molecular analysis of the pmo (particulate methane ... · utive open reading frames designated...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Mar. 2000, p. 966–975 Vol. 66, No. 3

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,† GRAHAM P. STAFFORD,ALLAN K. NIELSEN,‡ AND J. COLIN MURRELL*

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

Received 11 October 1999/Accepted 14 December 1999

The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II meth-anotrophs of the a-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have beencloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribedfrom a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, forMethylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for s70

promoters were identified, suggesting that these pmo genes are recognized by s70 and negatively regulatedunder low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of thesegenes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies ofpmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order todifferentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copyof 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 g-Proteobacteria, Methylococcus capsulatus Bath. The derivedproteins 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. Hybridizationexperiments 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.

Methane-oxidizing bacteria (methanotrophs) play an impor-tant part in the global carbon cycle, recycling up to 60% (680Tg) of total global methane production per year (25). Methaneis used as the sole source of carbon and energy by theseorganisms. It is oxidized to methanol by the key enzyme meth-ane monooxygenase (MMO). Methanol is further oxidized toformaldehyde. Formaldehyde is then either assimilated intocell biomass or oxidized via formate to carbon dioxide. Allknown methanotrophs possess the membrane-bound or partic-ulate form of MMO (pMMO), and some have a second en-zyme, the cytoplasmic, or soluble, MMO (sMMO).

Two types of methanotrophs can be distinguished on thebasis of biochemical and ultrastructural differences (3, 33).Genetic and biochemical work has been carried out mainly ontwo organisms, the type I methanotroph Methylococcus capsu-latus Bath, a g-proteobacterium, and the type II methanotrophMethylosinus trichosporium OB3b, an a-proteobacterium. An-other 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-con-taining monooxygenase which is oxygen and light sensitive. The26-kDa subunit of pMMO was labeled by acetylene, an inhib-

itor of MMO, indicating that it harbors the active site of theenzyme (6, 24). This subunit is encoded by pmoA, which hasbeen shown to be highly conserved among methanotrophs andcan be used to detect these organisms in a range of environ-ments (13, 16).

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

In methanotrophs possessing both pMMO and sMMO, thepMMO is expressed when copper/biomass ratios in the me-dium 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 thesMMO genes being transcribed under low-copper conditions.Under high-copper conditions, the transcription of sMMOgenes stops and the pMMO genes are transcribed. The pmogenes from M. capsulatus Bath are transcribed into a singlepolycistronic mRNA of 3.3 kb. In addition, smaller transcriptswere observed, representing monocistronic transcripts encod-ing pmoC, pmoA, and pmoB or translationally inactive degra-dation products (23). For M. trichosporium OB3b grown undernon-copper-limiting conditions, a pMMO-specific mRNA of4.0 kb was detected (23).

The cloning of pmo genes has been very difficult becauseparts of these genes seem to be toxic to Escherichia coli (21,

* Corresponding author. Mailing address: Department of BiologicalSciences, University of Warwick, Coventry, CV4 7AL, United King-dom. Phone: 44 (0) 2476 523553. Fax: 44 (0) 2476 523568. E-mail: [email protected].

† Present address: Horticulture Research International, Welles-bourne, Warwick CV35 9EF, United Kingdom.

‡ Present address: Department of Microbiology, Technical Univer-sity of Denmark, DK-2800 Lyngby, Denmark.

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30). The same has been observed for amo genes encoding arelated enzyme, ammonia monooxygenase (18). We reporthere the sequencing and comparative analysis of pmo genesfrom two type II methanotrophs belonging to the a-subclass ofthe Proteobacteria, M. trichosporium OB3b and Methylocystissp. strain M. Primer extension experiments revealed the tran-scriptional start site and putative promoter region of pMMOgenes for Methylocystis sp. strain M and provide evidence forthe genetic organization of these gene clusters.

MATERIALS AND METHODS

Bacterial strains and growth conditions. M. trichosporium OB3b was obtainedfrom the University of Warwick culture collection. Methylocystis sp. strain M waskindly supplied by H. Uchiyama, Tsukuba, Japan. Methanotrophs were grown onnitrate mineral salts medium (NMS) (33) in batch culture with a headspace ofmethane 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. Itwas grown on nutrient agar (Difco) or on Luria-Bertani medium in the presenceof ampicillin (final concentration, 50 mg ml21) where appropriate.

DNA manipulations. Preparation of plasmid DNA and standard DNA manip-ulations 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 frommethanotrophs was isolated as follows. One liter of batch culture (optical densityat 540 nm [OD540] 0.5 to 0.6) was pelleted and resuspended in 5 ml of solution1 (50 mM Tris [pH 8.0]–25% sucrose). Then 0.5 ml of lysozyme (20 mg ml21 in0.25 mM Tris [pH 8.0]) was added. After incubation for 30 min at 37°C, 1 ml of0.25 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 wasincubated 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). ForDNA-DNA hybridizations, nucleic acids were transferred to a nylon membrane(Hybond-N) using a blotting apparatus (Turboblotter; Schleicher & Schuell).Hybridizations were carried out in 63 SSC (13 SSC is 0.15 M NaCl plus 0.015M sodium citrate) at 55°C for oligonucleotide probes and at 60°C for DNAfragment probes. The initial wash was done at 55°C in 63 SSC for oligonucle-otide probes and at 60°C in 23 SSC for DNA fragment probes. The stringencywas then gradually raised by increasing the temperature and lowering the SSCconcentration. DNA probes were radiolabeled with [a-32P]CTP either by nicktranslation (26) or by random priming (9). Probes were generated by PCR withthe primers indicated. Tables 1, 2, and 3 contain information on the probes andprimers used in this study. Oligonucleotides were end labeled with [g-32P]ATPusing T4 kinase (26).

PCR. PCR was performed in 50-ml reaction mixtures in 0.5-ml microcentrifugetubes using a Hybaid Touchdown thermal cycling system. Taq polymerase (GibcoBRL) was used. After an initial denaturation step of 94°C for 5 min, the Taqpolymerase 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. Forexpected PCR products of less than 0.6 kb, cycles of 0.5 min at each temperatureand a final extension of 5 min were run. Reaction products were checked for sizeand 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 intopUC19 or by using the TA cloning kit (Invitrogen) according to the manufac-turer’s instructions. DNA sequencing was carried out using DyeDeoxy termina-tors by the University of Warwick Sequencing Facility, with Perkin-Elmer ABI373A and 377 automated sequencers. In all cases, double-stranded DNA se-quences were obtained by completely sequencing both strands of DNA.

Computer analysis. Analysis of DNA sequences and homology searches werecarried out with standard DNA sequencing programs and the BLAST server of

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

Primer or probe Clonegenerated Positions Use of

primer

M. trichosporium OB3bPrimer A BG3 2136–2155 ForwardPrimer B 1710–1728 ReversePrimer C 1230–1247 ProbePrimer D BC217 829–849 ForwardPrimer E 718–737 ReversePrimer F 656–674 ProbePrimer G BG114 3136–3156 ForwardPrimer H 2926–2947 ReversePrimer I 3239–3251 ProbePrimer J PV216 262–282 ForwardPrimer K 167–188 ReversePC6F 111–128 ProbePrimer Cfor C59 630–647 Forward

Methylocystis sp. strain MPrimer Arev C59 1810–1790 ReversePrimer Bfor MtB5 3514–3531 ForwardPrimer Brev 4102–4084 ReversePrimer L C1 1099–1118 ForwardPrimer M 1092–1071 ReversePrimer N 1025–1045 Probe

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

Probe Primer designations or locations Restriction site within the probe

M. trichosporium OB3bpmoA A189 and A682a SalI 1683, BclI 1954OB1 111–128, 478–461 BamHI 255OB2 656–674, 1042–1023 SalI 773OB3 2162–2180, 2567–2549 BglII 2266

Methylocystis sp. strain MpmoA A189 and A682a SalI 2132, BclI 2402pmoC C126 (126–145) and C572 (572–551)b EcoRI 1046, SalI 1150pC1 302–319, 651–634 HpaI 395pC59 1169–1185, 1589–1572 SalI 1146, BclI 1447, PstI 1578p286 2016–2033, 2418–2435 SalI 2127, BclI 2398

a See reference 13.b Based on pmo copy 1 (accession number L40804) from M. capsulatus Bath.

TABLE 3. Primers used in primer extension experiments

PrimerPosition inrespective

gene clusterSequence (59339)

O1A 118–101 TGG CGC CGC AAA ATG ACGO1 295–278 GCG CAG AAC GAG CGC TCCO2 414–397 TGT TGT TAC GCT CAT CTCO3 1529–1502 CTT CGA TGT AAA CAT CACO4 2481–2465 TCG CCA GTT CGG CCA TTCM1 651–634 GGA CGA ACA CCG ACG TCGM1A 680–663 ACA TCC AAT AAG GCC GCCM2 891–874 CAG CCG TGC TAG TCG TCGM3 1982–1965 CCG CTT TTC GAT TGT GACM4 2867–2851 GCG GCG AGC TTG ACT AGC

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the National Center for Biotechnology Information (NCBI) using the BLASTalgorithm (1, 2). Putative rho-independent terminators, codon usage tables andcodon preferences, and the hydrophobicities of proteins were calculated usingthe Genetics Computer Group (GCG) software package. The locations of pu-tative transmembrane-spanning regions were calculated using the TMHMMtool, 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 culturesto an OD540 of 0.4 to 0.5 (mid-exponential-growth phase). These cultures testednegative for sMMO by a colorimetric sMMO assay (4). The cells were pelletedby centrifugation at 3,500 3 g and stored at 280°C. In addition, fresh 1.5-mlaliquots from a chemostat culture of M. trichosporium OB3b at an OD540 of 7.5were used (dilution rate as described by Nielsen et al. [23]). The chemostatculture received copper sulfate to a final concentration of 50 mM 2 h prior tosampling, to ensure that the cells were expressing pMMO. The cell suspensionthen tested negative for sMMO (4). The cell pellet was resuspended in 200 ml ofsolution I (0.3 M sucrose–0.01 M sodium acetate [pH 4.5]) and 200 ml of solutionII (2% sodium dodecyl sulfate–0.01 M sodium acetate [pH 4.5]). The cell sus-pension was transferred to a blue Ribolyser tube (Hybaid), and 400 ml of phenol(saturated with 50 mM sodium acetate [pH 4.5]) was added. The cells were lysedusing a Hybaid Ribolyser at speed setting 6 for 20 to 40 s. After this step, cellswere kept on ice when possible. The suspension was centrifuged for 5 min, andthe aqueous phase was transferred to a fresh tube. Four hundred microliters ofphenol was added, and the tubes were incubated for 4 min at 65°C and thenfrozen in dry ice-ethanol for 10 s. The tubes were spun for 5 min, and theaqueous phase was transferred to a new tube. Four hundred microliters ofphenol-chloroform was added, mixed vigorously for 30 s, and spun for 5 min. Theaqueous phase was transferred to a new tube, and nucleic acid was precipitatedwith 40 ml of sodium acetate (pH 4.5) and 900 ml of 96% (vol/vol) ethanol at220°C for 20 min. The pellet was washed with 70% (vol/vol) ethanol, dried in avacuum drier, and resuspended in 40 ml of water. The RNA preparations werefinally treated with RNase-free DNase (Gibco BRL) for 30 min at 37°C andexamined using 1.5% (wt/vol) agarose gels. The concentration of nucleic acid insolutions was determined by measuring the A260 using a DU-70 spectrophotom-eter (Beckman).

Primer extension experiments. A 2.5-ml volume of RNA (5 to 10 mg) and 1 mlof [g-32P]ATP-labeled primer (5 ng) were heated for 1 min at 75°C in 1 ml ofhybridization buffer (4.53 hybridization buffer contains 250 mM HEPES [pH7.0] 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 ofavian myeloblastosis virus reverse transcriptase (Amersham) were added to eachprimer extension reaction mixture. The mixture was incubated at 45 or 50°C for30 min. The extension products were precipitated with 1 ml of sodium acetate(pH 4.5) and 20 ml of 96% (vol/vol) ethanol on ice, washed with 70% (vol/vol)ice-cold ethanol, dried, and resuspended in 6 ml of “stop solution” (Sequenase

version 2.0 DNA Sequencing Kit; USB). The extension products were preheatedat 75°C for 2 min and loaded onto an 8% (wt/vol) polyacrylamide gel alongsidea set of dideoxy sequencing products of the appropriate plasmid DNA templatewith the same primer. Sequencing reactions were carried out according to themanufacturer’s instructions (Sequenase version 2.0 DNA Sequencing Kit; USB).Primers O1, O1A, and O2 were used with plasmid BC217; primers O3 and O4were used with plasmid P236; primers M1, M1A, and M2 were used with plasmidC1; and primers M3 and M4 were used with plasmid P286 (see Table 3).

Nucleotide sequence accession numbers. The fully sequenced pmoCAB geneclusters have been deposited in the GenBank database under accession numbersAF186586 and AF186587 for Methylosinus trichosporium OB3b and Methylocystissp. strain M, respectively.

RESULTS

The pmo gene cluster from M. trichosporium OB3b. A South-ern blot of genomic DNA from M. trichosporium OB3b wasprobed with a pmoA probe derived from the known sequenceof M. capsulatus Bath (28) (Fig. 1a). In some digests, two bandswere identified, which suggested that two copies of pmoA werepresent in M. trichosporium OB3b. The 2.0-kb PstI fragmenthybridizing with pmoA was cloned into pUC19 to generateclone P236, and the insert was sequenced (Fig. 2). The se-quence contained regions of DNA which showed high identitywith pmoA and the 59 two-thirds of pmoB from M. capsulatusBath. Since this fragment had a BglII site at the end of theputative pmoA gene, it was concluded that one of the BglIIchromosomal DNA fragments (1.6, 4.8, and 5.2 kb) which hadhybridized to pmoA should contain the pmoA gene and se-quences 59 of pmoA, probably including pmoC.

Attempts to clone the 4.8-kb BglII fragment were unsuccess-ful, probably due to a toxic effect in E. coli. Similar observa-tions have been made previously during attempts to clone pmoand amo genes (18, 28). Instead, we used a PCR approachsimilar to that described by Stolyar et al. (30) to obtainpmoC. M. trichosporium OB3b genomic DNA was digestedwith BglII. The DNA was religated and digested with BclIwhich cut within the known pmoA sequence. This procedureyielded a linear fragment of DNA with unknown sequence inthe middle flanked by the known sequence of pmoA. Primers

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 OB3bat 65°C, 23 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, 23 SSC.

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targeted to the regions immediately next to the BclI site (prim-ers A and B; Table 1) were used in a PCR and amplified a1.2-kb fragment. This was cloned into the TOPO vector (In-vitrogen). The resulting clones were checked for the presenceof sequence upstream of the BglII site by probing with primerC (Table 1). Plasmid DNA from one of the positive clones,BG3, was prepared, and the insert was sequenced. Based onthe M. capsulatus Bath sequence, pmoC, the pmoC-pmoA in-tergenic region, and 214 bp of pmoA were identified. Since thesequence contained the putative start codon of pmoC but nosequence further upstream, two more PCR cloning experi-ments involving digestion with BclI and PvuI and primers D toF and J to K (Table 1) were carried out in order to obtain thesequence upstream of pmoC. In this way, clones BC217 andPV216 were generated, respectively (Fig. 2).

The 39 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 a590-bp fragment from M. trichosporium OB3b chromosomalDNA. This fragment, designated MtB5, was cloned and se-quenced. It contained the rest of the pmoB gene, as expected.It was impossible to get sequence 39 of pmoB using Methylo-cystis sp. strain M-based primers, probably because the se-quences diverge. Therefore, another PCR approach was usedinvolving digestion with BglII, PCR with primers G and H, andprobing with primer I. The insert of the resulting clone BG114was sequenced. It contained all of pmoB, as well as 800 bp 39of pmoB. The pmoB sequences obtained with the two methodswere identical, although it was subsequently determined thatthis downstream sequence originated from the second copy ofpmo genes (see “Duplication of pmo gene clusters” below).The downstream (39) sequence contained the start of anotheropen reading frame, orfD, identified by codon usage prefer-ence; the derived amino acid sequence (104 amino acids [aa])

showed good similarity (52% at the amino acid level) to apartial sequence, orf4, from Nitrosococcus and Nitrosospira spp.(accession numbers AF047705 and U92432). Orf4 is locateddownstream (39) of the amo genes and thus seems to be thehomologous gene in nitrifiers (15). Since clone BG114 wasderived from the second copy of pmo genes, orfD does notappear in Fig. 2.

The pmo gene cluster from Methylocystis sp. strain M. ASouthern blot of genomic DNA from Methylocystis sp. strain Mwas probed with a homologous pmoA probe generated by PCRusing primers A189 and A682 (Table 2) with Methylocystis sp.strain M chromosomal DNA as the template. At least twoDNA fragments were present in a number of different digests,e.g., with PstI, EcoRI, and BglII, suggesting that there were twocopies of pmoA in Methylocystis sp. strain M (Fig. 1b), as hadbeen found in M. capsulatus Bath and M. trichosporium OB3b.A 3.5-kb PstI fragment was cloned into pUC19 to generateP286, and the insert was sequenced (Fig. 3). The sequenceexhibited a high degree of identity with the pmoA and pmoBgenes from M. capsulatus Bath and M. trichosporium OB3b.The region downstream (39) of pmoB contained no open read-ing frames (based on analysis of codon usage preference) andshowed no significant homology to polypeptides in the data-base.

Based on the assumption that the pmo genes in M. tricho-sporium OB3b and Methylocystis sp. strain M have a high de-gree of similarity, the known M. trichosporium OB3b sequencewas used to PCR amplify the homologous sequence fromMethylocystis sp. strain M: a reverse primer, Arev, targeting theintergenic region pmoC-pmoA from Methylocystis sp. strain M,and a forward primer, Cfor, specific to the pmoC sequencefrom M. trichosporium OB3b, amplified a 740-bp fragmentfrom genomic DNA. This was cloned into the TOPO vector(Invitrogen) to produce clone C59. The insert was sequenced

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.



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and found to contain most of pmoC (positions 1004 to 1749 inthe final sequence [Fig. 3]). The start of pmoC was obtainedusing a PCR approach. Genomic DNA was digested with SalI,religated, and digested again with SstI. Primers targeting theregion at the SstI site (primers L and M [Table 1]) amplified afragment of 1.2 kb. It was cloned into the TOPO vector (In-vitrogen) to generate clone C1, and the insert was sequenced.It contained 45 bp of known pmoC sequence, the 59 end ofpmoC and 870 bp upstream of the pmoC gene (positions 1 to1048 in the final sequence [Fig. 3]).

Based on codon usage preference, the end of an open read-ing frame (orfX) was identified at the 59 end of the clonedsequence. The derived 48 aa were BLAST searched and turnedout to be 76% similar (56% identical) to cytochrome c551peroxidase (residues 301 to 344; complete length, 346 aa) fromPseudomonas aeruginosa.

Duplication of pmo gene clusters. It had been suggested thatmethanotrophs contain two very similar copies of pmo genes,with 13 differences over 3,183 bp in the two copies of pmoCABfrom M. capsulatus Bath being noted (30). However, thesedifferences lead to different restriction patterns, so that theindividual copies can be distinguished using hybridization ex-periments. In addition, the sequences upstream of pmoC fromM. 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) shouldbe unique and could be used as a point of reference. If a probespecific for the upstream region bound to the same fragment ina particular digest as a probe for the pmoC gene, they musthave originated from the same copy of pmo genes. A compar-ison 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, indicat-ing that clones BC217, BG3, and P236 originated from thesame pmo gene cluster. The same held true for the 6.5-kb SphIfragment. Both OB2 and OB3 bound to the same 1.7-kb BglIIfragment, a further indication that the clones were derivedfrom the same cluster. However, there was no 2.3-kb BglIIfragment hybridizing to probe OB3, which would be expectedif clones P236 and BG114 were derived from the same pmogene cluster.

For Methylocystis sp. strain M, probe pC1 bound to a 6.0-kbBamHI fragment (Table 5). Probes pC59 and p286 also boundto a 6.0-kb BamHI fragment and—weakly—to a 10.0-kb frag-ment, suggesting that all three clones originated from the samecopy and that the second copy was located on a 10.0-kb BamHIfragment. In the PstI digest, probes pC1 and pC59 bound to the

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.

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

Fragment

Size(s) of band(s) (kb)a with probeb:

OB1 OB2 OB3

Observed Expected Observed Expected Observed Expected

BamHI (1.8), 2.7, (.10.0) .0.155, 2.714 2.7 2.714 2.7, (4.2) 2.714BglII (1.9), 5.2 .0.527 1.7, (3.5) 1.639 1.7, (7.5, .9.0) 1.639, 2.282SalI 0.95, 1.65 .0.673 0.7, (0.95, 1.65, 1.7) 0.689, (.0.6) (0.7), 1.8 1.841SphI 6.5 .2.496 6.5 .2.964 6.5 .2.496

a Observed, after hybridization; Expected, calculated according to the restriction pattern (Fig. 2 and 3). Sizes given in parentheses indicate weak bands.b See Table 1 and Fig. 2 and 3 for positions of probes.



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same fragments, further indicating that clones C1 and C59originated from the same copy and that the second copy ofpmoC was located on a 2.5-kb PstI fragment.

If the upstream sequences were completely different in thetwo copies, probes OB1 and pC1 would have bound to onlyone fragment in each digest. Instead, two fragments had hy-bridized to the probes. However, one band in each digest wasconsiderably fainter than the other, leaving no doubt as towhich copy was most similar and thus providing a point ofreference. The cross-hybridization of probes OB1 and pC1binding to the upstream region of both pmo copies indicatesthat these regions share a higher degree of similarity thananticipated.

Two or three copies of pmoC? Genomic DNA of M. tricho-sporium OB3b and Methylocystis sp. strain M was digested,Southern blotted, and probed with homologous pmoC probes(probes OB2 and pC59, respectively) and with a Methylocystissp. strain M pmoC probe (generated using primers C126 andC572 [Table 2]). In each digest, two to four fragments hybrid-ized to each probe (Fig. 4). A comparison with the knownrestriction patterns strongly suggested that there are only twocopies of pmoC in both organisms, but the possibility that theremay be three copies, as have been found in M. capsulatus Bath(30), cannot be ruled out completely.

Sequence analysis and comparison of pmo gene clustersfrom M. trichosporium OB3b, Methylocystis sp. strain M, and

M. capsulatus Bath. In M. trichosporium OB3b, the pmo genesconsist of three open reading frames designated pmoC (771bp), pmoA (756 bp), and pmoB (1,296 bp). The intergenicsequences are 244 bp (pmoC-pmoA) and 174 bp (pmoA-pmoB)long. The derived amino acid sequences show that the pre-dicted proteins are highly hydrophobic and contain severaltransmembrane-spanning regions (Fig. 5). The locations oftransmembrane-spanning regions for the M. trichosporiumOB3b proteins as predicted by the TMHMM tool (available onthe Expasy website [http://www.expasy.ch/tools/#transmem])are as follows: for PmoC, aa 23 to 41, 63 to 85, 106 to 124, 150to 168, 175 to 197, and 216 to 238; for PmoA, aa 30 to 48, 67to 85, 113 to 135, 139 to 161, and 217 to 239; and for PmoB, aa197 to 215 and 242 to 260. (The first of the three transmem-brane-spanning regions for PmoB seen in Fig. 5 was only atheoretical one, since these residues were suggested to consti-tute a leader sequence [21]). The N termini of the proteinswere all predicted to be located in the cytosol.

The pmo genes from Methylocystis sp. strain M were identi-cal or very similar in length to pmoC (771 bp), pmoA (756 bp),and pmoB (1,260 bp). The intergenic sequences were 313 and121 bp for pmoC-pmoA and pmoA-pmoB, respectively. Like-wise, the predicted proteins contained several transmembrane-spanning regions (Fig. 5).

The putative Shine-Dalgarno sequences at about 7 bp up-stream of the respective start codons were very similar to the E.coli consensus sequence (59 AGGAGG [11]). The programTERMINATOR from the GCG package was used to identifyputative rho-independent terminators in the DNA sequence.For M. trichosporium OB3b, a putative terminator was identi-fied 60 bp downstream of pmoB. For Methylocystis sp. strain M,a good putative terminator was identified 60 bp downstream oforfX (although without the TCTG motif). The next putativeterminator downstream of pmoB was 500 bp 39 of pmoB.

The three sets of pmo gene sequences known so far showedhigh identities with each other (Table 6). The pmo genes fromthe a-Proteobacteria M. trichosporium OB3b and Methylocystissp. strain M were 84% identical to each other; the pmoC geneshad the highest identity value (86%), and the pmoB genes hadthe lowest (83%). They were about 70% identical to the pmogene sequences of the g-proteobacterium M. capsulatus Bath,and again, the pmoC genes had the highest identity (75%). Theintergenic sequences did not show significant identities amongthe three species.

Primer extension experiments. The 59 ends of pMMOmRNAs were mapped in primer extension experiments usingtotal RNA isolated from pMMO-expressing cells grown inexponential-growth batch cultures of M. trichosporium OB3band Methylocystis sp. strain M and from a chemostat culture ofM. trichosporium OB3b expressing pMMO. The locations ofprimers are indicated in Fig. 2 and 3 (for exact positions in the

FIG. 4. Southern blot of genomic DNA probed with a 400-bp pmoC probehomologous to Methylocystis sp. strain M. The wash conditions were 23 SSC at75°C. Lanes: 1, M. capsulatus Bath digested with SmaI; 2 through 5, M. trichos-porium OB3b DNA digested with BclI, EcoRI, NotI, and PstI, respectively; 7through 10, Methylocystis sp. strain M DNA digested with BclI, PstI, SalI, andXhoI, respectively.

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

Fragment

Size(s) of band(s) (kb)a with probeb:

pC1 pC59 p286

Observed Expected Observed Expected Observed Expected

BamHI 6.0 .3.425 6.0, .10.0 .3.425 6.0, (.10.0) .3.425BglII 7.5, (.10.0) .2.704 7.5, (.10.0) .2.704 7.5, (.10.0) .2.704PstI 1.7, (.10.0) 1.572 1.7, (2.5, .10.0) .1.572 3.5, 4.5 3.468XhoI 2.8, 2.9 .1.927 2.4, 2.8, 2.9 .1.927 1.4 1.431

a Observed, after hybridization; Expected, calculated according to the restriction pattern (Fig. 2 and 3). Sizes given in parentheses indicate weak bands.b See Table 1 and Fig. 2 and 3 for positions of probes.



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clusters, see Table 3). The primers targeted the regions imme-diately upstream of the pmo genes (O2 through O4 and M2through M4) and also the region further upstream of pmoC(O1 and O1A; M1 and M1A).

For Methylocystis sp. strain M, two potential start sites wereidentified with primer M1 (Fig. 6). The stronger signal mappedto A574, whereas the weaker signal mapped to T586. Just up-stream of both, two putative promoter-like sequences (Fig. 7)with good similarities to the s70 consensus sequence in E. coli(12) were identified. The 235 consensus was identical at 5 outof 6 positions and 4 out of 6 positions for A574 and T586,respectively, and the 210 consensus was met by 3 out of 6 for

both. In order to confirm that both 59 ends were present intotal mRNA, primer M1A was used, which bound in the sameregion as primer M1 (29 bp further downstream). This gaveidentical results. Thus, the mRNAs initiated 300 bp upstreamof the pmoC start codon. The other primers gave weak signalsof primer extension products (data not shown). Primer M2,binding at the start of pmoC, gave two primer extension prod-ucts mapping to C827 and T828, and with primers M3 and M4,two more putative start sites were found and mapped to G1916and T2789. However, these signals were very faint compared tothat of A574, and there were no obvious similarities to knownpromoters just upstream (59) of any of these. Therefore, it is

FIG. 5. Predicted topology of derived Pmo proteins from M. trichosporium OB3b and Methylocystis sp. strain M. The protein sequences were analyzed with theTMHMM tool (Expasy website [http://www.expasy.ch/tools/#transmem]). The shaded columns depict regions of high hydrophobicity (probability of transmembranelocation 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 forM. trichosporium OB3b proteins.

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

Comparison

pmoC pmoA pmoB

% ofDNA

identity

% of aasimilarity(identity)

% ofDNA

identity


% ofDNA

identity


M. trichosporium OB3b with Methylocystis sp. strain M 86 94 (86) 84 95 (87) 83 89 (81)M. trichosporium OB3b with M. capsulatus Batha 75 80 (60) 69 81 (59) 63 69 (46)Methylocystis sp. strain M with M. capsulatus Bath 75 80 (61) 70 78 (58) 62 68 (46)

a Copy 1 of pmo (accession number L40804) from M. capsulatus Bath was used for the analysis, but since copies 1 and 2 are nearly identical, the values are the samefor copy 2 except for pmoC2, which is slightly less similar (1% at both the DNA and the amino acid level).



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likely that these 59 ends were the result of processing of thelong transcript from A574.

Surprisingly, the same putative promoter sequence that wasidentified in Methylocystis sp. strain M was present in M. tri-chosporium OB3b (Fig. 7). However, several attempts withprimers O1A, O1, O2, O3, and O4 did not yield any primerextension products, probably indicating that the sequence so

far (500 bp upstream of the pmoC gene) does not contain thepmo promoter. Nielsen et al. (23) found a pMMO-specificmRNA of 4.0 kb during growth under high-copper conditionswhich disappeared during the switch to copper-limited condi-tions. Since the pmoCAB genes in M. trichosporium OB3b are3.2 kb in length, the promoter might be up to 800 bp upstream(59) of the pmoC gene. Alternatively, it is possible that theprimer extension does not work, for reasons unknown.

DISCUSSION

We report here the complete sequences of the pmo operonencoding the pMMO from two distinct genera of methanotro-phic bacteria, M. trichosporium OB3b and Methylocystis sp.strain M. Both fall in the a-subclass of the Proteobacteria. Theonly other pmo operon sequenced is that of M. capsulatusBath, a g-proteobacterium. We also found transcriptional startsites by primer extension experiments and identified putativepromoter sequences. The pmo cluster in both organisms con-sists of three genes, pmoCAB. Two copies of the clusters arepresent (Fig. 1 and 4) which are probably almost identical. Ourdata confirm this for M. trichosporium OB3b. The sequence forpmoB from clone MtB5 was identical to that for clone BG114,although the latter originated from the second copy of pmogenes. Furthermore, the sequence (711 bp) of the second copyof pmoA was identical to that of the first copy in M. trichospo-rium OB3b (data not shown). Southern hybridization experi-ments suggested that M. trichosporium OB3b and Methylocystissp. strain M contained two copies of pmoC instead of three asin M. capsulatus Bath (Fig. 4). Similarity between the pmo geneclusters from the three organisms was highest at the 59 end ofthe operon and decreased towards the 39 end. Both the inter-genic sequences and the regions outside the pmo clustershowed no significant homologies. This was not surprising,since there is no obvious evolutionary pressure on their con-servation. However, the intergenic sequences in the two pmooperons of M. capsulatus Bath were nearly as conserved as thegenes themselves, and the amoC-amoA intergenic sequences inNitrosomonas europaea were also nearly identical (14). Thisshould indicate that the duplication events have occurred ineach organism relatively recently, certainly after the separationof the ammonia-oxidizing lineage and the methanotroph lin-eage and also after the separation of different methanotrophicspecies. It seems unlikely that gene duplication occurred sep-arately in all these groups. Klotz and Norton (15) propose thatamo gene duplication occurred a long time ago. Thus, theintergenic regions might have an as yet undiscovered function.

The predicted pMMO polypeptides from M. trichosporiumOB3b and Methylocystis sp. strain M are very similar in se-quence and structure (Fig. 5). PmoC and PmoA are highlyhydrophobic proteins with six predicted transmembrane-span-ning regions, whereas PmoB is probably inserted into themembrane with only two helices. The predicted polypeptidescontain 17 histidine residues in total for M. trichosporiumOB3b and M. capsulatus Bath and 16 for Methylocystis sp. strain

FIG. 6. Primer extension analysis to identify the transcriptional start site forthe pmo genes in Methylocystis sp. strain M. The positions of the 235 and 210regions (boxed) and the transcriptional start sites (arrows) are indicated.

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. The235 and 210 motifs are overlined, and the start of transcription is indicated at 11.



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M, 12 of which are conserved for all three species and another3 of which are conserved only in M. trichosporium OB3b andMethylocystis sp. strain M. When the comparison is extended tothe subunits of the ammonia monooxygenase (Amo), threehistidine residues are conserved in PmoA and AmoA; His 30,His 48, and His 168. It has been proposed that these histidineresidues act as copper ligands and may be located at the activesite of the enzyme (8). Likewise, there are 4 conserved histi-dine residues each in PmoB and AmoB and in PmoC andAmoC, and although these polypeptides probably do not con-tain the active site of the enzyme, it is still possible that theyprovide ligands for copper ions at the active site. Sayavedra-Soto et al. (27) have proposed that aa 200 to 230 are importantfor the function of the AmoC and PmoC proteins because oftheir high degree of conservation. This motif is also apparentin the PmoC proteins from the two species under study here.

At the 59 end of the pmo region sequenced from Methylo-cystis sp. strain M, a codon usage preference analysis identifieda 150-bp open reading frame. The derived amino acid se-quence showed high similarity values to cytochrome c peroxi-dases 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 de-toxification, as the monooxygenase mechanism involves theactivation of oxygen. It seems unlikely that this gene is subjectto the same transcriptional regulation as the pmo genes, espe-cially since a good rho-independent terminator was identified60 bp downstream of orfX. Although we were hoping to findthe genes encoding the copper binding compounds found byDiSpirito et al. (7), there are no indications that they are partof the pmo operon.

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

As there are two copies of the pmo gene clusters, and sinceseveral of the clones were generated by PCR with primers thatwould not discriminate between the copies, it was necessary toconfirm that our clones originated from the same copy. Thiswas achieved by multiple probing of chromosomal digests (Ta-ble 2). The fragments hybridizing with the various probes werein accordance with the restriction pattern as shown in Fig. 2and 3.

Primer extension data suggested that the three genes in thepmo cluster were transcribed from a single promoter upstreamof pmoC. Previously, Northern blots of M. trichosporium OB3band M. capsulatus Bath had also revealed the presence of alarge transcript encoding the complete operon (23). In Methy-locystis sp. strain M, the RNA transcript was shown to initiate300 bp upstream of the pmoC start codon. We identified theputative promoter, which showed good similarity to the con-sensus sequence for s70 promoters in E. coli (Fig. 7). Thissuggests that the transcription of these genes is negativelyregulated under copper-limiting conditions. The observationthat low levels of pMMO seem to be present in sMMO-ex-pressing M. capsulatus Bath (34) can be explained by low basaltranscription and a leaking promoter. In M. trichosporiumOB3b, the same conserved motif is present, but surprisingly,transcription did not start here. There was no evidence for as54-like promoter as was found upstream of mmoX in thesMMO gene cluster, and there was no evidence for intergenicpromoters 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 4thFramework Programme. Graham Stafford was supported by a BBSRCstudentship.

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