Copper-Binding Compounds from Methylosinus trichosporium OB3b

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO^C) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L₁ and CBC-L₂, respectively. The amino acid sequence of CBC-L₁ is S?MYPGS?M, and that of CBC-L₂ is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L₁ showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g_⊥ = 2.087, g_∥ = 2.42 G, |A_∥| = 128 G). The EPR spectrum of CBC-L₂ was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu²⁺ center (g_⊥ = 2.073, g_∥ = 2.324 G, |A_∥| = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g_⊥ = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMO^C mutants PP319 and PP359 regardless of the copper concentration in the culture medium.In methanotrophs, the relationship between the concentration of copper and expression of the two different methane monooxygenases (MMOs) is well characterized (8, 11, 45, 49, 50). Under low copper-to-biomass ratios, methane oxidation activity is observed in the soluble fraction, and the enzyme is referred to as the soluble methane monooxygenase (sMMO). At higher copper-to-biomass ratios, methane oxidation activity is observed in the membrane fraction, and the enzyme is referred to as the membrane-associated or particulate methane monooxygenase (pMMO). The polypeptides and structural genes for both enzymes have been characterized (4, 18–22, 24, 25, 32, 34–40, 43–45, 47–49, 51, 62, 63). In addition to expression of the two MMOs, four other physiological traits have been identified in cells expressing the pMMO that are affected by the copper concentration in the culture medium. First, the concentration of copper in the culture media is directly related to pMMO activity in cell-free fractions, although the levels of expression of pMMO polypeptides vary in different methanotrophs (1, 8, 30, 36, 50, 63). For example, the expression levels of the three pMMO polypeptides in Methylococcus capsulatus Bath remained constant with varying copper concentrations (8, 36), whereas in Methylomicrobium albus BG8, the expression level of the putative pMMO polypeptides increased with increased copper in the culture medium (8). Second, the concentrations of membrane-associated copper and iron show a proportional increase as the copper concentration in the culture medium is increased (36, 63). Third, the formation and level of intracytoplasmic membranes in cells cultured in copper-supplemented media are dependent on the copper concentration in the culture media (8, 11, 40, 48). Lastly, the K_s for methane oxidation by pMMO is altered by the copper concentration in the culture media (33a).Berson and Lidstrom (1) have recently noted that in spite of the central role of copper in the physiology of methanotrophs, the mechanism(s) of copper acquisition remains vague. Although true, a few studies have suggested the existence of a specific copper acquisition system in M. capsulatus Bath and M. trichosporium OB3b. The first indication of a specific copper uptake system was provided from phenotypic characterization of the constitutive sMMO mutants (sMMO^C) isolated by Phelps et al. (42). Fitch et al. (17) found that in M. trichosporium OB3b, these sMMO^C mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Working with the same mutants, Téllez et al. partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4 × 10¹⁶ M⁻¹ (55). Other evidence for a specific copper uptake system was provided by the copper-binding cofactor (CBC) from M. capsulatus Bath (63). During the isolation of the pMMO from M. capsulatus Bath, CBC was identified in association with the purified enzyme, in the washed membrane fraction, and in the extracellular fraction. The CBC was determined to be a small polypeptide with a molecular mass of 1,232 Da. In M. capsulatus Bath, the cellular location of the CBC varied depending on the copper concentration in the culture medium and on the expression of the pMMO.This paper ties together and extends these observations on specific copper acquisition systems in M. trichosporium OB3b and M. capsulatus Bath. Here we describe the initial isolation and characterization of two copper-complexing agents, called CBC-L₁ and CBC-L₂, from the M. trichosporium OB3b wild type and sMMO^C mutant PP319. CBC-L₁ from M. trichosporium OB3b was identical to the CBC previously identified during the isolation of the pMMO from M. capsulatus Bath. This paper is also the first report of a second CBC, CBC-L₂, which may have been present as a contaminant in previous CBC preparations from M. capsulatus Bath. One or both of the CBCs appear to be the same copper-complexing agent partially purified by Téllez et al. (55). Lastly, this report describes the effect of the copper concentration in the culture medium on copper uptake, the expression of both MMOs, and extracellular concentration of the CBC in wild-type and sMMO^C mutant strains of M. trichosporium OB3b.     

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene.     

Degradation of dimethyl nitrosamine by Methylosinus trichosporium OB3b

The degradation of dimethyl nitrosoamine (DMNA) by a methanotroph, Methylosinus trichosporium OB3b, was studied using 14C-labelled DMNA. The organism was capable of assimilating DMNA-carbon and converting it to CO2. The rates of CO2 production (VCO2) from DMNA and its cellular uptake (VP) were linearly correlated with DMNA concentrations of 0.03-10 mM, which corresponded to approximately 3% of added DMNA metabolized in 24 h. These rates were two to three orders of magnitude less than the rate of uptake of methane (VCH4. VCH4 was suppressed when the concentrations of DMNA exceeded 0.3 mM. In the presence of 0.1 mM DMNA, VP and VCO2 were essentially the same in the presence or absence of methane in the first 8 h of incubation, but declined sharply thereafter only when methane was absent. These observations suggest that the metabolism of DMNA was carried out by methane monooxygenase (MMO), and that NADH, a cofactor for MMO, may be provided by the oxidation of the stored compounds in the cells when methane is not available.     

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b.

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene.     

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.     

Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b.

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.     

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.     

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.     

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.     

Growth of Methylosinus trichosporium OB3b on methane and poly-beta-hydroxybutyrate biosynthesis

Optimal conditions for batch cultivation of the obligate methanotroph Methylosinus trichosporium OB3b on methane without superatmospheric pressure were chosen. The yield of absolutely dry biomass after 120 h of growth reached 20 g/l. This biomass contained 30% poly-beta-hydroxybutyrate (PHB) with molecular weight 300 kDa. The growth process included the stages of biomass growth and PHB biosynthesis. The latter stage occurred under nitrogen-deficiency conditions. It was accompanied by an increase in the activity of PHB biosynthesis enzymes (beta-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase) and the main NAD(P)H producer, methylenetetrahydromethanopterin dehydrogenase. The activity of PHB depolymerase increased insignificantly.     

Carbon source regulation of gene expression in Methylosinus trichosporium OB3b

Applied Microbiology and Biotechnology - Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper regulating expression of...     

Cell growth and oxygen transfer in Methylosinus trichosporium OB3b cultures

Summary The steady-state concentration of M. trichosporium OB3b increased about two-fold in the continuous culture when the feeding medium was supplemented by ferrous sulphate (50mg/L) and citric acid (100mg/L) at a steady state. In batch and continuous cultures, the cell growth was significantly inhibited by excess N-sources (NH₄OH, NH₄Cl, NH₄NO₃, HNO₃, and NaNO₃) and ammonium N-sources were more inhibitory. Both volumetric O₂ transfer coefficient and specific O₂ uptake rate increased monotonously in an extensive range of air flow rate (0.1–7 vvm) and the methane interfered with the O₂ transfer even at very low flow rates (0.01–0.1 vvm).     

Trichloroethylene degradation and mineralization by pseudomonads and Methylosinus trichosporium OB3b

 To examine the trichloroethylene (C₂HCl₃)-degrading capability of five microorganisms, the maximum rate, extent, and degree of C₂HCl₃ mineralization were evaluated for Pseudomonas cepacia G4, Pseudomonas cepacia G4 PR1, Pseudomonas mendocina KR1, Pseudomonas putida F1, and Methylosinus trichosporium OB3b using growth conditions commonly reported in the literature for expression of oxygenases responsible for C₂HCl₃ degradation. By varying the C₂HCl₃ concentration from 5 μM to 75 μM, V _max and K _m values for C₂HCl₃ degradation were calculated as 9 nmol/(min mg protein) and 4 μM for P. cepacia G4, 18 nmol/(min mg protein) and 29 μM for P. cepacia G4 PR1, 20 nmol/(min mg protein) and 10 μM for P. mendocina KR1, and 8 nmol/(min mg protein) and 5 μM for P. putida F1. This is the first report of these Michaelis-Menten parameters for P. mendocina KR1, P. putida F1, and P. cepacia G4 PR1. At 75 μM, the extent of C₂HCl₃ that was degraded after 6 h of incubation with resting cells was 61%–98%; the highest degradation being achieved by toluene-induced P. mendocina KR1. The extent of C₂HCl₃ mineralization in 6 h (as indicated by concentration of chloride ion) was also measured and varied from 36% for toluene-induced P. putida F1 to 102% for M. trichosporium OB3b. Since C₂HCl₃ degradation requires new bio-mass, the specific growth rate (μ_max) of each of the C₂HCl₃-degradation microorganisms was determined and varied from 0.080/h (M. trichosporium OB3b) to 0.864/h (P. cepacia G4 PR1). Received: 1 May 1995/Received revision: 11 July 1995/Accepted: 26 July 1995     

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.     

Role of iron in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The effect of iron ions on particulate methane monooxygenase was studied by using the EDTA-treated membranes from Methylosinus trichosporium OB3b. When the membrane was treated with EDTA the activity remained 82% of the as-isolated membranes, and the activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, ferric, ferrous and cupric ions stimulated the activity, indicating those ions were needed for the activity. When propargylamine was added, pMMO activity decreased and also the iron ESR signal decreased. As the ESR signal involves the ferrous nitrosyl complex in EDTA-treated membranes, the active site of pMMO may contain a mononuclear non-heme iron.     

Degradation of Trichloroethylene by Methanol-Grown Cultures of Methylosinus trichosporium OB3b PP358

A soluble methane monooxygenase-constitutive mutant strain of Methylosinus trichosporium OB3b, strain PP358, was grown with methanol as the carbon source, and the kinetics of trichloroethylene (TCE) degradation were determined. PP358 exhibited high TCE degradation rates under both oxygen- and carbon-limiting conditions. The optimal pseudo first-order rate constant for TCE was comparable to the values measured for cells grown with methane. We found that growth under oxygen-limiting conditions results in increased accumulation of polyhydroxybutyrate, which in turn correlates with higher transformation capacities for TCE. It was also shown that methanol inhibits TCE degradation only at high concentrations. Thus, methanol-grown cultures of PP358 represent an efficient system for the biodegradation of chlorinated hydrocarbons.     

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.     

Redox behavior of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

The redox properties of the copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b were investigated. The ESR spectrum of the pMMO-containing membranes from M. trichosporium OB3b indicated a typical type II copper (II) signal (g = 2.24, A = 18.4 mT, g = 2.06, ₂= 0.84). By anaerobic addition of excess amounts of duroquinol, an optimum reductant of pMMO, the ESR spectra indicated that the copper cluster in membranes was reduced and successively oxidized by dioxygen, a substrate of pMMO. The result suggests that the copper is the active site of pMMO or an electron carrier. During the titration, the intensity of the type II copper signal decreased with decreasing potential and the multiple hyperfine structure at g = 2.06 appeared clearly. Although the copper signal did not change by treatment of the EDTA-treated membranes with duroquinol and dioxygen, the copper signal intensity decreased with decreasing potential in the redox titration. These results suggest that some redox mediators play a role as an electron carrier between the active site and a reductant, and the presence of at least two types of copper sites in pMMO- containing membranes. On the basis of the ESR spectra of the EDTA-treated membranes and the as-isolated membranes, it is concluded that one type of the copper sites functions as the active site of pMMO (A-site), and the other type of copper sites plays a role as an electron carrier (E-site)     

Properties of the membranes containing the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was partiallypurified and characterized by measuring the effects of reducing agents and additives, and the stability ofpMMO was studied. Duroquinol was a suitable reducing agent, and pMMO was stabilized by bovine serumalbumin (BSA). Among the additivies, the copper (II) ion stimulated pMMO at low concentration andinhibited at high concentration. The optimum conditions for pMMO activity were as follows: 45 ° C, pH 6.5and 55 mM 3-morpholinopropanesulfonic acid (MOPS) buffer, and the rate of propene epoxide formationwas 13.6 nmol min mg protein. ESR spectra indicate that the copper cluster in the membrane fraction isreduced by duroquinol and oxidized by dioxygen. The result suggests that the copper cluster is containedin the active site of pMMO.