INTRODUCTION

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Methanotrophic bacteria oxidize methane to methanol via the activity of methane monooxygenase (MMO), which, depending on the copper concentration in the cultivation medium, can be found in different fractions. At high copper concentrations, a particulate form of methane monooxygenase (pMMO) with high substrate specificity is expressed. At low copper concentration, the soluble form of the enzyme (sMMO), which possesses a very low substrate specificity and unique properties, is expressed (3). It has been shown that although all methanotrophic bacteria express the pMMO, only some strains, mainly of the type II (34, 39, 43) and type X (6) methanotrophs, are able to express the sMMO. So far, Methylomonas methanica 68-1 (24) and Methylomonas sp. strain GYJ3 (41) are the only type I methanotrophs in which the sMMO has been detected. This nonspecific enzyme oxidizes a variety of hydrocarbons, including aliphatics and aromatics (16), and seems to be highly conserved in its structure (33). Whereas little is known about the structure and mechanism of the pMMO (21, 35), the sMMO has been described and purified from Methylosinus trichosporium OB3b (11), Methylocystis sp. strain M (34), Methylobacterium sp. strain CRL-26 (38), and Methylococcus capsulatus Bath (15). The sMMO from these microorganisms are multicomponent proteins consisting of a hydroxylase, a reductase, and a regulatory protein (protein B, with the exception of the Methylobacterium strain) with similar relative molecular masses.

It was shown that the activity of the hydroxylase component can also be measured in the absence of the other components, with hydrogen peroxide as the oxygen and hydrogen donor (23, 27). The reductase is able to transfer electrons to an acceptor such as dichlorophenolindophenol or cytochrome c (5). In contrast, the activity of the third component, the regulatory protein B, can be measured only if the other components are present.

Methanotrophs able to express the sMMO are of special interest in bioremediation processes due to the extremely high oxidation potential of this enzyme system. Several research groups have studied the distribution of sMMO-possessing strains in various environments by using gene-specific probes (30, 32, 45). The sMMO from newly isolated microorganisms appear to be very highly conserved at the nucleotide sequence level compared to the well-studied sMMO from M. trichosporium and M. capsulatus (31).

The strain studied, WI 14, was isolated from swine manure and was classified as a type II methanotrophic bacterium (19). Originally, it was thought to belong to the genus Methylosinus (20), but subsequent partial sequencing of the 16S rDNA, which can be used for phylogenetic analysis (13, 14), grouped it within the genus Methylocystis (unpublished data).

In this study we describe the expression, purification, and characterization of the sMMO and the complete 16S rDNA and sMMO sequencing of isolate WI 14.

	MATERIALS AND METHODS

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Chemicals. DEAE-Sepharose, Blue-Sepharose, and Superose 12 were purchased from Pharmacia (Uppsala, Sweden), and methane and propylene were purchased from Messer Griesheim. Standard proteins for gel filtration and for sodium dodecyl sulfate (SDS)-gel electrophoresis were purchased from Merck (Darmstadt, Germany) and Boehringer (Mannheim, Germany), respectively. All other chemicals used were of analytical grade and were purchased from various manufacturers.

Growth conditions and cell disruption. Methylocystis sp. strain WI 14 was cultivated on two different mineral salts media. The standard medium contained (in milligrams per liter) the following: KH₂PO₄, 340; K₂HPO₄, 435; MgSO₄ · 7H₂O, 35.6; FeSO₄ · 5H₂O, 2.5; CaCl₂ · 2H₂O, 1.85; MnSO₄ · H₂O, 0.308; ZnSO₄ · 7H₂O, 0.220; and NaMoO₄ · 2H₂O, 0.126. (NH₄)₂SO₄ (0.47 g/liter) was used as the nitrogen source. The NMS medium described previously (12) was modified and contained (in milligrams per liter) the following: K₂HPO₄, 860; KH₂PO₄, 530; K₂SO₄, 170; MgSO₄ · 7H₂O, 37; CaCl₂, 7; FeSO₄ · 7H₂O, 5; ZnSO₄ · 7H₂O, 0.58; MnSO₄ · 7H₂O, 0.47; KI, 0.17; H₃BO₃, 0.12; CoCl₂ · 5H₂O, 0.10; and NaMoO₄ · 2H₂O. 0.10. NaNO₃ (0.85 g/liter) was used as the nitrogen source. Phosphate and iron solutions were autoclaved separately and added to the final medium. The pH of the complete medium was adjusted to 6.8 with 1 M NaOH. No external copper was added to the medium.

Enzyme assays. The activity of MMO was measured by gas chromatography (in a GC 5890 II instrument [Hewlett Packard] with a 25-m Ultra 2 capillary column 50°C isotherm, hydrogen carrier gas at 2.26 ml/min, and a flame ionization detector) with propylene as the substrate. The reaction mixture contained, in a final volume of 2 ml, 25 mM Tris-HCl buffer (pH 7.2), 5 to 10 mM NADH or NADPH, and a corresponding amount of active enzyme; it was placed in a vial with 10 ml of headspace which was sealed gas-tight. By using a gas-tight syringe, 2 ml of the gas phase was displaced by propylene. After incubation for 15 min at 30°C on a rotary shaker, the propylene oxide formed was quantified by using a calibration curve. Furthermore, the activity of the hydroxylase of sMMO was measured as described above with 10 mM H₂O₂ instead of the other components and NAD(P)H. The activity of sMMO was also measured with naphthalene. Solid naphthalene was added to the reaction mixture containing whole cells (optical density at 600 nm of 0.2) in 25 mM Tris-HCl buffer (pH 7.2) and incubated for 15 min at 30°C. The - or -naphthol formed was measured spectrophotometrically by monitoring the increase of absorption at 525 nm after addition of 0.1 µmol of Fast Blue B salt ( = 3.81 mmol¹ cm¹ [24]).

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Protein determination. The protein concentration was determined as described by Bradford (2) with bovine serum albumin as the standard. During purification of the components of sMMO, protein was determined by measuring the absorption at 280 nm.

Protein purification. All purification procedures were performed at 4°C on an LKB II FPLC system (Pharmacia, Uppsala, Sweden).

(i) Hydroxylase and component B. After concentration and desalting by ultrafiltration, the enriched hydroxylase and component B, respectively, were loaded onto two combined Superose 12 HR gel filtration columns (10 by 300 mm) equilibrated with 50 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl. The proteins were eluated with the same buffer and collected in 0.5-ml fractions. The flow rate was 0.4 ml/min. Active fractions were pooled and concentrated by ultrafiltration.

(ii) Reductase (component C). After concentration and desalting by ultrafiltration, the enriched reductase was loaded onto a Blue Sepharose column (10 by 100 mm) equilibrated with buffer A. The column was washed with 0.5 M NaCl in buffer A until no protein could be detected. Reductase was eluated with a linear gradient between 0.5 and 1 M NaCl in buffer A. Active fractions were pooled and concentrated by ultrafiltration.

Purification control and estimation of molecular mass. Gel electrophoresis was performed as described previously (40) in 10 or 12% polyacrylamide gels containing 0.1% SDS (25) by using the discontinuous buffer system originally described by Davis (7).

Determination of the amino terminal sequence. Purified components of sMMO were separated into subunits by SDS-gel electrophoresis and blotted to a polyvinylidene difluoride membrane (Bio-Rad). Amino-terminal amino acid sequence determination was performed with a sequencer (473A; Applied Biosystems) by the Edman method. Phenylthiohydantoin amino acids were analyzed by high-pressure liquid chromatography with a reversed-phase column (9). The determined N-terminal amino acids were used to assign the open reading frames to the appropriate components of sMMO.

Isoelectric focusing. The isoelectric point of the components of sMMO from Methylocystis sp. strain WI 14 was determined with the Multiphor II system from Pharmacia, using SERVALYT gels (pH 3 to 9) as specified by the manufacturer. The proteins were visualized by staining with Coomassie blue R-250.

Atomic absorption spectrometry. To determine the iron content of purified hydroxylase (1 mg was diluted in 10 mM HCl) and to determine the copper content of the cultivation media, an atomic absorption spectrum was recorded (Elan 5000; Perkin-Elmer).

Sequencing of the 16S rDNA. (i) Oligonucleotide primers. Primers were synthesized with a DNA synthesizer (380A; Applied Biosystems). The primers 27f (26), R5 (42), R8 (8), and 1492r (26) were used.

(ii) Isolation and PCR amplification of 16S rDNA. Total genomic DNA from WI 14 was obtained by boiling the cell suspension in sterilized aqua dest. for 5 min and removing the cell debris by centrifugation (29). PCR was carried out with primers 27f and 1492r in a thermal cycler (480; Perkin-Elmer Cetus). PCR mixtures were prepared in a total reaction volume of 50 µl and consisted of 5 µl of the supernatant fluid from the boiled bacterial suspension, 40 to 60 pmol (2 µl) of each primer, 200 µM (8 µl) deoxynucleoside triphosphates (Pharmacia), and ultrapure water (27.5 µl). The samples were overlaid with 75 µl of mineral oil, and the initial denaturation was done for 2 min at 100°C. Subsequently, after the mixture was chilled on ice, 2.5 U (0.5 µl) of Taq polymerase in 5 µl of 10× Taq DNA polymerase buffer (100 mM Tris-HCl [pH 9.0], 500 mM KCl, 15 mM MgCl₂) (Pharmacia) was added to each sample. The thermal profile included 30 cycles of 1 min at 94°C (denaturation), 1 min at 65°C (reannealing), and 1 min at 72°C (DNA extension) and denaturation. All water controls were subjected to the same PCR conditions as the samples. Following the last cycle, the sample block was kept at 4°C until electrophoresis was performed.

(iii) Cloning and DNA sequencing of PCR fragments by the cycle-sequencing reaction. The PCR product was separated by gel electrophoresis on 1% agarose (TAE 1×), cut out, and extracted from the gel with the QIAEX II gel extraction kit (Qiagen). Purified DNA was cloned in a pCR 2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen) in the competent E. coli strain TOP 10. Plasmid DNA was isolated with the Wizard Plus SV Miniprep system (Promega). A sequencing PCR with the sequencing primers mentioned above was carried out with the ABI PRISM dRhodamine Terminator cycle-sequencing kit (Applied Biosystems) in a GeneAmp PCR system 2400 (Perkin-Elmer). The thermal profile included 25 cycles of 30 s at 96°C (denaturation), 5 s at 50°C (reannealing), and 4 min at 60°C (DNA extension). The product of the sequencing PCR was purified with a CENTRI-SEP column (Princeton Separations) and analyzed in an automated fluorescence sequencer (373A; Applied Biosystems).

(iv) Phylogenetic analysis. 16S rDNAs for comparison were obtained from the GenBank sequence database of the National Center for Biotechnology Information, Bethesda, Md. Sequences were aligned with the ClustalW multiple-sequence alignment of the European Bioinformatics Institute, and the dendrogram was constructed with the programs SEQBOOT, DNAPARS, DNADIST, FITCH, NEIGHBOR, and CONSENSE from the PHYLIP version 3.5c package (10).

PCR amplification and sequencing of sMMO gene cluster. Total genomic DNA from Methylocystis sp. strain WI 14 was obtained as described above. The sMMO gene cluster was amplified in three discrete fragments. The PCR amplification and sequencing reaction conditions were identical to those described for the 16S rDNA amplification and sequencing reactions, unless stated otherwise. The primer sequences used for the sMMO amplification and sequencing reactions of the individual sMMO genes are listed in Table 1. All assigned nucleotide position numbers correspond to the sMMO gene cluster sequence of Methylocystis sp. strain M (accession no. U81594). The first fragment (2,012 bp), amplified with primers mmoXF2 and mmoYR2, starts at position 540 (the start codon A₅₄₀TG of mmoX) and ends at position 2551 (which lies within mmoY). The second fragment (1,564 bp), amplified with primers mmoYF and mmoZR2, starts at position 2440 (which lies within mmoY) and ends at position 4003 (which lies within mmoZ). The third fragment (2,349 bp), amplified with primers mmoBF and mmoCR2, starts at position 3523 (which includes mmoB) and ends at position 5871 (which includes the mmoC termination codon TGA₅₈₇₁). An annealing temperature of 50°C was used in the amplification of the first and third fragments, and an annealing temperature of 65°C was used for the second fragment. The PCR-amplified products were cloned as described above.

	RESULTS

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Sequencing and comparison of 16S rDNA. By using the universal primers 27f and 1492r, a DNA fragment from isolate WI 14 was amplified, cloned, and sequenced. Comparison of 16S rDNA revealed that strain WI 14 (accession no. AF153281) was very close to the Methylocystis group, in particular to Methylocystis sp. strain M and Methylocystis strain EB1 (Fig. 1).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Phylogenetic analysis of the 16S rDNA from Methylocystis sp. strain WI 14. The BOOTSTRAP values from 100 replicates are indicated.

Expression and properties of sMMO. Depending on growth conditions, Methylocystis sp. strain WI 14 is able to form pMMO or sMMO. If the strain grows at copper concentrations exceeding 0.1 µM, sMMO expression is strongly repressed (Table 2). The nitrogen source also seems to influence the expression of this enzyme. Table 2 shows that although the microorganism grew on several N sources such as some amino acids, sMMO activity could be detected only if nitrate or ammonium ions were used. As expected, growth did not occur with amino acids as the sole sources of nitrogen and carbon.

Substrate specificity. The substrate specificity of the sMMO from Methylocystis sp. strain WI 14 was determined by using ultracentrifuged crude extract and is presented in Table 3. sMMO converts a variety of substrates such as alkanes up to heptane, alkenes, cyclic hydrocarbons, aromatics, and halogenated aromatics to their corresponding hydroxylated or oxidized products. Interestingly, the alkanes were not hydroxylated in the 1 position. All the products detected were n-alcohols (minimum detection limit, 100 pg/µl). For the halogenated substrates (chlorobenzene, fluorobenzene, and bromobenzene), a new product peak, whose retention time did not correspond to phenol, resorcine, or pyrogallol, respectively, could be observed. Furthermore, no chloride, fluoride, or bromide ions were detected, indicating that no oxidative dehalogenation occured.

Purification of sMMO. The components of the sMMO from Methylocystis sp. strain WI 14 were separated by chromatography on DEAE. Hydroxylase, component B, and reductase were eluted with a step gradient of 0.15, 0.3, and 0.5 M NaCl, respectively.

(i) Component A (hydroxylase). Table 4 shows details of the purification of the hydroxylase. This component could be enriched about 10-fold, resulting in a specific activity of 0.52 U/mg and a yield of 21%. These results suggest that the hydroxylase makes up about 10% of the soluble cell protein. SDS-gel electrophoresis of the purified protein indicated three distinct bands (Fig. 2).

View larger version (93K): [in this window] [in a new window]   FIG. 2.   SDS-gel electrophoresis of the purified components of sMMO from Methylocystis sp. strain WI 14. St., protein standard as indicated; E., crude cell extract; A, B, and C: components of sMMO. Molecular weights are given (in thousands) to the right of the gels.

(ii) Component B. The activity of component B can be measured only if the other components are present. Since only small amounts of the other components were available in purified form and due to the low stability of the hydroxylase, a purification table could not be generated. However, component B was identified by the increase of propylene oxide formed when it was added to ultracentrifuged extracts (addition of 10 µg of component B increased the oxidation rate by 50%). The final purification step on Superose 12 was necessary to obtain an almost pure protein band in SDS-gel electrophoresis (Fig. 2).

(iii) Component C (reductase). Reductase was purified almost to completion by ion-exchange and affinity chromatography (Table 4). Gel electrophoresis led to the complete cessation of enzyme activity. In contrast to the hydroxylase, the 204-fold enrichment of reductase suggests that this protein makes up only about 0.5% of the soluble cell protein.

Molecular properties and stability of the components of sMMO. The molecular properties of the components of sMMO have been determined. The native molecular mass of the hydroxylase was determined by gel filtration to be 229 kDa. SDS-gel electrophoresis shows three distinct bands with molecular masses of 57, 43, and 23 kDa (Fig. 2). Together, these results suggest that the hydroxylase has an ₂₂₂ structure. The isoelectric point was found to be 4.3. Atomic absorption spectroscopy indicated the presence of 3.6 mol of iron per mol of protein. The hydroxylase is very unstable and splits into its inactive subunits if the protein is stored frozen even for short periods (data not shown).

Reconstitution of the components of sMMO. For low-level sMMO activity, only the hydroxylase (MMOH) and the reductase (MMOC) components were required. The presence of component B (MMOB) increased the sMMO activity by about 10-fold but was not essential for enzyme reaction.

Inhibition of sMMO. Metal ions such as Zn²⁺, Cu²⁺, or Ni²⁺ were particularly strong inhibitors of reductase and sMMO activity. In contrast, EDTA mainly affected the reconstituted sMMO activity and presumably the hydroxylase and component B as well. Hg²⁺ ions completely eliminated reductase activity and consequently the reconstituted sMMO activity.

sMMO gene sequences. sMMO genes from strain WI14 are clustered on the chromosome. Table 5 shows the high degree of homology (both at the nucleotide and amino acid level) to the corresponding genes from Methylocystis sp. strain M, Methylosinus trichosporium OB3b, and Methylococcus capsulatus (Bath). Until now, only the complete sMMO nucleotide sequence from these strains was available.

	DISCUSSION

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16S rRNA gene phylogenetic analysis revealed that isolate WI 14 possesses type II methanotroph sequences that are most closely related to the genus Methylocystis, in particular to Methylocystis sp. strain M and Methylocystis strain EB1. This is in agreement with the studies of the morphology (electron microscopy of membranes) and biochemistry (detection of hydroxypyruvate reductase and -ketoglutarate dehydrogenase activity as key enzymes of the type II methanotrophic bacteria) of strain WI 14 (19).

In methanotrophic bacteria, the oxidation of methane is always catalyzed by an MMO, which requires two reduction equivalents to split the O---O bond. As mentioned above, the enzyme exists in both particulate (pMMO) and soluble (sMMO) forms. Whereas all strains have the pMMO, only a few are able to express the sMMO (1, 33). The Methylocystis sp. strain WI 14 described in this study expresses the sMMO only if grown under copper limitation (0.1 µM causes a loss of about 60% of sMMO activity) and with ammonia or nitrate as the nitrogen source. If serine or cysteine is used instead, the strain grows but does not form the sMMO even though copper is absent. The influence of copper ions on sMMO expression is well known. It could be demonstrated that in M. trichosporium OB3b, the sMMO is completely repressed at copper concentrations of about 0.4 µM (22). It has been suggested that the sMMO-encoding genes localized in an operon are controlled by a regulatory copper-binding protein which interacts with DNA in the promoter region. Copper changes the affinity of the protein to DNA and represses sMMO expression (36, 37). However, the mechanism by which nitrogen regulates expression has not yet been described. Although it is known that strains such as Methylocystis sp. strain GB 25, which are not able to form the sMMO, cannot grow on nitrate as the nitrogen source (18), the effect of nitrogen on sMMO and pMMO expression has not been reported previously.

The sMMO investigated shows a very narrow substrate specificity and oxidizes some aliphatics, aromatics, and halogenated compounds only to their corresponding hydroxylated products. Interestingly, the oxidation of aliphatics always led to the n-alcohol, not the 1-alcohol. In contrast, Green and Dalton (16) showed that the sMMO of Methylococcus capsulatus (Bath) partially oxidizes pentane and hexane to 1-pentanol and 1-hexanol, respectively. Furthermore, in this study, the halogenated hydrocarbons were hydroxylated but not dehalogenated. For the sMMO of M. trichosporium OB3b, Sullivan and Chase (44) obtained similar results. 1,2,3-Trichlorobenzene was oxidized to 2,3,4- and 3,4,5-trichlorophenol.

Similar to nearly all sMMO so far isolated (except for the enzyme from Methylobacterium sp. strain CRL-26 [38]), the sMMO from Methylocystis sp. strain WI 14 has three components: a hydroxylase, a reductase, and a regulatory protein. The molecular properties of these components and the nucleotide sequences of the encoding genes possess particularly strong similarity to the sMMO from M. trichosporium OB3b and Methylocystis sp. strain M (Table 5). Reconstitution of the purified components showed that component B is not necessary for enzyme activity but that its presence results in a 10-fold increase in activity. For the sMMO from M. trichosporium and Methylocystis sp. strain M, it has been demonstrated that component B increases activity 10- and 11-fold, respectively (34), and that in the last strain it could be replaced by Fe²⁺ ions. In contrast, in the investigated strain, Fe²⁺ ions did not affect the hydroxylase and were not able to replace component B but were essential for maintenance and recovery of reductase activity after storage.

sMMO from Methylocystis sp. strain WI 14 is strongly inhibited by Cu²⁺, Zn²⁺, and Ni²⁺ ions. Although the inhibition of Zn²⁺ and Ni²⁺ ions is due mainly to the influence on the reductase, Cu²⁺ ions also seem to affect the other components. However, Hg²⁺ ions lead to a total loss of enzyme activity, suggesting that there are essential sulfhydryl groups involved in substrate hydroxylation and coenzyme oxidation, respectively. Green et al. (17) showed that Cu²⁺ and Zn²⁺ ions inhibit the reductase component of the sMMO from Methylococcus capsulatus (Bath), thereby inhibiting sMMO activity. On the other hand, Ni²⁺ ions had no effect on the reductase or on sMMO activity as a whole. In contrast, Jahng and Wood (22) reported that both Cu²⁺, Zn²⁺, and Ni²⁺ ions influenced sMMO activity.