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. 1998 Dec;180(24):6440–6445. doi: 10.1128/jb.180.24.6440-6445.1998

Cytochrome P460 Genes from the Methanotroph Methylococcus capsulatus Bath†

David J Bergmann 1, James A Zahn 1,‡, Alan B Hooper 2, Alan A DiSpirito 1,*
PMCID: PMC107742  PMID: 9851984

Abstract

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.

Autotrophic, nitrifying bacteria and methanotrophs can both oxidize ammonia to nitrite in a two-step process. In the first, energy-dependent step, ammonia is oxidized to hydroxylamine. In methanotrophs, this reaction is catalyzed by a membrane-bound methane monooxygenase (pMMO) and, in certain species, if copper is limiting, by a separate, soluble methane monooxygenase (sMMO) (7, 30, 33, 35). In autotrophic nitrifying bacteria, ammonia is oxidized to hydroxylamine by ammonia monooxygenase, a membrane-bound enzyme with considerable similarity in catalytic properties and primary structure to the pMMO of methanotrophs (7, 9, 11, 18, 21, 26, 34, 42). In the second step, hydroxylamine is oxidized to nitrite, releasing four electrons. In the nitrifying bacterium Nitrosomonas europaea, the oxidation of hydroxylamine is catalyzed by two periplasmic cytochromes, hydroxylamine oxidoreductase (HAO) and cytochrome P460 (8, 12, 21, 32). HAO is considerably more abundant than cytochrome P460 and supports a much higher rate of hydroxyamine oxidation than cytochrome P460 in vitro (12, 21, 28). HAO is a complex enzyme, consisting of three 63-kDa subunits, each of which contain seven c hemes and a unique heme P460 chromophore (3, 4, 21, 22). The heme P460 of HAO is named for its Soret absorbance maximum in the reduced state and constitutes the active site (20). Cytochrome P460 is considerably smaller, consisting of a dimer of 18-kDa subunits, each of which contains a single heme, also known as heme P460 (8, 14, 21, 28). The heme P460 chromophores of HAO and cytochrome P460 have quite similar spectral properties (2, 4, 21), and both consist of a modified c heme that is covalently attached to the polypeptide by three linkages, two of which are thioether linkages to cysteine residues of the polypeptide. However, the third covalent linkage between the polypeptide and the heme P460 in the two cytochromes differ in nature; the heme P460 in HAO is linked covalently to a tyrosine residue (3, 22), while in cytochrome P460 the heme appears to be linked to a lysine residue (5). It is interesting that despite the similarities of the P460 hemes of HAO and cytochrome P460, the two cytochromes have no similarity in amino acid sequence, apart from the presence of c-heme binding site motifs (CXXCH) (8, 32).

In the methanotroph Methylococcus capsulatus Bath, cytochrome P460 is responsible for the oxidation of hydroxlyamine to nitrite (43). Because of its similarities in size, subunit composition, and electron paramagnetic resonance spectra to the cytochrome P460 of N. europaea, the M. capsulatus Bath cytochrome was also named cytochrome P460 (43). In this study, we examined the gene encoding cytochrome P460 from M. capsulatus Bath and compared the deduced amino acid sequence to that of cytochrome P460 from N. europaea in an attempt to define additional characteristics of this unique class of cytochromes. The amino acid sequence of the M. capsulatus Bath cytochrome P460 indicates that the cytochromes P460 of nitrifiers and methanotrophs have a common, though very distant, ancestral form and may share important structural features.

MATERIALS AND METHODS

Culture conditions and protein purification.

Culture conditions for N. europaea, M. capsulatus Bath, Methylosinus trichosporium OB3b, Methylocystis parvus OBBP, Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2 were described previously (10, 26, 42, 43).

The potential induction of cytochrome P460 in M. capsulatus Bath by ammonia was carried out in a 5-liter Bioflow 3000 (New Brunswick Scientific, New Brunswick, N.J.) fermentor/chemostat. Cells were cultured in nitrate mineral salts medium at 42°C (40) under an atmosphere of 5% methane–95% (vol/vol) air. The cells were cultured to a wet cell density of 2 g/liter and were induced by the addition of 9.4 mM (NH4)Cl2 (40). An oxygen saturation level of 50% ± 3% and pH (7.0 ± 0.1) was controlled following the addition of ammonia. After the addition of ammonia, 35-ml cell samples were taken every 15 min for 4 h.

Cytochrome P460 was isolated by the method of Zahn et al. (43). Before N-terminal amino acid sequencing of cytochrome P460, samples were applied to a 6- by 10- by 0.5-cm preparative sodium dodecyl sulfate (SDS)-containing denaturing gel (25). After electrophoresis, the gel was briefly soaked in transfer buffer (25 mM Tris [pH 8.3], 193 mM glycine, 20% methanol, 0.05% SDS) and transferred to a polyvinylidene difluoride membrane, using a Panther semidry electrophoretic blotter (Owl Scientific Inc., Woburn, Mass.) at 10 V and 400 m A, for 1 h. The greenish cytochrome P460 band was cut out of the membrane and sequenced by Edman degradation in an Applied Biosciences 477A protein sequencer coupled to a 120A analyzer.

HAO was purified from N. europaea as described by Arciero and Hooper (3).

DNA methods.

Genomic DNAs from M. capsulatus Bath and N. europaea were isolated by the methods of Ausubel et al. (6) and McTavish et al. (26), respectively. Genomic DNA from M. trichosporium OB3b, M. parvus OBBP, M. marinus A45, M. albus BG8, and Methylomonas sp. strains MN and MM2 was isolated as described previously (15). Plasmid DNA was isolated by the alkaline lysis method (31).

Digestion of DNA with restriction endonucleases, dephosphorylation with calf alkaline phosphatase, and ligation with T4 DNA ligase were performed as directed by the manufacturers (Life Technologies, Gaithersburg, Md., and Promega Corporation, Madison, Wis.). Agarose gel electrophoresis of restriction fragments was conducted by standard methods (31). Restriction fragments in gels were transferred to positively charged nylon membranes (Magna Charge Plus; MSI, Wesboro, Mass.) after denaturation by capillary transfer (31). Degenerate oligonucleotide probes were prepared by the Iowa State University DNA Sequencing Facility and 5′ end labeled with [32P]ATP by using T4 polynucleotide kinase (31). Longer, double-stranded probes were prepared by the random hexamer priming technique (15), using a Prime-A-Gene kit (Promega). To hybridize Southern blots with degenerate oligonucleotide probes, membranes were prehybridized for 1 h and hybridized overnight in 6× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7])–1× Denhardt’s solution–0.5% SDS–10% polyethylene glycol (8,000 Da) at 42°C (31). To hybridize Southern blots with longer probes, membranes were prehybridized for 1 h and hybridized overnight in 6× SSPE–0.5% BLOTTO (31)–0.5% SDS at 55°C.

A clone bank of M. capsulatus Bath genomic fragments was prepared in the cosmid vector PVK102 (24). The vector was digested with SalI and treated with calf intestinal alkaline phosphatase. Partial and complete XhoI digests of M. capsulatus Bath genomic DNA were combined and size fractionated by sucrose density gradient ultracentrifugation (1) to yield fragments greater than 15 kbp. Approximately 9 μg of M. capsulatus Bath XhoI fragments was ligated to 3 μg of phosphatase-treated vector. Approximately 4 μg of the ligation mixture was packaged into phage capsids by a the Pack-A-Gene kit (Stratagene, La Jolla, Calif.) and used to infect Escherichia coli DH5α (Life Technologies). The resulting colonies were selected for tetracycline resistance and kanamycin susceptibility, transferred to microtiter plates, and imprinted on nylon membranes. Colonies were lysed in situ on nylon membranes, and DNA was denatured and fixed as described by Sambrook et al. (31) prior to hybridization with oligonucleotide probes. Restriction fragments of cosmid clones were subcloned into the plasmid vector pBluescript KS (Stratagene) for sequencing. Universal and custom oligonucleotide primers were used to sequence double-stranded plasmid DNA by the DNA Sequencing Facility at Iowa State University.

RNA methods.

Total RNA was isolated from a late-log-phase culture of M. capsulatus Bath by a modification of the method of Waechter-Brulla et al. (37). Ten milliliters of culture was centrifuged briefly at 3,000 × g and 5°C, and the cell pellet was resuspended in 3.3 ml of TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA). Hot lysis buffer (20 mM Tris-HCl [pH 7.5], 2% [wt/vol] SDS, 20 mM NaEDTA, 200 mM NaCl) was added, and the mixture was incubated for 3 min at 70°C. The solution was then extracted three times with phenol (pH 4.3) at 70°C, once with phenol-chloroform-isoamyl alcohol (25:24:1, pH 7.5) at 20°C, and once with chloroform-isoamyl alcohol (24:1) at 20°C. RNA was precipitated by addition of 1/10 volume of 3.0 M sodium acetate (pH 4.0) and 2 volumes of ethanol and incubation for over 12 h at −20°C; the pellet was resuspended in water with 0.1 M NaEDTA.

Electrophoresis of RNA in agarose gels containing formaldehyde and transfer to nylon membranes were performed as described by Nielsen et al. (27). The membranes were prehybridized in buffer (50% [vol/vol] formamide, 5× SSPE, 0.1% [wt/vol] SDS, 2× Denhardt’s solution) for 1 h at 42°C. The membranes were hybridized to a denatured double-stranded probe, previously labeled by random hexamer priming, in fresh buffer overnight at 42°C. Northern blots were rinsed at 20°C for 20 min in 1.0× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% SDS and were then rinsed in 0.2× SSC–0.1% SDS for 20 min at 20°C and at 42°C prior to exposure to a phosphorimager.

Mapping of the 5′ end of transcripts was performed by primer extension as described by Nielsen et al. (27). Primer extension products were separated by denaturing electrophoresis alongside samples of dideoxy sequencing reactions (Sequenase 2.0 kit; United States Biochemicals, Cleveland, Ohio) performed with the same primer and then visualized by autoradiography.

Preparation of antibodies against cytochrome P460 and HAO.

Antiserum against cytochrome P460 was raised in one New Zealand White rabbit by Animal Pharm Services, Inc. (Healdsburg, Calif.). Immunoglobin G was purified from the serum by using immobilized protein A (Pharmacia Biotech) according to the manufacturer’s instructions. The cytochrome P460 antibody fraction was purified from the immunoglobin G fraction by using immobilized cytochrome P460 bound to a 1-ml HiTrap affinity column as instructed by manufacturer (Pharmacia Biotech).

Antiserum against HAO from N. europaea was raised as previously described (42). The HAO antibody fraction was purified from the serum by using immobilized HAO as described above.

SDS-polyacrylamide gel electrophoresis and immunoblot analysis.

Electrophoresis was performed on SDS-containing denaturing gels by the method of Laemmli (25) or on Tricine gels as specified by the manufacturer (Novex Experimental Technologies, San Diego, Calif.). Gels were stained for total protein with Coomassie brilliant blue R or blotted for immunoassays.

Proteins were blotted onto nitrocellulose by using a Panther semidry electrophoretic blotter (Owl Scientific) according to the manufacturer’s directions. Following treatment with serum raised against purified protein, filter-bound antibodies were detected by an alkaline phosphatase assay as instructed by the manufacturer (Bio-Rad Laboratories, Hercules, Calif.).

Nucleotide sequence accession number.

DNA sequences were deposited in GenBank under accession no. AF091435.

RESULTS

Cloning and sequencing the cypA gene cluster of M. capsulatus.

The N-terminal amino acid sequence of cytochrome P460 from M. capsulatus, derived by Edman degradation, was EPAAAPNGISLPAGYKDWKMIGVSSRIEQNNLRAILGNDIAVKAAREGRTHPWPDGAIL. This sequence agrees with the much shorter sequence published earlier by Zahn et al. (43) and was used to synthesize a degenerate oligonucleotide probe with the sequence 5′-GGI-TAY-AAR-GAY-TGG-AAR-ATG-ATI-GG-3′, where I represents inosine and Y and R represent mixtures of pyrimidines and mixtures of purines, respectively. The probe was used to screen 2,300 cosmid clones, yielding a single clone that hybridized to the probe. An approximately 6-kbp PstI fragment of this clone was subcloned and sequenced (Fig. 1 and 2).

FIG. 1.

FIG. 1

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Map of the 6-kb PstI fragment that contains the cyp gene of M. capsulatus Bath. ORFs and certain restriction endonuclease sites (P, PstI; K, KpnI; H, HindIII; C, SacII; L, SalI; X, XhoI) are indicated. The circle denotes the location of the oligonucleotide probe, flags denote transcriptional start sites, and the dashed line denotes the region sequenced.

FIG. 2.

FIG. 2

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DNA sequence and translated amino acid sequence of the gene cluster encoding cyp and two ORFs upstream. The three ORF start sites and restriction endonuclease sites are indicated. A possible ς70 promoter sequence and likely RBSs are underlined. Putative signal peptide residues of ORF2 and the cyp gene products are shown in italics. Transcription start sites are marked with asterisks. Sequences of primers used for primer extension are underlined.

The gene encoding cytochrome P460 of M. capsulatus Bath, cyp, is the third open reading frame (ORF) in a gene cluster (Fig. 2). The first ORF extends from bases 265 to 1893 and is preceded by a probable ribosome binding site (RBS; AGGAAA) nine bases upstream. A second ORF extends from bases 1912 to 2310, with a likely RBS (AGGAGC) seven bases upstream. The third ORF, cyp, extends from bases 2359 to 2841 and is preceded by a probable RBS (AGGAGA) seven bases upstream. No ORFs are found within 500 bases downstream of cyp.

ORF1 encodes a putative 59,700-Da polypeptide that is predicted by the TopPred2 program (36) to have nine membrane-spanning helices. ORF2 has an N-terminal hydrophobic region which may represent a signal peptide 22 residues long (Fig. 2); if this were cleaved, a 12,200-Da periplasmic protein would be produced. A search of the GenBank database using the FASTA program (Genetics Computer Group, Madison, Wis.) did not reveal significant homology between the polypeptides encoded by ORF1 and ORF2 and any proteins in the database. However, a BLASTP search (1) did show weak homology (24% identity) between the ORF1 polypeptide and the sodium-hydrogen antiporter of Synechocystis sp. (GenBank accession no. 90914).

The amino acid sequence of cytochrome P460 from M. capsulatus Bath, derived from the sequence of cyp, contains an N-terminal signal peptide that is cleaved off in the mature polypeptide, which starts at glutamate 18. The mature holocytochrome, including heme, is predicted to have a mass of 16,210 Da, 180 Da less than estimated by mass spectroscopy (43). A single c-heme binding motif, CMGCH, is found near the C terminus of the polypeptide.

The sequence of cytochrome P460 from M. capsulatus Bath displays little similarity to cytochrome P460 from N. europaea, and only 23.6% of amino acid residues are identical (reference 8 and Fig. 3). However, some short regions of the M. capsulatus Bath cytochrome, such as residues 9 to 22, 23 to 34, 55 to 61, 79 to 95, and 134 to 138, have a greater proportion of similar and identical residues, indicating that these regions may have been conserved between the two cytochromes.

FIG. 3.

FIG. 3

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Comparison of amino acid sequences of the cyp gene products of M. capsulatus Bath (top) and N. europaea (bottom). Lines and dots lie between identical and similar amino acid residues, respectively.

Transcriptional start site mapping.

A Northern blot of total RNA from M. capsulatus Bath, probed with the 442-base SacII-HindIII fragment containing the cloned cyp gene, indicated that the cyp transcript was about 950 bases long (Fig. 4). The 5′ end of the cyp transcript was mapped by primer extension using primer TXCYP, the reverse complement of bases 2380 to 2401 (Fig. 2). Two adjacent transcriptional start sites were indicated at bases 2340 and 2341 (Fig. 5). No −35 and −10 ς70 consensus promoter sequences are located near the cyp transcriptional start sites. However, sequences with a weak resemblance to −24 and −12 ς54 consensus promoter sequences are located 15 bases upstream of the transcriptional start site (Fig. 2). The 5′ end of the transcript from ORF1 (and possibly ORF2) was also mapped by using three primers: TXORA, the reverse complement of bases 284 to 302; TXORB, the reverse complement of bases 181 to 199; and TXORC, the reverse complement of bases 105 to 121 (Fig. 2). Only TXORC yielded a major primer extension product, starting at base 47 (Fig. 5). Weak matches to E. coli ς70 −35 and −10 consensus promoter sequences are found at bases 6 to 11 and 29 to 34, respectively (Fig. 2).

FIG. 4.

FIG. 4

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Northern blot of total RNAs from E. coli DH5α (lane 1, negative control) and M. capsulatus Bath (lane 2). About 8 μg of RNA was loaded in each lane. Hybridization and washing conditions are described in Methods and Materials. Locations of RNA size markers are indicated on the left.

FIG. 5.

FIG. 5

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Primer extension analysis using primers TXCYP, upstream of the cyp translational start site (A), and TXORC, upstream of the ORF1 translational start site (B). In each panel, sequencing reactions with primer and plasmid DNA template of the cloned gene are shown in the left four (T, C, G, and A) lanes, with the primer extension products in the rightmost lane.

Southern blots.

To determine if close homologues to the cyp gene of M. capsulatus Bath might exist in other species of methanotrophs, genomic Southern blots of M. trichosporium OB3b, M. parvus OBBP, M. marinus A45, M. albus BG8, and Methylomonas sp. strains MN and MM2 were probed with a 442-base SacII-HindIII fragment of the cloned M. capsulatus Bath cyp gene (Fig. 6). In addition to hybridizing with M. capsulatus Bath restriction fragments, the cyp probe hybridized relatively strongly to single restriction fragments of M. trichosporium OB3b DNA and much more weakly to a single restriction fragment of M. parvus OBBP DNA. No hybridization of the M. capsulatus Bath cyp probe to the other methanotrophs was observed.

FIG. 6.

FIG. 6

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Southern blot of EcoRI digests of genomic DNA from methanotrophic bacteria probed with a 442-bp SacII-HindIII fragment containing the cyp gene of M. capsulatus Bath. Lane 1, M. capsulatus Bath; lane 2, M. albus BG8; lane 3, Methylomonas sp. strain MN; lane 4, Methylomonas sp. strain MM2; lane 5, M. marinus A45; lane 6, M. parvus OBBP; lane 7, M. trichosporium OB3b; lane 8, E. coli DH5α.

In two other experiments, we looked for potential homologues to the cyp or the hao gene from N. europaea by probing genomic Southern blots of the same eight species of methanotrophs with a 2.3-kbp BamHI-SmaI fragment containing the cloned N. europaea cyp gene, which encodes cytochrome P460 (8), and a 542-bp PvuII-EcoRI fragment containing a cloned N. europaea hao gene (30). However, no hybridization of either probe to DNA from any methanotroph was observed.

Immunoblotting.

Antisera to cytochrome P460 from M. capsulatus Bath and to HAO from N. europaea were used in immunoblotting experiments to determine if homologous proteins exist in selected methanotrophs and N. europaea. Except for cross-reactivity with a 16,000-Da polypeptide in cell extracts from M. capsulatus Bath, none of the polypeptides from whole-cell extracts from N. europaea, M. trichosporium OB3b, M. parvus OBBP, M. albus BG8, and Methylomonas sp. strains MN, and MM2 showed cross-reactivity to cytochrome P460 antiserum.

Regulation.

Ammonia induction experiments showed that the concentration of cytochrome P460 polypeptide remained constant over a 4-h observation period following the addition of 5 mM ammonia, although induction of a high-molecular-mass c-type cytochrome (10) was observed (Fig. 7). Repeated addition of ammonia also failed to alter the cellular concentration of cytochrome P460. In addition, the antiserum to cytochrome P460 was used in immunoblotting experiments to show that the cellular concentration of cytochrome P460 did not change with expression of the different MMOs (results not shown).

FIG. 7.

FIG. 7

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Ammonia induction of M. capsulatus Bath. Samples were analyzed for the concentration of ammonia oxidized ( Created by potrace 1.16, written by Peter Selinger 2001-2019 ) and nitrite concentration (■) (A), for total heme-staining polypeptides on an SDS–15% polyacrylamide gel (B), for high-molecular-mass heme-staining polypeptide on an SDS–7% polyacrylamide gel (C), and for cytochrome P460 by immunoblot analysis on SDS–15% denaturing gels (D). All whole-cell and protein samples were reduced with β-mercaptoethanol and heated to 95°C for 2 min prior to loading.

Oxidation of ammonia to nitrite.

Production of nitrite following ammonia addition to early-stationary-phase cultures of M. trichosporium OB3b, M. parvus OBBP, M. albus BG8, and Methylomonas sp. strains MN and MM2 was also examined. Nitrite production was observed in stoichiometric amounts to the ammonia oxidized in all strains tested except Methylomonas sp. strains MN and MM2.

DISCUSSION

The primary amino acid sequences of cytochromes P460 from N. europaea and M. capsulatus Bath are dissimilar, despite the similarity in overall amino acid composition noted by Zahn et al. (43). However, the presence of a small number of conserved amino acid residues throughout both cytochromes suggests derivation from a common ancestral form. Among these conserved residues are the C-terminal c-heme binding motif (ECXXCH) and a lysine residue (K61 in M. capsulatus Bath) which, in N. europaea, is believed to form a covalent cross-link to the heme (5).

The placement of the gene encoding cytochrome P460 was different in M. capsulatus Bath than in N. europaea. In N. europaea, cyp is not located near any other ORFs (8). In M. capsulatus Bath, cyp is part of a gene cluster with two other ORFs but appears to be transcribed separately. Neither of these ORFs encodes cytochrome c′, the likely electron acceptor of cytochrome P460 (44).

Cytochrome P460 appears to be constitutively expressed in M. capsulatus Bath. Ammonia induction experiments showed that the concentration of cytochrome P460 polypeptide remained constant over a 4-h observation period following the addition of ammonia. In addition, the polypeptide concentrations in extracts from cells expressing the pMMO and from cells expressing the sMMO were identical.

Southern blots indicated that a cytochrome P460 similar to that of M. capsulatus Bath (a type X methanotroph) probably exists in both type II methanotrophs tested, M. parvus OBBP and M. trichosporium OB3b, but not in any of the type I methanotrophs tested, M. marinus A45, M. albus BG8, and Methylomonas sp. strains MN and MM2 (17, 18). It is not known if a P460 cytochrome, distinct from both the N. europaea and M. capsulatus Bath P460 cytochromes, exists in the type I methanotrophs. Antisera to cytochrome P460 from M. capsulatus Bath did not cross-react with any of the other methanotrophs tested in immunoblot experiments. The results suggest high sequence variability between the methanotrophs tested for potential cytochromes P460, even among methanotrophs which appear to have cyp homologues.

Results of Southern blot and immunoblot experiments using a gene probe and antisera to HAO, respectively, were also negative for all methanotrophs tested. We do not know whether methanotrophs such as M. albus BG8 that show negative hybridization or cross-reactivity to both cytochrome P460 and HAO gene probes or antibodies, respectively, and oxidize ammonia to nitrite contain a heme P460-containing enzyme or a non-P460-containing HAO (22, 37). In addition, the existence of methanotrophs such as Methylomonas sp. strains MN and MM2 that show negative hybridization or cross-reactivity to both cytochrome P460 and HAO gene probes or antibodies and do not oxidize ammonia to nitrite raises the question of whether all strains of methanotrophs can oxidize ammonia to nitrite.

The presence of a gene similar to cyp of M. capsulatus Bath in type II but not type I methanotrophs is somewhat puzzling when one considers that M. capsulatus Bath, which belongs to the γ subfamily of the proteobacteria, is more closely related to the type I methanotrophs, which also belong to the γ subfamily, than to the type II methanotrophs, which belong to the α subfamily (17). The presence of a gene similar to cyp of M. capsulatus Bath in type II but not type I methanotrophs is even more surprising because the sequence of the 27,000-Da subunit of pMMO from M. capsulatus Bath is considerably more similar to those from the type I methanotrophs than to those from type II methanotrophs (19). The presence of genes encoding similar P460 cytochromes in M. capsulatus Bath and type II methanotrophs suggests that one or both of these groups may have acquired the cyp gene by horizontal gene transfer.

ACKNOWLEDGMENTS

We thank B. Voss (Iowa State University) and J. Nott (Iowa State University Protein Facility) for technical assistance.

This work was supported by Department of Energy grant 02-96ER20237 (A.A.D.).

Footnotes

†

Journal paper J-18098 from the Agriculture and Home Economics Experiment Station, Ames, Iowa (project 3252).

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