for the wild-type cells, the addition of copper to mutant cultures grown on methane and nitrate as the nitrogen source has no noticeable effect on the growth rate and sMMO expression. In vitro experiments indicated that the copper-resistant phenotype does not arise from an increased stability of sMMO to copper deactivation. Furthermore, the mutant cultures exhibit altered speciation of copper in the extracellular fluid and have substantially decreased levels of cell-associated copper. On the basis of these results, we propose that the mutant phenotype arises from defects in copper uptake and metabolism rather than from changes in sMMO expression or enzyme stability.     

Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.     

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 muM and remained constant upon further increases of the copper concentration to 120 muM. The cell growth rate (mu), on the other hand, decreased over the entire range (10-120 muM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 muM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.     

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.     

Residues near the N-terminus of protein B control autocatalytic proteolysis and the activity of soluble methane mono-oxygenase.

Soluble methane mono-oxygenase (sMMO) of Methylococcus capsulatus (Bath) catalyses the O2-dependent and NAD(P)H-dependent oxygenation of methane and numerous other substrates. During purification, the sMMO enzyme complex, which comprises three components and has a molecular mass in excess of 300 kDa, becomes inactivated because of cleavage of just 12 amino acids from the N-terminus of protein B, which is the smallest component of sMMO and the only one without prosthetic groups. Here we have shown that cleavage of protein B, to form the inactive truncated protein B', continued to occur when intact protein B was repeatedly separated from protein B' and all detectable contaminants, giving compelling evidence that the protein was cleaved autocatalytically. The rate of autocatalytic cleavage decreased when the residues flanking the cleavage site were mutated, but the position of cleavage was unaltered. Analysis of a series of incremental truncates showed that residue(s) essential for the activity of sMMO, and important in determining the stability of protein B, lay in the region Ser4-Tyr7. Protein B was shown to possess intrinsic nucleophilic activity, which we propose initiates the cleavage reaction via a novel mechanism. Proteins B and B' were detected in approximately equal amounts in the cell, showing that truncation of protein B is biologically relevant. Increasing the growth-medium copper concentration, which inactivates sMMO, did not alter the extent of in vivo cleavage, therefore the conditions under which cleavage of protein B may fulfil its proposed role as a regulator of sMMO remain to be identified.     

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.     

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.     

Batch cultivation of Methylosinus trichosporium OB3B: IV. Production of hydrogen-driven soluble or particulate methane monooxygenase activity

Batch culture conditions were established for the formation of H(2)-driven whole-cell soluble or particulate methane monooxygenase (sMMO or pMMO) activity in the obligate methanotroph, Methylosinus trichosporum Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H(2) to a nitrate-containing minimal salts growth medium or Ni and Mo to a nitrate-lacking growth medium (induces a nitrogenase that generates intracellular H(2)) markedly enhanced both the hydrogenase and the accompanying washed-cell H(2)-driven MMO activities of shake-flask cultured cells. For sMMO containing cells, H(2) provided in vitro reducing power for the oxidation of chlorinated solvents such as chloroform and trichloroethylene. Cell cultivations under N(2)-fixing conditions in a 5-L bioreactor, however, required an initial nitrate concentration of at least 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H(2)-driven sMMO or pMMO activity. Elevation of the initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalent growth yield. High nitrate also shortened the culture time for sMMO containing cells by approximately 25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting-state cells retained 90% and 70% of their H(2)-driven sMMO and pMMO activity, respectively. This makes their practical use quite feasible. (c) 1995 John Wiley & Sons, Inc.     

Biochemical characterization of MmoS, a sensor protein involved in copper-dependent regulation of soluble methane monooxygenase

Methane monooxygenase (MMO) enzymes catalyze the oxidation of methane to methanol in methanotrophic bacteria. Several strains of methanotrophs, including Methylococcus capsulatus (Bath), express a membrane-bound or particulate MMO (pMMO) at high copper-to-biomass ratios and a soluble MMO (sMMO) form when copper is limited. The mechanism of this "copper switch" is not understood. The mmoS gene, located downstream of the sMMO operon, encodes a sensor protein that is part of a two-component signaling system and has been proposed to play a role in the copper switch. MmoS from M. capsulatus (Bath) has been cloned, expressed, and purified. The purified protein is a tetramer of molecular mass 480 kDa. Optical spectra indicate that MmoS contains a flavin cofactor, identified as flavin adenine dinucleotide (FAD) by fluorescence spectroscopy and chromatographic analysis. The redox potential of the MmoS-bound FAD, which binds within the N-terminal PAS-PAC domains, is -290 +/- 2 mV at pH 8.0 and 25 degrees C. Despite extensive efforts, MmoS could not be loaded with Cu(I) or Cu(II), indicating that MmoS does not sense copper directly. These data suggest that MmoS functions as a redox sensor and provide new insight into the copper-mediated regulation of sMMO expression.     

Effects of copper on expression of methane monooxygenases,trichloroethylene degradation,and community structure in methanotrophic consortia

Copper plays a key role in regulating the expression of enzymes that promote biodegradation of contaminants in methanotrophic consortia (MC). Here, we utilized MC isolated from landfill cover to investigate cometabolic degradation of trichloroethylene (TCE) at nine different copper (Cu²⁺) concentrations. The results demonstrated that an increase in Cu²⁺ concentration from 0 to 15 μM altered the specific first‐order rate constant k_1,TCE, the expression levels of methane monooxygenase (pmoA and mmoX) genes, and the specific activity of soluble methane monooxygenase (sMMO). High efficiency TCE degradation (95%) and the expression levels of methane monooxygenase (MMO) were detected at a Cu²⁺ concentration of 0.03 μM. Notably, sMMO‐specific activity ranged from 74.41 nmol/(mg_cell h) in 15 μM Cu²⁺ to 654.99 nmol/(mg_cell h) in 0.03 μM Cu²⁺, which contrasts with cultures of pure methanotrophs in which sMMO activity is depressed at high Cu²⁺ concentrations, indicating a special regulatory role for Cu²⁺ in MC. The results of MiSeq pyrosequencing indicated that higher Cu²⁺ concentrations stimulated the growth of methanotrophic microorganisms in MC. These findings have important implications for the elucidation of copper‐mediated regulatory mechanisms in MC.     

TrzN from Arthrobacter aurescens TC1 Is a zinc amidohydrolase

TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce multimilligram quantities of functionally folded, recombinant TrzN. The TrzN obtained from Escherichia coli (trzN) cells cultured with increasing zinc in the growth medium showed corresponding increases in specific activity, and enzyme obtained from cells grown with 500 muM zinc showed maximum activity. Recombinant TrzN contained 1 mole of Zn per mole of TrzN subunit. Maximally active TrzN was not affected by supplementation with most metals nor by EDTA, consistent with previous observations (E. Topp, W. M. Mulbry, H. Zhu, S. M. Nour, and D. Cuppels, Appl. Environ. Microbiol. 66:3134-3141, 2000) which had led to the conclusion that TrzN is not a metalloenzyme. Fully active native TrzN showed a loss of greater than 90% of enzyme activity and bound zinc when treated with the metal chelator 8-hydroxyquinoline-5-sulfonic acid. While exogenously added zinc or cobalt restored activity to metal-depleted TrzN, cobalt supported lower activity than did zinc. Iron, manganese, nickel, and copper did not support TrzN activity. Both Zn- and Co-TrzN showed different relative activities with different s-triazine substrates. Co-TrzN showed a visible absorption spectrum characteristic of other members of the amidohydrolase superfamily replaced with cobalt.     

Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus.

The nickel-dependent chemolithoautotrophic growth of Alcaligenes eutrophus is apparently due to a requirement of nickel for active hydrogenase formation. Cells grown heterotrophically with fructose and glycerol revealed a specific activity of soluble and membrane-bound hydrogenase which was severalfold higher than the normal autotrophic level. The omission of nickel from the medium did not affect heterotrophic growth, but the soluble hydrogenase activity was reduced significantly. In the presence of ethylenediaminetetraacetic acid (EDTA), almost no hydrogenase activity was detected. The addition of nickel allowed active hydrogenase formation even when EDTA was present. When chloramphenicol was added simultaneously with nickel to an EDTA-containing medium, almost no hydrogenase activity was found. This indicates that nickel ions are involved in a process which requires protein synthesis and not the direct reactivation of a preformed inactive protein. The formation of the membrane-bound hydrogenase also appeared to be nickel dependent. Autotrophic CO2 assimilation did not specifically require nickel ions, since formate was utilized in the presence of EDTA and the activity of ribulosebisphosphate carboxylase was not affected under these conditions.