INTRODUCTION

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Methane monooxygenase (MMO) catalyzes the initial attack on methane by methanotrophic bacteria. This task is sufficiently difficult that nature tolerates an unusual lack of specificity in the substrates that MMO oxygenates; MMO can also initiate the aerobic (cometabolic) transformation of a variety of haloorganic compounds, including trichloroethylene, chloroform, dichloromethane, dichloroethane, trichloroethane, trifluoroethylene, and tribromoethylene among others (6, 9, 17).

Some species (type II species, some type X species, and recently a type I methanotroph) produce both a soluble MMO (sMMO) and a particulate (membrane-associated) MMO (pMMO) (1, 14, 24). Even a minuscule amount of available Cu(II) (0.85 to 1.0 µmol/g [dry weight] of cells) selects for pMMO at the expense of sMMO expression (13). Significantly, sMMO is better suited for catalyzing cometabolic transformations than pMMO is (5, 19, 24).

Copper is thought to be, along with iron, part of the catalytic site of pMMO from Methylococcus capsulatus (Bath) (10). Active pMMO complexes contain as many as 14.5 copper atoms per 99-kDa enzyme complex (25). Most of the copper, however, is loosely bound and may perform secondary functions, such as enzyme stabilization, copper storage, or maintenance of a specific redox state. At Cu concentrations ranging from 1 to 20 µM, the levels of membrane-associated Cu and Fe and the specific activity of pMMO are directly related to the external copper concentration (25).

Because copper is found in many polluted environments (8), capable organisms are frequently unable to produce sMMO when they are grown under conditions relevant to in situ bioremediation. A recent investigation by Bowman et al. (2) of a trichloroethylene-contaminated aquifer showed that methane injection stimulated growth of indigenous sMMO-producing methanotroph populations. However, the sMMO activity was 41 to 67% lower in the polluted groundwater (with aqueous-phase copper levels of less than 1 µM) than in copper-free, nitrate-salts media (NSM).

Copper has a central regulatory role in Methylosinus trichosporium OB3b, affecting the synthesis of sMMO and pMMO, membrane organization, and the growth rate (11, 15, 18, 23). To overcome the problem of sMMO suppression, Phelps et al. (21) developed mutant strains (sMMO^c) that grew well and expressed sMMO in the presence of 12 µM copper. These mutants exhibited no pMMO activity and lacked characteristic cytoplasmic membrane structures that are normally present when cells are grown in copper-sufficient media. Fitch et al. (7) reported that sMMO^c mutants solubilized extracellular copper but were not capable of copper assimilation.

Here we describe experiments designed to illuminate physiological aspects of the external, copper-binding ligands (CBLs) produced by Methylosinus trichosporium wild-type strain OB3b and by strain PP358, an sMMO^c mutant. Our results provide information about the physiology of MMO selection in sMMO-producing methanotrophs and the nature of mutations that are responsible for stable sMMO^c phenotypes.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. Wild-type Methylosinus trichosporium OB3b (= ATCC 35070), designated RTR, and sMMO^C mutant PP358 (= ATCC 55315) were provided by G. Georgiou of the University of Texas at Austin. The mutant strain was described by Phelps et al. (21) and Fitch et al. (7).

sMMO activity. Culture sMMO activity was examined by using the naphthalene colorimetric assay of Brusseau et al. (3). To obtain a quantitative estimate of sMMO activity, several 0.5-ml aliquots of a cell-formate suspension were mixed with 0.5 ml of the saturated naphthalene preparation and incubated at 30°C. These preparations were sacrificed at 10-min intervals and used to develop and measure color at 528 nm. A₅₂₈ values were converted to concentrations of the naphthol-azo dye by using an assumed extinction coefficient of 38,000 M¹ cm¹ (3).

Analytical techniques. In all experiments, growth was monitored by measuring light scattering at a wavelength of 600 nm (A₆₀₀; 1-cm cuvette) with a Shimadzu model UV160U recording spectrophotometer.

Paper chromatography. Ascending paper chromatography was performed as described by Smith and Seakins (22) with a solvent mixture consisting of acetic acid, n-butanol, and water (3:12:5, by volume). Fractions of the size exclusion HPLC (SE-HPLC) peak eluting near 25 ml (see below) were pooled for analysis. A 5-µl aliquot was applied to the chromatographic paper (8 by 20 cm; type 3MM; Whatman) and dried with a hair drier at the lowest setting. In order to increase the amount of sample applied to the paper, the application procedure was repeated 10 times. Chamber equilibration with the solvent occurred overnight. The prepared paper was then introduced into the solvent chamber to initiate the separation. Runs were terminated after 4 h. After the chromatogram was removed from the chamber, it was dried at 100°C in an oven for 10 min and sprayed with a 5-mg/ml solution of ninhydrin (Sigma) in acetone for staining. Chromatographs were analyzed under visible and/or UV light.

SE-HPLC. Normally, samples were obtained for SE-HPLC directly from cell cultures, as described previously. Alternatively, lyophilized filtrate was suspended in Tris buffer (0.02 M Tris [Sigma], 0.1 M NaCl; pH 7.5) at a concentration of 30 mg/ml for use in SE-HPLC. The solution was passed through a type SE-100 17-µm column (30 by 1 cm; Innovata, Sweden). A 200-µl sample loop was used for all SE-HPLC experiments. Cu(II) (5 µl of a 10 mM CuSO₄ stock solution) was added to some samples to mark fractions with a high affinity for copper and to establish the binding capacity of the ligand present. All samples were eluted at a flow rate of 0.4 ml/min by using the same Tris buffer. Fractions (0.5 ml) were collected and assayed spectrophotometrically (A₂₈₀). The copper was measured in each sample before and after spiking with CuSO₄ and in eluent fractions.

Molecular weight determination. The molecular weight standards used included bovine serum albumin (molecular weight, 67,000), ovalbumin (43,000), RNase A (13,700), horse heart cytochrome c (12,400), eledoisin-related peptide (700), and tryptophan (204) (Sigma). Urea (3 M) was added to the Tris buffer to eliminate potential hydrophobic interactions between solutes and the SE-100 HPLC column material. The elution volume was then correlated with the molecular weight of the standards by performing a linear regression analysis.

Characterization of CBL binding strength. In competition experiments, the biochelator (CBL) was mixed with copper and a chelator of known affinity and binding strength for copper. The chelators used (which were obtained from Sigma) included triethylenetetraamine (TRIEN), EDTA, nitrilotriacetic acid (NTA), ethylenediamine (EN), diethylenetriamine (DEN), iminodiacetic acid (IDA), and tryptophan. In order to isolate CBL for competition experiments, lyophilized cell filtrate from PP358 cultures grown in medium containing 5 µM copper was prepared for SE-HPLC as described previously. Fractions of the peak eluting near 25 ml (designated the peak of interest [POI]) were pooled, and 120 µl of the combined fractions was mixed with 70 µl of SE-HPLC Tris buffer and 50 µl of a 50 µM stock solution of a second (competing) copper chelator prepared in SE-HPLC Tris buffer. The molar concentration of the competing ligand was approximately equal to the concentration of copper and, as such, approximately equal to the copper binding capacity of the POI added in these blends. The resulting mixture was stored at room temperature for 24 h before components were again separated by SE-HPLC. The copper in the eluted fractions was measured to establish the distribution of this element in the biochelator and the competing ligand. Additional analyses were performed by using DEN mixed with pooled fractions of the POI at three DEN/Cu molar ratios. The amount of POI was kept constant (130 µl from the pooled fractions), and the amount of DEN was varied to produce DEN/Cu molar ratios of 0.5, 1.0, and 2.0. SE-HPLC was again used to separate the mixtures, and the copper in the fractions was measured to determine its distribution in DEN and the CBL.

(1)

(2)

CBL specificity for copper. POI solutions were obtained by pooling fractions from the SE-HPLC 25-ml peak, after the original PP358 sample was spiked with copper as described above. Portions (300 µl) of the resultant solutions were mixed with copper and nickel stock solutions to give final liquid phase metal concentrations of 5 µM (Cu) and 100 µM (Ni). Citrate was added to each mixture to a final concentration of 200 µM to complex excess metal ions not bound to the CBL. We prepared a control solution in which 300 µl of HPLC Tris buffer replaced the POI solution. After storage at room temperature for 24 h (for attainment of equilibrium), the mixtures were separated by SE-HPLC. Copper and nickel analyses were performed to determine which metal ion was bound to the CBL.

CBL-Cu uptake experiments. The CBL-Cu uptake experiments were designed to determine the manner in which Cu affects the production and utilization of the CBL and the nature of the sMMO^C mutation in PP358. Both strain RTR and strain PP358 were grown without copper in 1-liter Erlenmeyer flasks to optical densities (A₆₀₀) of 1.0 to 1.5. Then the cultures were divided, and copper (10 µM in the RTR culture, 5 µM in the PP358 culture) was added to one-half of the reactor contents. In each case the second half of the culture was left undisturbed. Incubation and growth were continued for both fractions. The periodic measurements obtained included measurements of A₆₀₀, sMMO activity (quantitative assay), the POI area (as determined by SE-HPLC), and the aqueous- and particulate-phase Cu concentrations. For Cu measurements, 1.5-ml culture samples were centrifuged for 10 min at 6,600 × g (Eppendorf model 5415 Centrifuge). The concentrate was passed through a 0.22-µm-pore-size type GS filter (Millipore) before the soluble Cu in the filtrate was measured. The cell pellet was resuspended in 1.5 ml of a 10 mM EDTA solution (pH 4.8) and incubated for 10 min with constant agitation. The resultant solution was again centrifuged, and the Cu in the filtered concentrate was measured to determine the amount of EDTA-extractable, particulate Cu. A second pellet was resuspended in 1.5 ml of deionized water to measure the particulate, nonextractable copper. The entire procedure was carried out twice by using two sets of independently grown RTR and PP358 cultures.

Culture and medium transfer experiments. Another set of experiments was designed to see if the PP358 phenotype was related to a defect in the CBL produced by that strain. Our objectives were pursued by transferring (i) wild-type (RTR) cells to cell-free media containing CBL produced by the mutant strain and (ii) PP358 cells to cell-free media including CBL that had previously supported growth of strain RTR.

	RESULTS

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sMMO activity. PP358 cultures expressed sMMO during growth on NSM containing methane with and without 5 µM Cu, as shown by naphthol production from naphthalene. As expected, sMMO activity was absent in RTR cultures containing 5 µM Cu, although sMMO was expressed in RTR cultures without copper. In copper-amended cultures, the ratio of total copper to cell dry weight at the maximum cell density was about 5.8 µmol/g of cell dry weight (CDW), a value that should have prevented sMMO expression in the RTR culture (13).

SE-HPLC. Representative chromatograms derived from the four cultures tested (PP358 and RTR, each with and without 5 µM Cu) are shown in Fig. 1 and 2. Each of the curves was developed without concentrating aqueous-phase constituents by lyophilization. Although the results of replicate experiments are not presented, the chromatograms were reproducible. Copper-complexing agents were detectable at 280 nm, as shown by the coelution of high copper levels and chromatogram peaks. The general features of the chromatograms derived from the mutant cultures grown with and without copper were similar (Fig. 1). In each case, copper was associated primarily with a single peak (the POI) at an elution volume of about 25 ml. The medium blank produced no such peak. Thus, in the presence or absence of external copper, the mutant strain excreted one or more (coeluting) ligands with affinity for copper. The POI was also present in the wild-type cultures with and without copper, but accumulated to only modest levels when 5 µM Cu was provided (Fig. 2). There was evidence of copper accumulation in a second peak at an elution volume of about 12 ml in chromatograms derived from the copper-free RTR culture.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Typical SE-HPLC chromatograms and corresponding copper analysis results for filtrates derived from batch PP358 cultures. The copper concentrations in the original growth media and the culture optical densities at the points of analysis were as follows: 0.25 µM Cu (copper free) and A600 = 1.69 (A); and 5 µM Cu and A600 = 1.06 (B). , copper concentration; , A280. A dashed line indicates an absence of data in a region. Abs., absorbance.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Typical SE-HPLC chromatograms and corresponding copper analysis results for filtrates derived from batch RTR cultures. The copper concentrations in the original growth media and the culture optical densities at the points of analysis were as follows: 0.25 µM Cu (copper free) and A600 = 1.15 (A); and 5 µM Cu and A600 = 1.12 (B). , copper concentration; , A280. A dashed line indicates an absence of data in a region. Abs., absorbance.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Growth (A600) and relative POI area (obtained from HPLC chromatograms) as a function of time in a PP358 batch culture grown without copper. Symbols: , culture A600; , relative POI area. (Inset) Relative POI area () as a function of the first moment of bacterial growth (area under bacterial growth curve). The relative POI area is the area of the SE-HPLC peak at a 25-ml elution volume divided by a representative peak area derived from the PP358 cultures (A600 = 1.06; 5 µM Cu). The line is a manual fit to the data. The experiment was repeated, and the same qualitative results were obtained. Abs, absorbance.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Growth (A600) and relative POI area (obtained from HPLC chromatograms) as a function of time in an RTR batch culture grown with 5 µM Cu. Symbols: , culture A600; , relative POI area. (Inset) Relative POI area () as a function of the first moment of bacterial growth (area under bacterial growth curve). The relative POI area is the area of the SE-HPLC peak at a 25-ml elution volume divided by a representative peak area derived from the PP358 cultures (A600 = 1.06; 5 µM Cu). The line is a manual fit to the data. The experiment was repeated, and the same qualitative results were obtained. Abs, absorbance.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   SE-HPLC chromatograms developed from fractions that comprised the POI at 25 ml (PP358 culture grown with 5 µM Cu). - - -, SE-HPLC obtained with Tris as the elution buffer; , SE-HPLC obtained with Tris-3 M urea as the elution buffer; , copper concentration in fractions derived from Tris eluent containing no urea.

Qualitative analysis of CBL functional groups. POI compounds, including the CBL, failed to produce color with chromogenic reagents designed to react with sugars, histidine, tryptophan, and phenols. In the UV range, pooled fractions from the SE-HPLC peak at 25 ml showed a peak at about 270 nm. There were no other distinguishing spectral features.

Molecular mass determination. The POI elution time in Tris eluent containing 3 M urea suggested that molecules contributing to the POI have a molecular mass of about 500 Da (Fig. 5 and 6). The peaks at elution volumes of 13 to 14 ml (Fig. 5) corresponded approximately to the molecular masses of POI multimers.

View larger version (44K): [in this window] [in a new window]   FIG. 6.   SE-HPLC volumes for elution of standards having known molecular weights. The eluent consisted of Tris buffer plus 3 M urea. The calibration curve was used to estimate the molecular weight of the CBL, as shown. RNS A, RNase A; BSA, bovine serum albumin.

Characterization of CBL binding strength. TRIEN and EDTA had greater affinity for the free cupric ion than the external chelator produced by Methylosinus trichosporium OB3b did (Table 2). Conversely, NTA, EN, tryptophan, and IDA bound copper less effectively than the CBL. The association constant for the CBL-Cu complex apparently is between the association constants for NTA (log K₁ = 13.1) (20) and EDTA (log K₁ = 18.8) and close to the association constant for DEN (log K₁ = 16.0). The residual copper complexed with NTA, tryptophan, and IDA represents a stoichiometric excess (above the capacity of the biochelator). Similarly, the residual copper associated with the POI in experiments involving TRIEN, EDTA, and DEN suggested that the capacity of the competing ligands to bind copper was exceeded slightly in these experiments or that equilibrium was not completely attained over the 24-h incubation period that preceded the separation.

1

CBL specificity for copper. Analyses of HPLC fractions from the POI-Cu-Ni mixture revealed that copper remained bound to the CBL even when the total nickel concentration was 20 times the total copper concentration (data not shown); that is, the complexation of copper with a ligand(s) that comprised the POI was not affected by excess nickel. Furthermore, under the conditions used, the distribution of Ni in chromatograms was not affected by the presence of the biochelator.

CBL disappearance and copper uptake: copper mass balance. The effect of adding copper to an RTR culture that was grown under copper-free conditions is illustrated by the results of a comparison of copper-amended and unamended fractions (Fig. 7). In the unamended (copper-free) culture, CBL accumulated in the medium to levels that were similar to those observed previously (Table 1). As expected, the sMMO activity in the unamended fraction was not affected by splitting the culture. Conversely, the sMMO activity in the Cu-amended, wild-type culture decreased dramatically after copper was added, and the POI concentration decreased until the POI was virtually undetectable. Growth was slightly faster in the Cu-amended fraction.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Response of RTR batch cultures grown without copper to the addition of 5 µM copper. (A) Growth of cultures with and without copper. (B) sMMO activity (as determined by the naphthalene assay). (C) Copper and CBL accumulation and uptake. The original culture was split, and copper was added to one aliquot at the point shown. Symbols: , culture to which copper was added; , copper-free culture. The lines represent manual fits to the data. The entire experiment was carried out two times, and the same qualitative results were obtained. The relative POI area is the area of the SE-HPLC peak at a 25-ml elution volume divided by a representative peak area derived from the PP358 cultures (A600 = 1.06; 5 µM Cu). Abs, absorbance.

Culture-medium transfer experiments. RTR cells grown in copper-free NSM were washed and resuspended in cell-free media derived from the PP358 culture containing 5 µM Cu. Under these conditions, extracellular copper was taken up by the wild-type organism, and the extracellular CBL concentration declined proportionally (Table 4). Furthermore, the sMMO activity in copper-starved wild-type cells decreased immediately with no interruption of growth.

	DISCUSSION

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Our observations are consistent with the following speculative picture. Both strain RTR and mutant derivatives of Methylosinus trichosporium OB3b excrete an extracellular CBL in the presence and absence of copper. Wild-type cells recover CBL and internalize copper only when copper is bound by the CBL. Cells with the sMMO^c mutant phenotype cannot reacquire the CBL or copper. Our results suggest that the CBL plays a role in copper assimilation by the wild-type organism.

Several lines of evidence (data from sequential SE-HPLC separations and urea addition experiments, changes in ionic strength, data from ligand competition experiments and paper chromatography) suggest that a single primary compound was responsible for the SE-HPLC peak at an elution volume of 25 ml. There is reason to suspect that ligands produced by strains RTR and PP358 are functionally, if not structurally, identical. CBLs from the mutant and wild-type organisms both elute at 25 ml during SE-HPLC separations. Both readily bind copper, both promote copper uptake by wild-type cells, and neither can supply copper to the mutant.

In general, our findings support hypotheses concerning the origin of the sMMO^c phenotype that were proposed by Fitch et al. (7) (i.e., that the mutant phenotype resulted from a defect in the mechanism of copper acquisition by Methylosinus trichosporium OB3b). Unlike wild-type cells, PP358 was Cu starved in the presence or absence of an external source of copper. It is evident that the mutant phenotype arises from the cells' inability to take up complexed copper, perhaps due to the absence of or a defect in a membrane-bound receptor protein, but certainly not due to a defect in the CBL.

During growth of the wild-type culture without copper and in cultures of PP358 under all copper conditions investigated, the rate of CBL accumulation was about proportional to cell density (Fig. 3). Our results suggest that the ligand was produced at a rate that was proportional to cell number and that reinternalization of CBL was negligible under the growth conditions used. The early stabilization of the CBL concentration in the wild-type culture supplemented with copper (Fig. 4) suggests that CBL production was matched by uptake during exponential growth. Assuming that the intracellular copper concentration is about 1 µmol of Cu per g of CDW in Methylosinus trichosporium OB3b (under copper-sufficient conditions), the calculated rate of CBL production and copper acquisition is about 500 molecules · (cell · min)¹. This value is based on an apparent doubling time of 20 h and a conversion factor of 10¹² cells per g of CDW and is of the same order of magnitude as the calculated specific rate of CBL accumulation in the mutant culture (Fig. 3). The calculation and comparison suggest that CBL is produced at a rate that nearly balances the cells' need for copper under maximum-growth conditions and accounts for the lack of CBL accumulation in wild-type cultures grown with copper.

The presence of a peak near 280 nm in the POI UV spectrum suggests that the biochelator has at least one aromatic functional group. The results of ninhydrin chromogenic reactions are not conclusive with respect to the peptidic nature of the biochelator. The other qualitative tests performed indicated that histidine, tryptophan, and phenol derivatives do not contribute to the CBL structure.

Unlike other UV-absorbing peaks in Fig. 1 and 2, elution of the CBL was delayed significantly by hydrophobic interactions with the size exclusion gel. Higher eluent ionic strength (concentration range, 0.05 to 0.25 M) increased the elution volume of the POI but did not significantly modify the elution pattern of other components in the mixture (data not shown). Addition of 3 M urea to the elution buffer accelerated the appearance of the POI (Fig. 5), suggesting that hydrophobic interactions significantly retarded transport of the Cu-ligand complex. Urea is commonly added to elution buffers to eliminate hydrophobic interactions between the gel matrix and the analytes to be separated.

Although the effectiveness of CBL for copper detoxification has not been tested, the strength of copper complexation by the isolated ligand suggests that copper acquisition is its primary function. A weaker ligand might suffice for metal detoxification. Moffett and Brand (16) reported that the binding constant for complexes involving Cu and a biochelator produced by a marine bacterium in response to copper stress is between 10¹² and 10¹³ M¹. Our results indicate that the binding constant for the Cu-CBL complex is greater by 3 to 4 orders of magnitude.

A detailed mechanistic explanation for the role of the CBL in copper acquisition remains to be established. Our observations could be explained, for example, by uptake of the copper-CBL complex, by degradation of CBL (accompanied by copper uptake), or by sudden changes in the balance of uptake and excretion. Hypotheses in this area remain speculation. When complete, the emerging physiological picture of copper acquisition by Methylosinus trichosporium OB3b may enable us to promote bioremediation efforts that are now inhibited by copper-mediated pMMO selection. Experiments designed to better establish biochelator function in Methylosinus trichosporium OB3b are in progress.

In summary, we identified and partially characterized a biochelator that accumulates in mutant cultures (sMMO^c, PP358) of Methylosinus trichosporium OB3b in the presence and absence of external copper. The ligand is produced constitutively during periods of growth by both mutant and wild-type cultures. The wild-type cells are able to utilize the CBL when external copper is present. However, under no circumstances does the mutant reacquire the biochelator. The mutation in PP358 seems to be unrelated to possible defects in the CBL since CBL produced by PP358 was readily utilized by a growing RTR culture. Furthermore, CBL produced by strain RTR did not support copper uptake by the mutant. The biochelator is apparently a low-molecular-weight, hydrophobic molecule with high affinity and selectivity for copper. It seems to have some aromatic character. We hypothesize that the biochelator is part of the normal mechanism of copper uptake by Methylosinus trichosporium OB3b. Metal detoxification remains an alternative, although much less likely, physiological explanation for ligand production.