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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,
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
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ABSTRACT |
The particulate methane monooxygenase gene clusters,
pmoCAB, from two representative type II methanotrophs of
the 
-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 
70 promoters were identified, suggesting that these
pmo genes are recognized by 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 
-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.
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INTRODUCTION |
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
-proteobacterium, and the type II methanotroph Methylosinus trichosporium OB3b, an -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
-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.
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MATERIALS AND METHODS |
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 ml1 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 [-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
[-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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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 [-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.
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RESULTS |
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.
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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.
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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
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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
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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.
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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
-
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
-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.
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 A
574, whereas the weaker
signal mapped to T
586. Just upstream
of both, two putative
promoter-like sequences (Fig.
7) with
good
similarities to the
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
A
574 and T
586, 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
C
827 and T
828, and with primers M3 and M4, two
more
putative start sites were found and mapped to G
1916
and T
2789.
However, these signals were very faint compared
to that of A
574,
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
A
574.
View larger version (15K):
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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.
|
|
View larger version (8K):
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[in a new window]
|
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 |
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 -subclass of the
Proteobacteria. The only other pmo operon sequenced is that of M. capsulatus Bath, a
-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 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 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.
Present address: Horticulture Research International, Wellesbourne,
Warwick CV35 9EF, United Kingdom.
Present address: Department of Microbiology, Technical University
of Denmark, DK-2800 Lyngby, Denmark.
| |
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Applied and Environmental Microbiology, March 2000, p. 966-975, Vol. 66, No. 3
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Abstract
Introduction
