Microbiology (2003), 149, 1785–1795
DOI 10.1099/mic.0.26061-0
Genes involved in the copper-dependent
regulation of soluble methane monooxygenase of
Methylococcus capsulatus (Bath): cloning,
sequencing and mutational analysis
Róbert Csáki,1,2 Levente Bodrossy,1,23 József Klem,1,2 J. Colin Murrell3
and Kornél L. Kovács1,2
1
Department of Biotechnology, University of Szeged, Szeged, Hungary
Correspondence
Kornél L. Kovács
2
[email protected]
3
Institute of Biophysics, Biological Research Center, Szeged, Hungary
Received 17 October 2002
Revised
26 February 2003
Accepted 20 March 2003
Department of Biological Sciences, University of Warwick, Coventry, UK
The key enzyme in methane metabolism is methane monooxygenase (MMO), which catalyses the
oxidation of methane to methanol. Some methanotrophs, including Methylococcus capsulatus
(Bath), possess two distinct MMOs. The level of copper in the environment regulates the
biosynthesis of the MMO enzymes in these methanotrophs. Under low-copper conditions, soluble
MMO (sMMO) is expressed and regulation takes place at the level of transcription. The structural
genes of sMMO were previously identified as mmoXYBZ, mmoD and mmoC. Putative
transcriptional start sites, containing a s70- and a sN-dependent motif, were identified in the
59 region of mmoX. The promoter region of mmoX was mapped using truncated 59 end regions
fused to a promoterless green fluorescent protein gene. A 9?5 kb region, adjacent to the
sMMO structural gene cluster, was analysed. Downstream (39) from the last gene of the operon,
mmoC, four ORFs were found, mmoG, mmoQ, mmoS and mmoR. mmoG shows significant
identity to the large subunit of the bacterial chaperonin gene, groEL. In the opposite orientation,
two genes, mmoQ and mmoS, showed significant identity to two-component sensor–regulator
system genes. Next to mmoS, a gene encoding a putative sN-dependent transcriptional activator,
mmoR was identified. The mmoG and mmoR genes were mutated by marker-exchange
mutagenesis and the effects of these mutations on the expression of sMMO was investigated.
sMMO transcription was impaired in both mutants. These results indicate that mmoG and mmoR
are essential for the expression of sMMO in Mc. capsulatus (Bath).
INTRODUCTION
Methanotrophs are a subgroup of methylotrophic bacteria,
which have the distinctive property of utilizing methane as
their sole source of carbon and energy. The key enzyme in
methane metabolism is methane monooxygenase (MMO),
which catalyses the oxidation of methane to methanol.
Some methanotrophs, including Methylococcus capsulatus
(Bath), possess two distinct MMOs (Lipscomb, 1994). The
3Present address: Department of Biotechnology, Division of Environmental and Life Sciences, Austrian Research Center, Seibersdorf,
Austria.
Abbreviations: (E)GFP, (enhanced) green fluorescent protein; IHF,
integration host factor; (p)(s)MMO, (particulate) (soluble) methane
monooxygenase; NMS, nitrate mineral salts; Gm, gentamicin; Km,
kanamycin.
The GenBank accession number for the sequence reported in this
paper is AF525283.
0002-6061 G 2003 SGM
biosynthesis of the MMO enzymes in these methanotrophs
is regulated by the level of copper available to the cells. When
methanotrophs are grown at a high copper-to-biomass
ratio, the membrane-bound, particulate MMO (pMMO) is
synthesized. pMMO (Zahn & Dispirito, 1996) is present in
all known methanotrophs (reviewed by Hanson & Hanson
1996). At a low copper-to-biomass ratio, some methanotrophs produce a soluble, cytoplasmic methane-oxidizing
enzyme (sMMO), e.g. Mc. capsulatus (Bath) (Stanley et al.,
1983), Methylosinus trichosporium OB3b (Lipscomb, 1994)
and Methylomonas (Shigematsu et al., 1999).
Regulation of copper-dependent MMO expression takes
place at the level of transcription (Nielsen et al., 1997;
Murrell et al., 2000). The intriguing and strong copper
regulation of the expression of MMO enzymes has been the
subject of several studies, but details of the mechanism of
regulation remain to be elucidated. A DNA fragment was
isolated from Mc. capsulatus (Bath) (Stainthorpe et al.,
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R. Csáki and others
1990), containing the gene cluster encoding sMMO,
consisting of mmoXYBZDC. It occurs in a single copy in
the genome of Mc. capsulatus (Bath) and its transcription is
initiated from a putative promoter possessing s70 and sN
recognition sites (Nielsen et al., 1997). pMMO is encoded by
the pmoCAB gene cluster. Mc. capsulatus (Bath) harbours
two almost identical sets of the pmoCAB cluster (Semrau
et al., 1995). In addition, a third pmoC gene has been
detected (Stolyar et al., 1999). The physiological significance
of multiple gene copies is unclear. However, the expression
and activity of pMMO in Mc. capsulatus (Bath) depends on
the availability of copper (Stanley et al., 1983; Nielsen et al.,
1997; Stolyar et al., 1999). The upstream (59) regions of
the pmo gene clusters appear to contain s70-binding sites
(Stolyar et al., 2001; Gilbert et al., 2000). In addition to
down-regulation of the biosynthesis of sMMO, copper ions
also inhibit the activity of sMMO in the cell (Green et al.,
1985; Jahng & Wood, 1996). A completely copper-deficient
environment, which would favour the expression of sMMO,
rarely exists in nature (Forstner & Wittman, 1979), therefore
pMMO is likely to be functional in most natural environments (Phelps et al., 1992; Hanson & Hanson, 1996). This is
disadvantageous for potential bioremediation applications
since sMMO has much wider substrate specificity than
pMMO (Burrows et al., 1984). sMMO is capable of degrading a number of recalcitrant and carcinogenic hazardous
chemicals (reviewed by Hanson & Hanson, 1996), hence for
biotechnological exploitation, control over the copperregulated expression of MMOs would be desirable. In this
study we aimed to identify the genes involved in the
copper-dependent regulation of sMMO expression in
Mc. capsulatus (Bath). mmoG, mmoQ, mmoS and mmoR
were identified downstream (39) from mmoXYBZDC, and
the phenotypic characterization of mutant strains deficient
in mmoG and mmoR genes was carried out.
METHODS
DNA manipulations. DNA manipulations were performed according to Sambrook et al. (1989). Dynazyme polymerase (Finnzymes)
was used in routine PCR experiments and Pfu polymerase
(Fermentas) was used when required (due to its proof-reading capability), according to the manufacturers’ instructions. The identities
of cloned PCR products were confirmed by DNA sequencing.
Sequencing was done on both strands with an Applied Biosystems
373 Stretch automatic DNA sequencer.
In silico analysis. Homology searches in the GenBank database
were carried out using the BLAST, BLASTX and BLASTP programs
(http://www.ncbi.nlm.nih.gov/). Protein motifs were identified by
PROSCAN (http://npsa-pbil.ibcp.fr). Promoters were predicted by
Neural Network Promoter Prediction (http://www.fruitfly.org/seq_
tools/promoter.html). Amino acid sequences were aligned using
CLUSTAL W software (Thompson et al., 1997). Homology searches in
the TIGR unfinished microbial genomes database were carried out
using the TIGR BLAST search engine for unfinished microbial
genomes: http://tigrblast.tigr.org/ufmg/ Preliminary sequence data
were obtained from the TIGR website at http://www.tigr.org.
Membrane topologies of polypeptides were predicted by the TMHMM
(version 2.0) server (http://www.cbs.dtu.dk/services/TMHMM/).
Isolation of total RNA. Total RNA was isolated from 4 ml aliquots
Growth media and strains. Bacterial strains used in this work are
listed in Table 1. Strains were routinely grown in nitrate mineral
salts (NMS) medium (Whittenbury & Dalton, 1981) containing
5?0 mM CuSO4. Low-copper medium was prepared without adding
CuSO4. Growth of Mc. capsulatus (Bath) under these low-copper
conditions allows sMMO expression in Mc. capsulatus (Bath). Solid
medium was prepared by the addition of agar (Gibco) (1?5 %, w/v)
and plates were incubated in anaerobic jars under a CH4/air/CO2
(50 : 48 : 2 %, by vol.) gas mixture for 5–7 days. Liquid cultures
(50 ml) were grown in 500 ml flasks fitted with rubber Suba Seals.
The headspace was filled with the same gas mixture. All cultures of
Mc. capsulatus (Bath) were grown at 45 uC. Escherichia coli strain
DH5a was used for propagating plasmids. Antibiotic resistance was
selected by the addition of kanamycin (Km, 25 mg ml21) or gentamicin (Gm, 10 mg ml21), as required.
Conjugation and construction of mutant strains. For conjugation, the E. coli S17-1 lpir mobilizer strain (Herrero et al., 1990) was
used as the donor strain after transformation with the appropriate
plasmid. Reporter constructs and plasmids for mutagenesis were
introduced into Mc. capsulatus (Bath) by conjugation. Donor
(E. coli) and recipient (Mc. capsulatus) cells were harvested when the
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cell densities reached 56108 cells ml21. Two millilitres of donor
and 30 ml recipient culture were mixed and resuspended in NMS
medium. Cells were then pelleted (13 000 g, 4 uC, 3 min) and resuspended in 200 ml NMS. The resulting cell suspension was plated as a
spot on a 0?2 mm pore size nitrocellulose filter disc (Reanal) and
placed onto an NMS plate containing 0?02 % (w/v) proteose peptone. Plates were incubated for 16 h at 37 uC under a CH4/air/CO2
(50 : 48 : 2 %, by vol.) gas mixture. Transconjugants were subsequently selected on NMS agar plates, supplied with the appropriate antibiotics (Gm, 10 mg ml21; Km, 25 mg ml21). Correct insertion
of the Gm resistance gene (aacC1) was tested by Southern blotting
and DNA–DNA hybridization. Genomic DNA from Km-sensitive
and Gm-resistant clones was purified and digested with appropriate
restriction enzymes. Southern blotting (Sambrook et al., 1989) was
used to transfer the DNA onto Hybond-N+ (Amersham) membrane. DNA–DNA hybridizations were carried out with gene-specific
and vector-specific probes as described by Sambrook et al. (1989).
The correct insertion and recombination events were confirmed by
PCR, using primers specific for the appropriate DNA fragments
(Table 1).
of Mc. capsulatus (Bath) batch cultures grown to a cell density of
108 ml21. Cells were centrifuged at 12 000 g and resuspended in
300 ml SET buffer [20 % (w/v) sucrose, 50 mM EDTA (pH 8?0),
50 mM Tris/HCl (pH8?0)]. Lysis buffer (300 ml) [20 % (w/v) SDS,
1 % (w/v) (NH4)2SO4, pH 4?8] was added and tubes were briefly
mixed by inversion until the solution become clear. Cell debris and
most of the DNA was precipitated by the addition of 300 ml saturated
NaCl solution, followed by centrifugation (12 000 g for 30 min).
RNA was precipitated from the supernatant by the addition of 500 ml
2-propanol (Sigma) and collected by centrifugation at 12 000 g for
30 min. The pellet was washed twice with 70 % (v/v) ethanol, resuspended in H2O and analysed on a 1?5 % (w/v) agarose gel.
RT-PCR. Total RNA extracts from Mc. capsulatus (Bath) were treated
with DNase I (Amersham). Total RNA (1 mg) was added to 30 pmol
reverse primer mxatgrev (Table 1) in 9 ml water. Samples were incubated at 70 uC for 15 min then cooled on ice. Four microlitres
dNTPs, 4 ml M-MLV 56 buffer, 0?5 ml RNasin ribonuclease inhibitor (Promega) and 1 U M-MLV reverse transcriptase (Promega)
were mixed with 11 ml DNase-treated RNA and incubated at 37 uC
for 1 h, followed by 5 min at 70 uC to inactivate the enzymes. A
sample (2?5 ml) of the reverse transcription reaction was used as
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Microbiology 149
Putative regulatory genes of sMMO
Table 1. Bacterial strains, plasmids and primers used in this study
Strain/plasmid/primer
Strains
Mc. capsulatus (Bath)
E. coli S17-1 lpir
E. coli DH5a
Plasmids
pBluescript2SK+
pK18mob
pK18mobsac
p34S-Gm
pPROBE-NT
pBBR1MCS-4
pCH4
pBX2
pMCS441
pKPS
pKPSg
pKHK
pKHKg
pKHKg2
pKHKg4
pKHKg6
pMX1
pMX2
pMX3
pMX4
pKmoG
pKmoR
Primers
MXr
MXf1
MXf2
MXf3
MXf4
pmoRf
pmoRr
mxatgrev
mxatgfw
HKfw
HKrev
Characteristics
recA thi pro hsdR2 RP4-2-Tc : : Mu Km : : Tn7 lpir
recA1 endA1 gyrA96 thi-1 relA1 hsdR17 (supE44 DlacU169) (W80lacZDM15)
Whittenbury et al. (1970)
Herrero et al. (1990)
Hanahan (1983)
ApR cloning vector
Kmr RP4mob, mobilizable cloning vector
Kmr RP4mob, mobilizable cloning vector with sacB gene for positive selection
ApR GmR, source of GmR cassette
KmR, promoter-probe vector with EGFP
ApR broad host range, mobilizable cloning vector
pBR325 with 12?5 kb EcoRI insert
pBluescript2SK+ with 1?1 kb Bsp120I insert
pBBR1MCS-4 with 4?2 kb Bsp120I insert
pK18mob with 1?0 kb EcoRI–BglII insert
pK18mob with 27 kb HindIII insert
pK18mob with 0?7 kb insert
pK18mob with 4?7 kb insert
pK18mob with 1?0 kb insert
pK18mob with 2?3 kb insert
pK18mob with 1?4 kb insert
pPROBE-NT with 475 bp HindIII–BamHI insert
pPROBE-NT with 335 bp HindIII–BamHI insert
pPROBE-NT with 258 bp HindIII–BamHI insert
pPROBE-NT with 165 bp HindIII–BamHI insert
pK18mobsac with 1?9 kb mmoG : : aacC1 insert
pK18mobsac with 1?5 kb mmoR : : aacC1 insert
Stratagene
Schäfer et al. (1994)
Schäfer et al. (1994)
Dennis & Zylstra (1998)
Miller et al. (2000)
Kovach et al. (1995)
Stainthorpe et al. (1989)
This work
This work
This work
This work
This work
This work
This work
This work
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This work
This work
59-CAGGATCCATGATGAATGCCCGATGA-39
59-CTAAGCTTCTGCCGCAGTTCGTCGC-39
59-CTAAGCTTTCCGCGCAGCCCGATTC-39
59-CTAAGCTTGAGTTATGGCGGCCCAGTA-39
59-CTAAGCTTCGGAGCATTCGGATAACG-39
59-AGTGGAAGTGCGCGATTCGG-39
59-CTGGATTGCGGCGCAGTCTA-39
59-TTCTTCTGTTCCGCCGCCT-39
59-ATGGCACTTCGCACCGCAACC-39
59-GTGGGTCAGATAATAGGCTCAG-39
59-ATCCGCCAGATACTCACCAAC-39
mmoX
mmoX
mmoX
mmoX
mmoX
mmoR
mmoR
mmoX
mmoX
mmoS
mmoS
template in the PCR reaction (one cycle of 96 uC for 1 min, followed
by 30 cycles of 96 uC for 30 s; 60 uC for 30 s; 72 uC for 30 s followed
by a final extension of 10 min at 72 uC). PCR products were analysed
by agarose gel electrophoresis.
SDS-PAGE and Western hybridization. Cells were harvested at
13 000 g for 2 min, resuspended in 1 ml 50 mM Tris/HCl, pH 7?0,
and were broken by repeated freeze–thaw cycles in liquid nitrogen.
Intact cells were removed by centrifugation (13 000 g, 15 min). The
cell-free extract was separated into membrane and soluble fractions
by centrifugation at 100 000 g for 1 h. The soluble fraction was
removed and the membrane fraction was resuspended in 100 ml
50 mM Tris/HCl, pH 7?0. Samples were stored at 220 uC. Protein
concentration was determined using the Bio-Rad protein assay kit.
Samples were analysed on 12 % (w/v) SDS-polyacrylamide gels
http://mic.sgmjournals.org
Reference
promoter
promoter
promoter
promoter
promoter
region
region
region
region
region
stained with Coomassie brilliant blue. Western blots (Sambrook
et al., 1989) were challenged with an antiserum raised against the
hydroxylase component of sMMO.
sMMO activity measurement. Approximately 50 mg crushed
naphthalene crystals was added to a 3 ml batch culture of
Mc. capsulatus (Bath) and this mixture was shaken at 45 uC for 1 h.
The cells were removed by centrifugation at 12 000 g for 2 min;
50 ml 5 mg o-dianisidine ml21 solution (Sigma) was added to the
supernatant and colour formation was monitored by measuring the
absorbance change at 530 nm. All measurements were done in triplicate. The sMMO activity of Mc. capsulatus (Bath) colonies was
visualized by the addition of naphthalene crystals to the plates.
Plates were incubated at 45 uC for 1 h and then 50 mg o-dianisidine
ml21 (Sigma) solution was layered onto the surface of the plate. The
deep purple colour of the colonies indicated sMMO activity.
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Green fluorescent protein (GFP) activity measurement. GFP
fluorescence was measured using a Quanta Master QM-1 fluorimeter
(Photon Technology International) at an excitation wavelength of
490 nm, an emission wavelength of 510 nm and emission/excitation
slit widths of 8 nm. Intensity readings are represented by arbitrary
units and were normalized to a cell density of 108 cells ml21. The
mean±SD values of three independent assays are indicated in the
Results.
Cloning and sequencing of smmo gene downstream region.
The 5?4 kb sequence downstream from mmoC was already cloned
on pCH4 (Stainthorpe et al., 1989, 1990). pCH4 was digested
with Bsp120I and the resulting 1?1 and 4?2 kb fragments were
isolated. The 1?1 kb fragment was ligated into Bsp120I-digested
pBluescript2SK+, resulting in pBX2. The 4?2 kb fragment was
ligated into Bsp120I-digested pBBR1MCS-4, resulting in pMCS441
(Fig. 1). Nucleotide sequences were determined using universal
sequencing primers. Additional sequencing was performed on both
strands by primers complementary to the ends of the known
sequences. The upstream (59) region of mmoQ was cloned from
genomic DNA of Mc. capsulatus (Bath). The restriction map of the
unknown genomic region was constructed by Southern hybridization analysis of genomic DNA digested by AccI, Bsp120I, BamHI,
HindIII, KpnI, PaeI, SalI and XhoI. The labelled DNA fragment
amplified from pCH4 by PCR, using primers HKfw and HKrev
(Table 1, Fig. 1), was used as the probe in Southern hybridization.
An approximately 27 kb genomic fragment was cloned into pK18mob
as follows. The 991 bp EcoRI–BglII fragment from the upstream region
of mmoX was cloned into EcoRI/BamHI-digested pK18mob and
designated pKPS. pKPS was introduced into Mc. capsulatus (Bath) via
conjugation. The vector inserted into the mmoX upstream region by
homologous recombination. The Km-resistant Mc. capsulatus (Bath)
colonies were selected and proper insertion was tested by diagnostic
PCR. Genomic DNA was purified, digested with HindIII and selfligated, resulting in pKPSg (Fig. 1). pKHKg was constructed on a
similar basis. The DNA fragment of mmoS was amplified by PCR using
primers HKfw and HKrev. The PCR product was ligated into HincIIdigested pK18mob. The proper orientation of the insert was selected
and this plasmid was named pKHK.
The genomic DNA, which contained the inserted pKHK, was digested
with BamHI, ligated and named pKHKg (Fig. 1). pKHKg was subcloned by digestion with EcoRI, PaeI, PstI and KpnI. The fragments which contained the vector were isolated and self-ligated,
resulting in pKHKg1, pKHKg2, pKHKg4 and pKHKg6 respectively
(Table 1, Fig. 1).
Construction of plasmids. The pPROBE-NT promoter-probe
vector (Miller et al., 2000) was used for testing the promoter activity
of the mmoX 59 region. Four promoter regions of mmoX (Fig. 2)
were amplified by PCR using the MXf1, MXf2, MXf3 and MXf4 forward primers and the MXr reverse primer (Table 1). The primers
were designed to place restriction sites at each end of the PCR product to facilitate insertion into the promoter-probe vector pPROBENT containing enhanced GFP (EGFP) as a reporter gene, resulting
in plasmids pMX1, pMX2, pMX3 and pMX4 (Fig. 2). Vectors for
inactivation of mmoG and mmoR in Mc. capsulatus (Bath) by markerexchange mutagenesis were constructed as follows. The 1128 bp
Bsp120I fragment of pCH4 was ligated into Bsp120I-digested
pBluescript2SK+, resulting in plasmid pBX2. A 314 bp fragment
was deleted from pBX2 by digestion with NcoI and BstEII. The
3755 bp fragment containing the whole vector and the 59 and 39 end
of the mmoG insert was isolated. The non-compatible ends of this
fragment were filled in with Klenow polymerase and ligated with the
855 bp SmaI fragment containing the Gm resistance gene, aacC1,
from p34S-Gm (Dennis & Zylstra, 1998), resulting in pBXGm1.
pBXGm1 was digested with XbaI and PvuII (Fig. 1). The 1844 bp
fragment was ligated into XbaI/SmaI-cut pK18mobsac (Schäfer et al.,
1994), resulting in plasmid pKmoG.
For the marker-exchange mutagenesis of mmoR, pKmoR was
constructed. pKHKg2 was digested with Eco147I and SalI, then filled
in with Klenow polymerase. The 4346 bp fragment was isolated and
ligated with the SmaI fragment of p34S-Gm containing the Gm
resistance gene, resulting in pKmoR (Fig. 1).
Fig. 1. Physical map of the chromosomal region of Mc. capsulatus (Bath) analysed in this work. Only restriction enzyme sites
discussed in this study are shown. The ORFs are indicated by large arrows. Black arrows indicate regions sequenced
previously (Stainthorpe et al., 1989, 1990). Clones used for sequencing are indicated by thick lines. The vector part of each
plasmid is not shown. The sizes of the inserts are shown in parentheses. HKfw and HKrev show the locations of primer-binding.
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Putative regulatory genes of sMMO
Fig. 2. Analysis of mmoX promoter region and construction of promoter-probe vectors. (a) Sequence upstream of mmoX.
Putative recognition sites described in the text are indicated by boxes for sN”24, sN”12, IHF and ribosome-binding site
(RBS). Consensus nucleotides are in bold type. Sequences used for the MXf1, MXf2, MXf3, MXf4 and MXr primers are
indicated by underlining. ‘+1’ indicates the start of transcription of the mmo cluster; M indicates the first methionine of MmoX.
(b) Copper-dependent expression of GFP from truncated promoters of mmoX in Mc. capsulatus (Bath). Black arrows indicate
the primers used for annealing the promoter fragments, which were tested in the promoter-probe vector pPROBE-NT. The
GFP activities were measured and the mean±SD of three independent experiments is shown. The lengths of the fragments
are shown in parentheses. The putative recognition sites for sN, IHF and RBS are indicated by boxes. T, Transcription
terminator; B, BamHI; H, HindIII; hatched bars, low copper; black bars, high copper.
RESULTS
Organization of the groEL, mmoQ, mmoS and
mmoR gene cluster
The approximately 9?5 kb region downstream (39) from the
gene cluster harbouring mmoXYBZDC (Stainthorpe et al.,
1989, 1990) was analysed in this study. Of this nonsequenced region 5?5 kb had been already cloned on pCH4
(Stainthorpe et al., 1989, 1990). An ORF was found oriented
in the same direction as mmoC and adjacent to it. This
hypothetical gene was named mmoG. Further downstream
from mmoG, another ORF was identified, which was
oriented in the opposite orientation. The putative product
of this gene (mmoQ) resembles the regulator part of a twocomponent sensor–regulator system (Stock et al., 2000).
Next to mmoQ, a sequence with a high degree of identity to
the 39 end of the sensor part of the two-component signal
transduction system was found at the end of the fragment.
The complete mmoS gene was isolated from the genome of
Mc. capsulatus (Bath). Upon sequencing this newly isolated
http://mic.sgmjournals.org
DNA fragment, the missing part of mmoS and a gene
encoding a putative sN-dependent transcriptional activator
(mmoR) (soluble methane monooxygenase regulator) were
found (Fig. 1).
Analysis of the mmoX promoter region
Previous analysis of the transcription of the mmo genes in
Mc. capsulatus (Bath) has revealed that the transcription
starts from a single promoter upstream (59) from mmoX. In
the mmoX promoter region, sequences resembling both
s70- and a weak sN-binding site were identified and a
s70-type promoter was predicted to be the likely promoter
(Nielsen, 1996).
Upon closer examination, a sN recognition site (Reitzer &
Schneider, 2001; Humberto et al., 1999) was found 12 bp
upstream from the transcriptional start site in the mmoX
promoter region: TGGCAC-N6-TGCNNt (Fig. 2a). Further
upstream (59) an integration host factor (IHF)-binding site
(WATCAA-N4-TTR) could be recognized, which is typical
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R. Csáki and others
for sN-type promoters (Collado-Vides et al., 1991). Genes
encoding IHF have been identified in the unfinished genome
of Mc. capsulatus (Bath) (TIGR; http://www.tigr.org); they
show 77 % sequence identity to those of E. coli. It is also a
general property of sN promoters that they have a distant
upstream (59) activating region to bind a sN transcription
activator protein, which then regulates the start of transcription by assuming an active or an inactive conformation
(Buck et al., 2000). The presence of such a distant activator
region was tested using a promoter-probe vector. To design
the necessary construct, a suitable reporter gene had to be
selected, which was not available for Mc. capsulatus (Bath).
GFP has been shown to be applicable in various systems
(Siegle et al., 2000). It requires only oxygen and Ca2+ for
activity. The pPROBE-NT vector is known to function in
a wide range of hosts, including Mc. capsulatus (Bath)
(R. Csáki and others, unpublished results). Various lengths
of the upstream region of mmoX were amplified by PCR and
cloned into a transcription fusion vector (Fig. 2b). The GFP
activities of cultures grown in the presence and absence of
copper were compared. The results (Fig. 2) clearly indicate
that a distant upstream region between 2358 and 2280 bp
(from the ATG codon of mmoX) contains the upstream
activator sequence for a sN-dependent transcription activator and is necessary for copper regulation of the mmoX
promoter. Taken together, the analysis of the upstream
region suggests that a sN-type promoter governs transcription of the sMMO structural gene cluster.
Sequence analysis of the new ORFs
MmoG, the putative chaperonin. mmoG encodes a putative protein of 559 aa with a predicted molecular mass
of 59?5 kDa. MmoG (methane-monooxygenase-associated
GroEL) has 39 % identity and 60 % similarity to the large
subunit of the cpn60-type chaperonins (GroEL) (Braig 1998;
Ranson et al., 1998), including GroEL of Mc. capsulatus
(Bath). Usually the gene encoding the small subunit of
the complex (GroESL) is located immediately upstream
from groEL, encoding the large subunit (Segal & Ron,
1996). However, no groES-like sequence was identified
39 or 59 to mmoG in Mc. capsulatus (Bath). A gene with
significant identity to mmoG has also been found in
Ms. trichosporium OB3b (Stafford et al., 2003). The
putative MmoG of Ms. trichosporium OB3b has 41 % identity and 59 % similarity to MmoG from Mc. capsulatus
(Bath). There was a putative Shine–Dalgarno sequence
(CGGAG) 10 bp upstream (59) from the ATG start codon
of mmoG. A typical s promoter motif was not recognizable. A characteristic component of heat-shock gene promoters is the CIRCE (controlling inverted repeat of
chaperone expression) motif, the binding site for the HrcA
protein, which regulates the expression of the chaperone.
Such a CIRCE motif (TTAGCACTC-N9-GAGTGCAA;
Lemos et al., 2001) was not present in the 351 bp region
separating mmoC and mmoG, although it was found
in both groESL operons identified elsewhere in the
Mc. capsulatus (Bath) genome (TIGR unfinished microbial
genomes, http://tigrblast.tigr.org/ufmg/). r-independent transcription terminal signals were missing from the intergenic
region between mmoC and mmoG. It should also be noted
that at 90 bp downstream (39) from the mmoG stop codon
there is a potential high energy (220?2 kcal=84?5 kJ)
stem–loop structure, which may serve as a r-independent
terminator signal. These properties suggest that mmoG
and mmoC might be co-transcribed and thus MmoG may
play a significant role in the maturation or assembly of
the sMMO complex or may be a transcription factor
required for the initiation of transcription of sMMO.
MmoS-MmoQ, the putative two-component signal
transduction system. The genes of the sensor–regulator
system, mmoS and mmoQ, were transcribed in the
opposite orientation to mmoG (Fig. 1). A putative s70
promoter motif was identified 59 to the consensus Shine–
Dalgarno sequence (Fig. 3). The putative gene product of
mmoS was 1177 aa long (predicted molecular mass of
128?6 kDa). The putative regulator protein MmoQ was
634 aa long (69?8 kDa). The start codon of mmoQ
overlaps with the stop codon of mmoS. The putative polypeptides encoded by mmoS and mmoQ showed approximately 50 % identity to two-component regulatory systems,
composed of a sensor and a regulator protein (Stock et al.,
2000). In these systems, the environmental signal detected
by the sensor protein is transmitted to the regulator
protein via transphosphorylation (reviewed by West &
Stock, 2001).
Fig. 3. Nucleotide sequence of the mmoS-mmoR intergenic region. Putative s70 ”35 and ”10 sequences are indicated by
underlining; the start codons of MmoS and MmoR are shown in open boxes; the 267 bp internal sequence between the two
putative promoters is not shown.
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Microbiology 149
Putative regulatory genes of sMMO
Two PAS-PAC domains (Ponting & Aravind, 1997;
Anantharaman et al., 2001) were detected in the N-terminal
sensor region of MmoS (Fig. 4a). These domains are usually
found in sensor proteins responding to the redox state of the
environment (Taylor et al., 1999). Predicted membrane
topology (Sonnhammer et al., 1998) suggested that MmoS
from Mc. capsulatus (Bath) contains a transmembrane region
between the 19th and 41st amino acids. Such a transmembrane arrangement is also characteristic of the PAS domains
of redox-sensing two-component systems (Bauer et al.,
1999). In addition, there was a GAF domain (Aravind &
Ponting, 1997) adjacent to the PAS-PAC domain in MmoS
of Mc. capsulatus (Bath). A GAF domain is characteristic
for sensor proteins binding small molecules, e.g. cGMP
(Anantharaman et al., 2001). The occurrence of both kinds
of domains within a single sensor molecule is a rare
phenomenon (Anantharaman et al., 2001). The typical
metal-binding motif MxCxxC was not recognizable in the
N-terminal region of MmoS.
A typical sensor protein element, the His-kinase phosphoacceptor domain, is present in MmoS. MmoS from
Mc. capsulatus (Bath) also contains two receiver domains
(REC), which receive the phosphoryl group at a conserved
Asp residue. Interestingly, the sensor contains a Hiscontaining phosphotransfer domain, which is usually
present on a separate protein (West & Stock, 2001).
The domain organization of the putative MmoQ is distinct
from the typical regulator proteins. The regulator protein
also contains a receiver domain, the phosphorylation of
which changes the activity of the regulator. Most regulators
are transcription factors. Their DNA-binding activity
changes upon phosphorylation, thereby controlling the
promoter activity and thus the biosynthesis of the participating protein(s) in response to the environmental
stimulus (Reitzer & Schneider, 2001). Some regulators
do not contain a DNA-binding domain. These regulators
transfer their phosphate groups to a target protein, thereby
changing its activity (Bourret et al., 1989; Robinson et al.,
2000; Stock et al., 2000). The putative MmoQ protein of Mc.
capsulatus (Bath) does not display any DNA-binding motifs.
The C-terminal region of MmoQ contains a GGDEF
sequence element, which is associated with the receiver
domain (Stock et al., 2000). In fact, the predicted
N-terminal protein sequence of MmoQ from Mc. capsulatus
(Bath) shows no significant similarity to any other proteins
in current databases.
MmoR, the putative sN-dependent transcription activator.
The mmoR gene encodes a 581 aa polypeptide of 63?4 kDa
carrying every feature described for sN-dependent transcription activators (Stock et al., 2000). In addition to the
sN activator domain and the ATPase motif, a DNAbinding helix–turn–helix domain is located towards the C
Fig. 4. Domain organization of the putative two-component phosphorelay system, MmoS-MmoQ. (a) The multi-component
phosphorelay system often begins with a hybrid histidine protein kinase. The transmembrane segment is indicated by Tm. The
HATPase domain catalyses the ATP-dependent autophosphorylation (thick black arrow) of a specific conserved His residue in
the HisKA domain. Dashed arrows indicate the putative phosphotransfer scheme when a specific environmental signal is
sensed by the PAS-PAC and GAF domains. The phosphoryl group is then transferred to a specific Asp residue located within
the receiver (REC) domain. The His-containing phosphotransfer domain (HPT) (usually located on a separate protein) receives
the phosphate moiety at a conserved His residue; (b) This phosphate moiety is then transferred to the receiver domain of the
regulator protein, MmoQ. X indicates a putative effector molecule(s), which may influence the transcription of the mmo operon.
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1791
R. Csáki and others
terminus. The mmoR gene is preceded by s70- and
ribosome-binding elements (Fig. 3). MmoR also lacks the
metal-binding MxCxxC motif (Koch et al., 1987), indicating that, as with MmoS, MmoR probably does not bind
copper ions directly. MmoR from Mc. capsulatus (Bath)
has 40 % identity and 54 % similarity to MmoR from
Ms. trichosporium OB3b (Stafford et al., 2003).
(a) 1
_
1
+
2_
2
+
3
_
3
+
2
_
3
+
M
a
b
Phenotypic characterization of DmmoG, DmmoR
mutants. DmmoG and DmmoR mutants of Mc. capsulatus
(Bath) were constructed to investigate the influence of
MmoG and MmoR on the expression of sMMO. The
growth rate of the DmmoG and DmmoR mutant strains
was indistinguishable from the wild-type controls on
copper-containing NMS plates, but both mutants grew
more slowly on ‘copper-free’ plates. Unlike the wild-type
Mc. capsulatus (Bath), the DmmoG and DmmoR mutants
showed no measurable naphthalene-oxidizing sMMO
activity (data not shown) under any growth conditions.
In line with the activity assays, the mutant strains did not
contain detectable amounts of sMMO hydroxylase polypeptides when analysed by blotting and probing with a
serum specific for the sMMO hydroxylase components of
Mc. capsulatus (Bath) (Fig. 5a). The complete absence of
these sMMO proteins was confirmed using cells grown in
liquid culture, both in the presence and absence of
copper ions. Finally, the mutants were subjected to RTPCR analysis to detect the transcripts of the mmo genes.
The PCR primers mxatgfw and mxatgrev (Table 1), which
amplified the N-terminal of mmoX, were used. As shown
in Fig. 5(b), mmoX transcripts were only present in wildtype Mc. capsulatus (Bath) cells grown in the absence
of copper.
The pMX1 promoter-probe plasmid, with the functional
mmoX promoter, was introduced into the DmmoG and
DmmoR mutants. On copper-containing medium, GFP was
not expressed in any of these strains. On low-copper
medium, only the wild-type strain with plasmid pMX1
showed GFP expression, while the mutants did not (data
not shown).
These various, independent methods unequivocally demonstrated that there was no functional sMMO enzyme (neither
transcribed nor translated gene products) produced in the
DmmoG and DmmoR mutant strains of Mc. capsulatus
(Bath). These results indicate that both MmoG and MmoR
are indispensable for the correct biosynthesis of sMMO as
has also been recently demonstrated in Ms. trichosporium
OB3b (Stafford et al., 2003).
The sMMO-deficient mutant strains were also capable of
growing on low-copper medium, apparently without
possessing functional sMMO. Hence, these cells had no
perceptible sMMO activity to support their growth on
methane. The growth rates of the mutants did not show
significant differences compared to the wild-type cells
expressing either sMMO or pMMO (data not shown).
1792
c
(b)
G
bp
1
+
1
_
2
+
3
_
750
500
250
Fig. 5. (a) The presence of polypeptides of the hydroxylase
component of sMMO was tested by Western hybridization.
Lanes: 1, Mc. capsulatus (Bath); 2, DmmoG; 3, DmmoR
strains. + indicates the results of cultures grown with copper
ions; ” indicates results of cultures grown in the absence of
copper ions; M indicates the purified hydroxylase component of
Mc. capsulatus (Bath). (b) mmoX-specific transcripts were
amplified by RT-PCR using mxatgfw and mxatgrev primers. G
indicates the genomic control where genomic DNA was used
as a template in the PCR reaction; + and ” show the presence or absence of copper in the medium. Lanes: 1, Mc. capsulatus (Bath); 2, DmmoG; 3, DmmoR. The sizes of the marker
DNA bands are shown on the left.
Presumably the mutant cells were scavenging Cu2+ and
pMMO sustained their growth on low-copper medium.
DISCUSSION
Four genes (mmoG, Q, S and R) immediately downstream
(39) from the smmo gene cluster were identified, sequenced
and characterized. The fact that an IHF recognition site is
present in the promoter region of mmoX and that there are
known IHF genes in the Mc. capsulatus (Bath) genome (data
obtained from the TIGR website), suggest that the expression of sMMO may not be regulated by a simple copper
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Microbiology 149
Putative regulatory genes of sMMO
switch. It has been demonstrated that an activator region at
about 335 bp upstream (59) from the start codon of mmoX
is required for copper-dependent activity of the promoter.
The presence of the IHF-binding site and the requirement
of an upstream activator region suggest the existence of a
sN-dependent promoter. The requirement of sN for the
expression of sMMO of Ms. trichosporium OB3b was also
demonstrated by the analysis of an rpoN mutant of
Ms. trichosporium OB3b which was also unable to express
sMMO (Stafford et al., 2003). Several attempts were made in
our study to mutate rpoN of Mc. capsulatus (Bath) without
any success.
Deletion mutagenesis was applied to generate ‘knock-out’
mutants of both mmoG and mmoR. Analysis of these
mutants provided evidence that these genes are required for
the expression of active sMMO. The data do not provide
straightforward evidence for the participation of these genes
in the copper-dependent regulation of sMMO expression,
although mutation analysis indicates an sMMO2 phenotype
under ‘low-copper’ growth conditions. DmmoR, and
interestingly the DmmoG strain with the pMX1 plasmid,
did not show GFP activity under ‘low-copper’ growth
conditions. This indicates that the transcriptional activation
of sMMO was abolished in these mutants. It has also been
found that on copper-deficient medium, these mutants are
unable to express sMMO and the promoter activity of
mmoX is impaired without any significant effect on the
growth rate as compared to the wild-type strain. MmoG
may therefore participate in the maturation and assembly of
the sMMO polypeptides or in the biosynthesis of a protein
regulating the expression of the sMMO structural gene
cluster (e.g. MmoR).
The MmoR protein likely participates in sN-dependent
promoter activation, although this promoter is coppersensitive and MmoR contains no copper-binding motifs.
Thus the exact role of MmoR needs further investigation. If
MmoR does indeed activate the expression of smmo genes,
additional proteins (possibly MmoQ and MmoS), which
may sense the changes in copper concentration in the cell
environment and mediate this information to MmoR, must
be involved (Fig. 6). Low-level transcription of pmoCAB
operons was detected by Stolyar et al. (2001) under sMMO
expressing conditions. We presume that traces of copper ions
in the medium may allow active pMMO formation. Under
laboratory conditions, where methane is supplied in vast
excess, this low amount of pMMO is sufficient for growth.
The only other system where the putative regulatory
genes mmoR and mmoG have been studied is that of
Ms. trichosporium OB3b, a Type II methanotroph found in
the a-subdivision of the Proteobacteria (Stafford et al., 2003).
A comparison with our results reveals several similarities
and differences. In both cases the mmoR and mmoG genes
are found in the neighbourhood of the smmo operon,
although the arrangement of the genes relative to the structural genes is dissimilar: mmoR and mmoG are positioned 59
to mmoX in Ms. trichosporium OB3b. mmoR has a putative
s70-like promoter in Mc. capsulatus (Bath), but there is no
recognizable promoter motif for mmoR in Ms. trichosporium
OB3b. No promoter-like sequences can be identified for
mmoG either in Mc. capsulatus (Bath) or in Ms. trichosporium
OB3b (Stafford et al., 2003). The deduced amino acid
sequences of the MmoR and MmoG proteins in the two
bacteria show a considerably high degree of identity (40 and
40 %, respectively) and similarity (50 and 55 %, respectively).
Mutation of mmoR or mmoG yields the same phenotype in
both strains, thus it is reasonable to assume that the functions
of MmoR and MmoG are the same in Mc. capsulatus (Bath)
and Ms. trichosporium OB3b.
It intends to say that the MmoR sequences in the two
bacteria show 40 % identity (50 % similarity) and the MmoG
Fig. 6. Model for the regulation of the mmo
operon. The model involves an unknown
mechanism for sensing copper ions. MmoS
transduces this signal by changing the phosphorylation stage of MmoQ. This signal is
then transferred from MmoQ to MmoR,
which activates the transcription of the mmo
operon via sN. A hypothetical signal pathway
is indicated by dotted arrows. The exact
function of MmoG cannot be determined
from the data obtained to date. It may be
required for the correct folding of MmoR
and/or for the maturation of active sMMO
(indicated by hatched arrows).
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R. Csáki and others
sequences show the same 40 % identity (55 % similarity)
in the two strains compared.
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