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Applied and Environmental Microbiology, March 2000, p. 966-975, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Molecular Analysis of the pmo (Particulate Methane Monooxygenase) Operons from Two Type II Methanotrophs

Bettina Gilbert, Ian R. McDonald, Ruth Finch,dagger Graham P. Stafford, Allan K. Nielsen,Dagger and J. Colin Murrell*

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Received 11 October 1999/Accepted 14 December 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha -Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma 70 promoters were identified, suggesting that these pmo genes are recognized by sigma 70 and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma -Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Methane-oxidizing bacteria (methanotrophs) play an important part in the global carbon cycle, recycling up to 60% (680 Tg) of total global methane production per year (25). Methane is used as the sole source of carbon and energy by these organisms. It is oxidized to methanol by the key enzyme methane monooxygenase (MMO). Methanol is further oxidized to formaldehyde. Formaldehyde is then either assimilated into cell biomass or oxidized via formate to carbon dioxide. All known methanotrophs possess the membrane-bound or particulate form of MMO (pMMO), and some have a second enzyme, the cytoplasmic, or soluble, MMO (sMMO).

Two types of methanotrophs can be distinguished on the basis of biochemical and ultrastructural differences (3, 33). Genetic and biochemical work has been carried out mainly on two organisms, the type I methanotroph Methylococcus capsulatus Bath, a gamma -proteobacterium, and the type II methanotroph Methylosinus trichosporium OB3b, an alpha -proteobacterium. Another well-studied type II organism, Methylocystis sp. strain M, was isolated from a trichloroethylene-degrading mixed culture (20, 32). The sMMOs of these bacteria are very similar (5, 10, 20), and their sMMO gene sequences are highly conserved (17).

Recently, the pMMO was purified from M. capsulatus Bath (21, 34) and M. trichosporium OB3b (31). It is a copper-containing monooxygenase which is oxygen and light sensitive. The 26-kDa subunit of pMMO was labeled by acetylene, an inhibitor of MMO, indicating that it harbors the active site of the enzyme (6, 24). This subunit is encoded by pmoA, which has been shown to be highly conserved among methanotrophs and can be used to detect these organisms in a range of environments (13, 16).

The pmo genes from M. capsulatus Bath have been cloned and sequenced (28, 30). The cluster consists of three consecutive open reading frames designated pmoC, pmoA, and pmoB. There are two virtually identical copies (13 base pair changes over 3,183 bp of pmoCAB) present in the genome of M. capsulatus Bath, and a third copy of pmoC has also been identified (30). Analysis of mutants constructed by deleting each of these pmo genes has shown that the duplicate copies of each of these genes can partly complement each other (30). Further regulatory studies would be facilitated by working with the pmo genes from M. trichosporium OB3b because, unlike M. capsulatus Bath, it can be grown on methanol as well as on methane, and it is generally more amenable to genetic manipulations (19).

In methanotrophs possessing both pMMO and sMMO, the pMMO is expressed when copper/biomass ratios in the medium are high (29). Northern analysis has shown that in M. capsulatus Bath the sMMO and pMMO are under copper-dependent reciprocal transcriptional regulation (23), with the sMMO genes being transcribed under low-copper conditions. Under high-copper conditions, the transcription of sMMO genes stops and the pMMO genes are transcribed. The pmo genes from M. capsulatus Bath are transcribed into a single polycistronic mRNA of 3.3 kb. In addition, smaller transcripts were observed, representing monocistronic transcripts encoding pmoC, pmoA, and pmoB or translationally inactive degradation products (23). For M. trichosporium OB3b grown under non-copper-limiting conditions, a pMMO-specific mRNA of 4.0 kb was detected (23).

The cloning of pmo genes has been very difficult because parts of these genes seem to be toxic to Escherichia coli (21, 30). The same has been observed for amo genes encoding a related enzyme, ammonia monooxygenase (18). We report here the sequencing and comparative analysis of pmo genes from two type II methanotrophs belonging to the alpha -subclass of the Proteobacteria, M. trichosporium OB3b and Methylocystis sp. strain M. Primer extension experiments revealed the transcriptional start site and putative promoter region of pMMO genes for Methylocystis sp. strain M and provide evidence for the genetic organization of these gene clusters.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and growth conditions. M. trichosporium OB3b was obtained from the University of Warwick culture collection. Methylocystis sp. strain M was kindly supplied by H. Uchiyama, Tsukuba, Japan. Methanotrophs were grown on nitrate mineral salts medium (NMS) (33) in batch culture with a headspace of methane and air (1:5) at 30°C or on NMS agar plates under the same conditions. E. coli TOP10 (Invitrogen) was used as the host in DNA cloning experiments. It was grown on nutrient agar (Difco) or on Luria-Bertani medium in the presence of ampicillin (final concentration, 50 µg ml-1) where appropriate.

DNA manipulations. Preparation of plasmid DNA and standard DNA manipulations were carried out according to the method of Sambrook et al. (26). Small-scale preparation of plasmid DNA from E. coli TOPO was performed using a kit (Qiaprep Spin Miniprep Kit; Qiagen). Chromosomal DNA from methanotrophs was isolated as follows. One liter of batch culture (optical density at 540 nm [OD540] 0.5 to 0.6) was pelleted and resuspended in 5 ml of solution 1 (50 mM Tris [pH 8.0]-25% sucrose). Then 0.5 ml of lysozyme (20 mg ml-1 in 0.25 mM Tris [pH 8.0]) was added. After incubation for 30 min at 37°C, 1 ml of 0.25 M EDTA (pH 8.0) was added, followed by incubation for 30 min at 37°C. Finally, Sarkosyl was added to a final concentration of 1%, and the mixture was incubated at 37°C for 30 min and at 60°C for 5 to 30 min until lysis was complete. The lysate was subjected to CsCl gradient centrifugation for 16 h (26). For DNA-DNA hybridizations, nucleic acids were transferred to a nylon membrane (Hybond-N) using a blotting apparatus (Turboblotter; Schleicher & Schuell). Hybridizations were carried out in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 55°C for oligonucleotide probes and at 60°C for DNA fragment probes. The initial wash was done at 55°C in 6× SSC for oligonucleotide probes and at 60°C in 2× SSC for DNA fragment probes. The stringency was then gradually raised by increasing the temperature and lowering the SSC concentration. DNA probes were radiolabeled with [alpha -32P]CTP either by nick translation (26) or by random priming (9). Probes were generated by PCR with the primers indicated. Tables 1, 2, and 3 contain information on the probes and primers used in this study. Oligonucleotides were end labeled with [gamma -32P]ATP using T4 kinase (26).

                              
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  		TABLE 1.   Primers and probes used for cloning the pmo gene clusters from M. trichosporium OB3b and Methylocystis sp. strain M


                              
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  		TABLE 2.   Probes for hybridization experiments and the positions of primers that were used to generate the probes by PCR


                              
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  		TABLE 3.   Primers used in primer extension experiments

PCR. PCR was performed in 50-µl reaction mixtures in 0.5-ml microcentrifuge tubes using a Hybaid Touchdown thermal cycling system. Taq polymerase (Gibco BRL) was used. After an initial denaturation step of 94°C for 5 min, the Taq polymerase was added. Then 28 cycles of 92°C for 1 min, 55 or 60°C for 1 min, and 72°C for 1 min were run, followed by a final extension of 10 min at 72°C. For expected PCR products of less than 0.6 kb, cycles of 0.5 min at each temperature and a final extension of 5 min were run. Reaction products were checked for size and purity on 1% (wt/vol) agarose gels after staining with ethidium bromide. Primers were purchased from Gibco BRL.

DNA cloning and sequencing. Putative pmo gene fragments were cloned into pUC19 or by using the TA cloning kit (Invitrogen) according to the manufacturer's instructions. DNA sequencing was carried out using DyeDeoxy terminators by the University of Warwick Sequencing Facility, with Perkin-Elmer ABI 373A and 377 automated sequencers. In all cases, double-stranded DNA sequences were obtained by completely sequencing both strands of DNA.

Computer analysis. Analysis of DNA sequences and homology searches were carried out with standard DNA sequencing programs and the BLAST server of the National Center for Biotechnology Information (NCBI) using the BLAST algorithm (1, 2). Putative rho-independent terminators, codon usage tables and codon preferences, and the hydrophobicities of proteins were calculated using the Genetics Computer Group (GCG) software package. The locations of putative transmembrane-spanning regions were calculated using the TMHMM tool, available at the Swiss Institute of Bioinformatics' Expasy website (http: //www.expasy.ch/tools/#transmem).

Isolation of total RNA. Total RNA was isolated from 50-ml aliqots of M. trichosporium OB3b and Methylocystis sp. strain M cells grown in batch cultures to an OD540 of 0.4 to 0.5 (mid-exponential-growth phase). These cultures tested negative for sMMO by a colorimetric sMMO assay (4). The cells were pelleted by centrifugation at 3,500 × g and stored at -80°C. In addition, fresh 1.5-ml aliquots from a chemostat culture of M. trichosporium OB3b at an OD540 of 7.5 were used (dilution rate as described by Nielsen et al. [23]). The chemostat culture received copper sulfate to a final concentration of 50 µM 2 h prior to sampling, to ensure that the cells were expressing pMMO. The cell suspension then tested negative for sMMO (4). The cell pellet was resuspended in 200 µl of solution I (0.3 M sucrose-0.01 M sodium acetate [pH 4.5]) and 200 µl of solution II (2% sodium dodecyl sulfate-0.01 M sodium acetate [pH 4.5]). The cell suspension was transferred to a blue Ribolyser tube (Hybaid), and 400 µl of phenol (saturated with 50 mM sodium acetate [pH 4.5]) was added. The cells were lysed using a Hybaid Ribolyser at speed setting 6 for 20 to 40 s. After this step, cells were kept on ice when possible. The suspension was centrifuged for 5 min, and the aqueous phase was transferred to a fresh tube. Four hundred microliters of phenol was added, and the tubes were incubated for 4 min at 65°C and then frozen in dry ice-ethanol for 10 s. The tubes were spun for 5 min, and the aqueous phase was transferred to a new tube. Four hundred microliters of phenol-chloroform was added, mixed vigorously for 30 s, and spun for 5 min. The aqueous phase was transferred to a new tube, and nucleic acid was precipitated with 40 µl of sodium acetate (pH 4.5) and 900 µl of 96% (vol/vol) ethanol at -20°C for 20 min. The pellet was washed with 70% (vol/vol) ethanol, dried in a vacuum drier, and resuspended in 40 µl of water. The RNA preparations were finally treated with RNase-free DNase (Gibco BRL) for 30 min at 37°C and examined using 1.5% (wt/vol) agarose gels. The concentration of nucleic acid in solutions was determined by measuring the A260 using a DU-70 spectrophotometer (Beckman).

Primer extension experiments. A 2.5-µl volume of RNA (5 to 10 µg) and 1 µl of [gamma -32P]ATP-labeled primer (5 ng) were heated for 1 min at 75°C in 1 µl of hybridization buffer (4.5× hybridization buffer contains 250 mM HEPES [pH 7.0] and 500 mM KCl), followed by gradual cooling to 30°C over a 60-min period. Three microliters of extension mix (260 mM Tris HCl [pH 8.4], 20 mM MgCl2, 20 mM dithiothreitol, 0.2 mM each deoxynucleoside triphosphate) and 1.6 U of avian myeloblastosis virus reverse transcriptase (Amersham) were added to each primer extension reaction mixture. The mixture was incubated at 45 or 50°C for 30 min. The extension products were precipitated with 1 µl of sodium acetate (pH 4.5) and 20 µl of 96% (vol/vol) ethanol on ice, washed with 70% (vol/vol) ice-cold ethanol, dried, and resuspended in 6 µl of "stop solution" (Sequenase version 2.0 DNA Sequencing Kit; USB). The extension products were preheated at 75°C for 2 min and loaded onto an 8% (wt/vol) polyacrylamide gel alongside a set of dideoxy sequencing products of the appropriate plasmid DNA template with the same primer. Sequencing reactions were carried out according to the manufacturer's instructions (Sequenase version 2.0 DNA Sequencing Kit; USB). Primers O1, O1A, and O2 were used with plasmid BC217; primers O3 and O4 were used with plasmid P236; primers M1, M1A, and M2 were used with plasmid C1; and primers M3 and M4 were used with plasmid P286 (see Table 3).

Nucleotide sequence accession numbers. The fully sequenced pmoCAB gene clusters have been deposited in the GenBank database under accession numbers AF186586 and AF186587 for Methylosinus trichosporium OB3b and Methylocystis sp. strain M, respectively.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The pmo gene cluster from M. trichosporium OB3b. A Southern blot of genomic DNA from M. trichosporium OB3b was probed with a pmoA probe derived from the known sequence of M. capsulatus Bath (28) (Fig. 1a). In some digests, two bands were identified, which suggested that two copies of pmoA were present in M. trichosporium OB3b. The 2.0-kb PstI fragment hybridizing with pmoA was cloned into pUC19 to generate clone P236, and the insert was sequenced (Fig. 2). The sequence contained regions of DNA which showed high identity with pmoA and the 5' two-thirds of pmoB from M. capsulatus Bath. Since this fragment had a BglII site at the end of the putative pmoA gene, it was concluded that one of the BglII chromosomal DNA fragments (1.6, 4.8, and 5.2 kb) which had hybridized to pmoA should contain the pmoA gene and sequences 5' of pmoA, probably including pmoC.


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  		FIG. 1.   Southern blot of genomic DNA from M. trichosporium OB3b (a) and Methylocystis sp. strain M (b) probed with a pmoA probe. (a) Lanes: 1, BamHI; 2, BglII; 3, EcoRI; 4, HpaI; 5, HindIII; 6, KpnI; 7, PstI; 8, SalI; 9, XhoI. The blot was probed with a PCR-amplified fragment of pmoA (550 bp) from M. trichosporium OB3b at 65°C, 2× SSC. (b) Lanes: 1, molecular mass standard, 1-kb ladder (Gibco BRL); 2, PstI; 3, HindIII; 4, EcoRI; 5, BamHI; 6, KpnI; 7, XhoI; 8, BglII; 9, HpaI; 10, SalI. The blot was probed with a PCR-amplified fragment of pmoA (550 bp) from Methylocystis sp. strain M at 70°C, 2× SSC.


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  		FIG. 2.   Physical and genetic map of the pmo genes in M. trichosporium OB3b and the overlapping cloned DNA fragments. The binding regions for probes OB1, OB2, and OB3 and for primers O1, O2, O3, and O4 used in primer extension experiments are also shown. See Tables 2 and 3 for exact positions.

Attempts to clone the 4.8-kb BglII fragment were unsuccessful, probably due to a toxic effect in E. coli. Similar observations have been made previously during attempts to clone pmo and amo genes (18, 28). Instead, we used a PCR approach similar to that described by Stolyar et al. (30) to obtain pmoC. M. trichosporium OB3b genomic DNA was digested with BglII. The DNA was religated and digested with BclI which cut within the known pmoA sequence. This procedure yielded a linear fragment of DNA with unknown sequence in the middle flanked by the known sequence of pmoA. Primers targeted to the regions immediately next to the BclI site (primers A and B; Table 1) were used in a PCR and amplified a 1.2-kb fragment. This was cloned into the TOPO vector (Invitrogen). The resulting clones were checked for the presence of sequence upstream of the BglII site by probing with primer C (Table 1). Plasmid DNA from one of the positive clones, BG3, was prepared, and the insert was sequenced. Based on the M. capsulatus Bath sequence, pmoC, the pmoC-pmoA intergenic region, and 214 bp of pmoA were identified. Since the sequence contained the putative start codon of pmoC but no sequence further upstream, two more PCR cloning experiments involving digestion with BclI and PvuI and primers D to F and J to K (Table 1) were carried out in order to obtain the sequence upstream of pmoC. In this way, clones BC217 and PV216 were generated, respectively (Fig. 2).

The 3' end of pmoB was obtained in two ways. Firstly, primers based on the end of pmoB in Methylocystis sp. strain M (primers Bfor and Brev; Table 1) were used to amplify a 590-bp fragment from M. trichosporium OB3b chromosomal DNA. This fragment, designated MtB5, was cloned and sequenced. It contained the rest of the pmoB gene, as expected. It was impossible to get sequence 3' of pmoB using Methylocystis sp. strain M-based primers, probably because the sequences diverge. Therefore, another PCR approach was used involving digestion with BglII, PCR with primers G and H, and probing with primer I. The insert of the resulting clone BG114 was sequenced. It contained all of pmoB, as well as 800 bp 3' of pmoB. The pmoB sequences obtained with the two methods were identical, although it was subsequently determined that this downstream sequence originated from the second copy of pmo genes (see "Duplication of pmo gene clusters" below). The downstream (3') sequence contained the start of another open reading frame, orfD, identified by codon usage preference; the derived amino acid sequence (104 amino acids [aa]) showed good similarity (52% at the amino acid level) to a partial sequence, orf4, from Nitrosococcus and Nitrosospira spp. (accession numbers AF047705 and U92432). Orf4 is located downstream (3') of the amo genes and thus seems to be the homologous gene in nitrifiers (15). Since clone BG114 was derived from the second copy of pmo genes, orfD does not appear in Fig. 2.

The pmo gene cluster from Methylocystis sp. strain M. A Southern blot of genomic DNA from Methylocystis sp. strain M was probed with a homologous pmoA probe generated by PCR using primers A189 and A682 (Table 2) with Methylocystis sp. strain M chromosomal DNA as the template. At least two DNA fragments were present in a number of different digests, e.g., with PstI, EcoRI, and BglII, suggesting that there were two copies of pmoA in Methylocystis sp. strain M (Fig. 1b), as had been found in M. capsulatus Bath and M. trichosporium OB3b. A 3.5-kb PstI fragment was cloned into pUC19 to generate P286, and the insert was sequenced (Fig. 3). The sequence exhibited a high degree of identity with the pmoA and pmoB genes from M. capsulatus Bath and M. trichosporium OB3b. The region downstream (3') of pmoB contained no open reading frames (based on analysis of codon usage preference) and showed no significant homology to polypeptides in the database.


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  		FIG. 3.   Physical and genetic map of the pmo genes in Methylocystis sp. strain M and the overlapping cloned DNA fragments. The binding regions for probes pC1, pC59, and p286 and for primers M1, M2, M3, and M4 used in primer extension experiments are also shown. See Tables 2 and 3 for exact positions.

Based on the assumption that the pmo genes in M. trichosporium OB3b and Methylocystis sp. strain M have a high degree of similarity, the known M. trichosporium OB3b sequence was used to PCR amplify the homologous sequence from Methylocystis sp. strain M: a reverse primer, Arev, targeting the intergenic region pmoC-pmoA from Methylocystis sp. strain M, and a forward primer, Cfor, specific to the pmoC sequence from M. trichosporium OB3b, amplified a 740-bp fragment from genomic DNA. This was cloned into the TOPO vector (Invitrogen) to produce clone C59. The insert was sequenced and found to contain most of pmoC (positions 1004 to 1749 in the final sequence [Fig. 3]). The start of pmoC was obtained using a PCR approach. Genomic DNA was digested with SalI, religated, and digested again with SstI. Primers targeting the region at the SstI site (primers L and M [Table 1]) amplified a fragment of 1.2 kb. It was cloned into the TOPO vector (Invitrogen) to generate clone C1, and the insert was sequenced. It contained 45 bp of known pmoC sequence, the 5' end of pmoC and 870 bp upstream of the pmoC gene (positions 1 to 1048 in the final sequence [Fig. 3]).

Based on codon usage preference, the end of an open reading frame (orfX) was identified at the 5' end of the cloned sequence. The derived 48 aa were BLAST searched and turned out to be 76% similar (56% identical) to cytochrome c551 peroxidase (residues 301 to 344; complete length, 346 aa) from Pseudomonas aeruginosa.

Duplication of pmo gene clusters. It had been suggested that methanotrophs contain two very similar copies of pmo genes, with 13 differences over 3,183 bp in the two copies of pmoCAB from M. capsulatus Bath being noted (30). However, these differences lead to different restriction patterns, so that the individual copies can be distinguished using hybridization experiments. In addition, the sequences upstream of pmoC from M. capsulatus Bath in the two copies diverge (30). Therefore, the sequence upstream of pmoC in M. trichosporium OB3b (clone BC217) and Methylocystis sp. strain M (clone C1) should be unique and could be used as a point of reference. If a probe specific for the upstream region bound to the same fragment in a particular digest as a probe for the pmoC gene, they must have originated from the same copy of pmo genes. A comparison of the fragments hybridizing with the restriction pattern (Fig. 2 and 3) further verified the origin of clones.

For M. trichosporium OB3b (Table 4), probes OB1, OB2, and OB3 bound to the same 2.7-kb BamHI fragment, indicating that clones BC217, BG3, and P236 originated from the same pmo gene cluster. The same held true for the 6.5-kb SphI fragment. Both OB2 and OB3 bound to the same 1.7-kb BglII fragment, a further indication that the clones were derived from the same cluster. However, there was no 2.3-kb BglII fragment hybridizing to probe OB3, which would be expected if clones P236 and BG114 were derived from the same pmo gene cluster.

                              
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  		TABLE 4.   Hybridization experiments showing the origins of the cloned regions of the pmo gene cluster for M. trichosporium OB3b

For Methylocystis sp. strain M, probe pC1 bound to a 6.0-kb BamHI fragment (Table 5). Probes pC59 and p286 also bound to a 6.0-kb BamHI fragment and---weakly---to a 10.0-kb fragment, suggesting that all three clones originated from the same copy and that the second copy was located on a 10.0-kb BamHI fragment. In the PstI digest, probes pC1 and pC59 bound to the same fragments, further indicating that clones C1 and C59 originated from the same copy and that the second copy of pmoC was located on a 2.5-kb PstI fragment.

                              
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  		TABLE 5.   Hybridization experiments showing the origins of the cloned regions of the pmo gene cluster for Methylocystis sp. strain M

If the upstream sequences were completely different in the two copies, probes OB1 and pC1 would have bound to only one fragment in each digest. Instead, two fragments had hybridized to the probes. However, one band in each digest was considerably fainter than the other, leaving no doubt as to which copy was most similar and thus providing a point of reference. The cross-hybridization of probes OB1 and pC1 binding to the upstream region of both pmo copies indicates that these regions share a higher degree of similarity than anticipated.

Two or three copies of pmoC? Genomic DNA of M. trichosporium OB3b and Methylocystis sp. strain M was digested, Southern blotted, and probed with homologous pmoC probes (probes OB2 and pC59, respectively) and with a Methylocystis sp. strain M pmoC probe (generated using primers C126 and C572 [Table 2]). In each digest, two to four fragments hybridized to each probe (Fig. 4). A comparison with the known restriction patterns strongly suggested that there are only two copies of pmoC in both organisms, but the possibility that there may be three copies, as have been found in M. capsulatus Bath (30), cannot be ruled out completely.


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  		FIG. 4.   Southern blot of genomic DNA probed with a 400-bp pmoC probe homologous to Methylocystis sp. strain M. The wash conditions were 2× SSC at 75°C. Lanes: 1, M. capsulatus Bath digested with SmaI; 2 through 5, M. trichosporium OB3b DNA digested with BclI, EcoRI, NotI, and PstI, respectively; 7 through 10, Methylocystis sp. strain M DNA digested with BclI, PstI, SalI, and XhoI, respectively.

Sequence analysis and comparison of pmo gene clusters from M. trichosporium OB3b, Methylocystis sp. strain M, and M. capsulatus Bath. In M. trichosporium OB3b, the pmo genes consist of three open reading frames designated pmoC (771 bp), pmoA (756 bp), and pmoB (1,296 bp). The intergenic sequences are 244 bp (pmoC-pmoA) and 174 bp (pmoA-pmoB) long. The derived amino acid sequences show that the predicted proteins are highly hydrophobic and contain several transmembrane-spanning regions (Fig. 5). The locations of transmembrane-spanning regions for the M. trichosporium OB3b proteins as predicted by the TMHMM tool (available on the Expasy website [http://www.expasy.ch/tools/#transmem]) are as follows: for PmoC, aa 23 to 41, 63 to 85, 106 to 124, 150 to 168, 175 to 197, and 216 to 238; for PmoA, aa 30 to 48, 67 to 85, 113 to 135, 139 to 161, and 217 to 239; and for PmoB, aa 197 to 215 and 242 to 260. (The first of the three transmembrane-spanning regions for PmoB seen in Fig. 5 was only a theoretical one, since these residues were suggested to constitute a leader sequence [21]). The N termini of the proteins were all predicted to be located in the cytosol.


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  		FIG. 5.   Predicted topology of derived Pmo proteins from M. trichosporium OB3b and Methylocystis sp. strain M. The protein sequences were analyzed with the TMHMM tool (Expasy website [http://www.expasy.ch/tools/#transmem]). The shaded columns depict regions of high hydrophobicity (probability of transmembrane location on the y axis) for amino acid residues (x axis) which are predicted to form transmembrane helices. See the text for the locations of transmembrane helices for M. trichosporium OB3b proteins.

The pmo genes from Methylocystis sp. strain M were identical or very similar in length to pmoC (771 bp), pmoA (756 bp), and pmoB (1,260 bp). The intergenic sequences were 313 and 121 bp for pmoC-pmoA and pmoA-pmoB, respectively. Likewise, the predicted proteins contained several transmembrane-spanning regions (Fig. 5).

The putative Shine-Dalgarno sequences at about 7 bp upstream of the respective start codons were very similar to the E. coli consensus sequence (5' AGGAGG [11]). The program TERMINATOR from the GCG package was used to identify putative rho-independent terminators in the DNA sequence. For M. trichosporium OB3b, a putative terminator was identified 60 bp downstream of pmoB. For Methylocystis sp. strain M, a good putative terminator was identified 60 bp downstream of orfX (although without the TCTG motif). The next putative terminator downstream of pmoB was 500 bp 3' of pmoB.

The three sets of pmo gene sequences known so far showed high identities with each other (Table 6). The pmo genes from the alpha -Proteobacteria M. trichosporium OB3b and Methylocystis sp. strain M were 84% identical to each other; the pmoC genes had the highest identity value (86%), and the pmoB genes had the lowest (83%). They were about 70% identical to the pmo gene sequences of the gamma -proteobacterium M. capsulatus Bath, and again, the pmoC genes had the highest identity (75%). The intergenic sequences did not show significant identities among the three species.

                              
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  		TABLE 6.   Comparison of pmo genes from M. trichosporium OB3b, Methylocystis sp. strain M, and M. capsulatus Bath

Primer extension experiments. The 5' ends of pMMO mRNAs were mapped in primer extension experiments using total RNA isolated from pMMO-expressing cells grown in exponential-growth batch cultures of M. trichosporium OB3b and Methylocystis sp. strain M and from a chemostat culture of M. trichosporium OB3b expressing pMMO. The locations of primers are indicated in Fig. 2 and 3 (for exact positions in the clusters, see Table 3). The primers targeted the regions immediately upstream of the pmo genes (O2 through O4 and M2 through M4) and also the region further upstream of pmoC (O1 and O1A; M1 and M1A).

For Methylocystis sp. strain M, two potential start sites were identified with primer M1 (Fig. 6). The stronger signal mapped to A574, whereas the weaker signal mapped to T586. Just upstream of both, two putative promoter-like sequences (Fig. 7) with good similarities to the sigma 70 consensus sequence in E. coli (12) were identified. The -35 consensus was identical at 5 out of 6 positions and 4 out of 6 positions for A574 and T586, respectively, and the -10 consensus was met by 3 out of 6 for both. In order to confirm that both 5' ends were present in total mRNA, primer M1A was used, which bound in the same region as primer M1 (29 bp further downstream). This gave identical results. Thus, the mRNAs initiated 300 bp upstream of the pmoC start codon. The other primers gave weak signals of primer extension products (data not shown). Primer M2, binding at the start of pmoC, gave two primer extension products mapping to C827 and T828, and with primers M3 and M4, two more putative start sites were found and mapped to G1916 and T2789. However, these signals were very faint compared to that of A574, and there were no obvious similarities to known promoters just upstream (5') of any of these. Therefore, it is likely that these 5' ends were the result of processing of the long transcript from A574.


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  		FIG. 6.   Primer extension analysis to identify the transcriptional start site for the pmo genes in Methylocystis sp. strain M. The positions of the -35 and -10 regions (boxed) and the transcriptional start sites (arrows) are indicated.


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  		FIG. 7.   Alignment of the promoter region in Methylocystis sp. strain M and the equivalent region in M. trichosporium OB3b. The identity is 62% over 58 bp. The -35 and -10 motifs are overlined, and the start of transcription is indicated at +1.

Surprisingly, the same putative promoter sequence that was identified in Methylocystis sp. strain M was present in M. trichosporium OB3b (Fig. 7). However, several attempts with primers O1A, O1, O2, O3, and O4 did not yield any primer extension products, probably indicating that the sequence so far (500 bp upstream of the pmoC gene) does not contain the pmo promoter. Nielsen et al. (23) found a pMMO-specific mRNA of 4.0 kb during growth under high-copper conditions which disappeared during the switch to copper-limited conditions. Since the pmoCAB genes in M. trichosporium OB3b are 3.2 kb in length, the promoter might be up to 800 bp upstream (5') of the pmoC gene. Alternatively, it is possible that the primer extension does not work, for reasons unknown.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We report here the complete sequences of the pmo operon encoding the pMMO from two distinct genera of methanotrophic bacteria, M. trichosporium OB3b and Methylocystis sp. strain M. Both fall in the alpha -subclass of the Proteobacteria. The only other pmo operon sequenced is that of M. capsulatus Bath, a gamma -proteobacterium. We also found transcriptional start sites by primer extension experiments and identified putative promoter sequences. The pmo cluster in both organisms consists of three genes, pmoCAB. Two copies of the clusters are present (Fig. 1 and 4) which are probably almost identical. Our data confirm this for M. trichosporium OB3b. The sequence for pmoB from clone MtB5 was identical to that for clone BG114, although the latter originated from the second copy of pmo genes. Furthermore, the sequence (711 bp) of the second copy of pmoA was identical to that of the first copy in M. trichosporium OB3b (data not shown). Southern hybridization experiments suggested that M. trichosporium OB3b and Methylocystis sp. strain M contained two copies of pmoC instead of three as in M. capsulatus Bath (Fig. 4). Similarity between the pmo gene clusters from the three organisms was highest at the 5' end of the operon and decreased towards the 3' end. Both the intergenic sequences and the regions outside the pmo cluster showed no significant homologies. This was not surprising, since there is no obvious evolutionary pressure on their conservation. However, the intergenic sequences in the two pmo operons of M. capsulatus Bath were nearly as conserved as the genes themselves, and the amoC-amoA intergenic sequences in Nitrosomonas europaea were also nearly identical (14). This should indicate that the duplication events have occurred in each organism relatively recently, certainly after the separation of the ammonia-oxidizing lineage and the methanotroph lineage and also after the separation of different methanotrophic species. It seems unlikely that gene duplication occurred separately in all these groups. Klotz and Norton (15) propose that amo gene duplication occurred a long time ago. Thus, the intergenic regions might have an as yet undiscovered function.

The predicted pMMO polypeptides from M. trichosporium OB3b and Methylocystis sp. strain M are very similar in sequence and structure (Fig. 5). PmoC and PmoA are highly hydrophobic proteins with six predicted transmembrane-spanning regions, whereas PmoB is probably inserted into the membrane with only two helices. The predicted polypeptides contain 17 histidine residues in total for M. trichosporium OB3b and M. capsulatus Bath and 16 for Methylocystis sp. strain M, 12 of which are conserved for all three species and another 3 of which are conserved only in M. trichosporium OB3b and Methylocystis sp. strain M. When the comparison is extended to the subunits of the ammonia monooxygenase (Amo), three histidine residues are conserved in PmoA and AmoA; His 30, His 48, and His 168. It has been proposed that these histidine residues act as copper ligands and may be located at the active site of the enzyme (8). Likewise, there are 4 conserved histidine residues each in PmoB and AmoB and in PmoC and AmoC, and although these polypeptides probably do not contain the active site of the enzyme, it is still possible that they provide ligands for copper ions at the active site. Sayavedra-Soto et al. (27) have proposed that aa 200 to 230 are important for the function of the AmoC and PmoC proteins because of their high degree of conservation. This motif is also apparent in the PmoC proteins from the two species under study here.

At the 5' end of the pmo region sequenced from Methylocystis sp. strain M, a codon usage preference analysis identified a 150-bp open reading frame. The derived amino acid sequence showed high similarity values to cytochrome c peroxidases from P. aeroginosa, Helicobacter spp., and Aquifex spp. Zahn et al. (35) purified a cytochrome c peroxidase from M. capsulatus Bath. They discussed its possible importance in detoxification, as the monooxygenase mechanism involves the activation of oxygen. It seems unlikely that this gene is subject to the same transcriptional regulation as the pmo genes, especially since a good rho-independent terminator was identified 60 bp downstream of orfX. Although we were hoping to find the genes encoding the copper binding compounds found by DiSpirito et al. (7), there are no indications that they are part of the pmo operon.

It was necessary to clone the pmo gene clusters in several fragments, as they seem to be toxic in E. coli (18). In particular, it seems to be impossible to obtain a clone containing both the pmoC promoter and the gene itself. This suggests that it is controlled by a promoter that is active in E. coli and that the overexpression of pmoC is lethal to E. coli.

As there are two copies of the pmo gene clusters, and since several of the clones were generated by PCR with primers that would not discriminate between the copies, it was necessary to confirm that our clones originated from the same copy. This was achieved by multiple probing of chromosomal digests (Table 2). The fragments hybridizing with the various probes were in accordance with the restriction pattern as shown in Fig. 2 and 3.

Primer extension data suggested that the three genes in the pmo cluster were transcribed from a single promoter upstream of pmoC. Previously, Northern blots of M. trichosporium OB3b and M. capsulatus Bath had also revealed the presence of a large transcript encoding the complete operon (23). In Methylocystis sp. strain M, the RNA transcript was shown to initiate 300 bp upstream of the pmoC start codon. We identified the putative promoter, which showed good similarity to the consensus sequence for sigma 70 promoters in E. coli (Fig. 7). This suggests that the transcription of these genes is negatively regulated under copper-limiting conditions. The observation that low levels of pMMO seem to be present in sMMO-expressing M. capsulatus Bath (34) can be explained by low basal transcription and a leaking promoter. In M. trichosporium OB3b, the same conserved motif is present, but surprisingly, transcription did not start here. There was no evidence for a sigma 54-like promoter as was found upstream of mmoX in the sMMO gene cluster, and there was no evidence for intergenic promoters as found in the sMMO operon (22).


    ACKNOWLEDGMENTS

We thank H. Uchiyama for Methylocystis sp. strain M.

B. Gilbert was supported by the Deutsche Forschungsgemeinschaft. This work was also supported by grants from the NERC and EC 4th Framework Programme. Graham Stafford was supported by a BBSRC studentship.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom. Phone: 44 (0) 2476 523553. Fax: 44 (0) 2476 523568. E-mail: cm{at}dna.bio.warwick.ac.uk.

dagger Present address: Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom.

Dagger Present address: Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark.


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Discussion
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Applied and Environmental Microbiology, March 2000, p. 966-975, Vol. 66, No. 3
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