ELSEVIER
FEMS Microbiology
Letters 132 (1995) 203-208
Evidence that particulate methane monooxygenase and ammonia
monooxygenase may be evolutionarily related
Andrew J. Holmes ‘, Andria Costello b, Mary E. Lidstrom b, J. Colin Murrell ‘.*
’
Department
h Environmental
of Biological
Science.
Engineering
Science, Unir,er.si& of Warwick,
138-78. California
Institute
Cowntry
of Technology
CV4 7AL. UK
Pasadena,
CA 91125, USA
Received 19 July 1995; revised 16 August 1995; accepted I6 August 1995
Genes encoding particulate methane monooxygenase
and ammonia monooxygenase
share high sequence identity.
Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa
polypeptides,
which are believed to contain the active sites, of particulate methane monooxygenase
and ammonia
monooxygenase.
A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA
and umoA) from a
variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of
the phylogenetic groups of both methanotrophs (a- and y-Proteobacteria)
and ammonia-oxidizing
nitrifying bacteria ( /3and -y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong
conservation of both primary and secondary structure. Nitrosococcus oceonus AmoA showed higher identity to PmoA
sequences from other members of the y-Proteobacteria than to AmoA sequences. These results suggest that the particulate
methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.
Keywords:
Methane monooxygenase:
Ammonia
monooxygenase;
Methanotroph:
1. Introduction
Methanotrophs
ria capable
represent
of growth
using
a unique
methane
group
of bacte-
as sole carbon
and energy source. They are a key component of the
global methane cycle and have been exploited in
biotechnology
for their ability to co-oxidise many
important environmental
contaminants
[ 1,2]. Both
the methane oxidation and co-oxidation properties of
methanotrophs are due to methane monooxygenase.
* Corresponding
author. Tel.: +44 (1203) 523 553; Fax: +44
(1203) 523 568; E-mail: [email protected].
0378.1097/95/$09.50
SSDI 0378.1097(95)0031
0
1995 Federation
l-8
of European
Microbiological
Nitrifier
There are two distinct types of this enzyme, the
cytoplasmic
soluble
methane
monooxygenase
(sMMO)
and the membrane-bound
particulate
methane monooxygenase
(pMM0). These two enzymes show no genetic or structural homology despite their similar function in the cell. Only the
pMM0 is present universally in all methanotrophs.
In those strains which possess both types of MMO,
the availability of copper ions (Cu’+) is believed to
regulate a switch between expression of sMM0 or
pMM0 [3].
pMM0 shares many similarities with ammonia
monooxygenase
(AMO), found only in ammoniaoxidizing nitrifying bacteria. Despite the very differSocieties. All rights reserved
204
A.J. Holmrs et ~1./ FEMS Microhiolo~~ Lettrn
ent physiologies of methanotrophs (pMM0) and nitrifiers (AMO), these enzymes may be considered
analogous in that they have a crucial role in cell
metabolism for both groups of bacteria. The two
enzymes are thought to consist of at least two membrane-associated
polypeptides of approximately
27and 4.5~kDa, they share broadly similar substrate and
inhibitor profiles, and may contain a trinuclear copper centre at the active site [4]. Neither AM0 nor
pMM0 has been reproducibly purified in active form.
The genes pmoA and pmoB, encoding the putative 27- and 45kDa polypeptides,
respectively, of
pMM0 of Methylococcus cupsulatus have recently
been cloned and sequenced [5]. Similarly, amoA and
amoB, encoding similar sized polypeptides of the
AM0 of Nitrosomonas europaea, have also been
cloned and sequenced [6]. A high level of identity
was found between the two predicted pMM0 gene
products and the corresponding AM0 gene products.
All of these polypeptides are membrane-associated
and their inferred sequences contain several hydrophobic membrane spanning regions. The evidence
for the identity of these polypeptides as components
of these enzymes is, as yet, indirect. The putative
pMM0
polypeptides
are only produced
during
growth conditions where pMM0 activity is detected.
In both MC. cupsulatus and Nm. europaea, the
27-kDa polypeptide is believed to contain the active
site and can be labelled by [‘5C]acetylene, a potent
inhibitor of these enzymes [7]. Using both pmo and
amo gene probes, specific DNA fragments were
identified in Southern blots of chromosomal DNA
digests
of all methanotrophs
and not nonmethanotrophic controls (A.J. Holmes, E. Kenna and
J.C. Murrell, unpublished data).
Sequence information for pmo and umo genes is
only available from one Type I (y-Proteobacteria)
methanotroph [5] and one /3-subdivision nitrifier [6l.
Cloning of these genes from Type II (a-Proteobacteria) methanotrophs
or y-subdivision
nitrifiers
has not been reported. To confirm the ubiquity of
these genes in methanotrophs and ammonia-oxidizing nitrifiers and their identity as components of the
particulate methane and ammonia monooxygenases,
cloning of pmo and amo genes from a phylogenetitally diverse range of organisms is essential. Analysis of sequences from a diverse range of organisms
may also yield information on the possible evolu-
132 IIYYSI 203-208
tionary relatedness of these enzymes. We have exploited the conserved nature of the presently available pMM0 and AM0 sequences to design broad
specificity oligonucleotide
primers suitable for PCR
amplification
of both these monooxygenase
genes.
Representatives
of all phylogenetic
groups of
methanotrophs and ammonia-oxidizing
nitrifiers were
included in the study.
2. Materials and methods
2. I. Bacterial strains
The following methanotroph strains from the University of Warwick Culture Collection were used in
this study: Methylococcus
capsulatus Bath, Methylomonas methanica S I, Methylobacter albus BG8,
Methylosinus trichosporium OB3b, and Methylocystis purc’us OBBP. Chromosomal
DNAs were prepared as described by Oakley and Murrell [8]. Nitrosomonus europaea NCIMB 11850, Nitrosospiru sp.
Np22, Nitrosolobus multiformis NCIMB 1 1849, and
Nitrosococcus
oceanus NCIMB 1 1848 were obtained from J. Prosser (Aberdeen) and DNA was
extracted from these organisms as described by Giovannoni [9].
2.2. PCR ampl$‘cation
PCR was performed in 50-p] reaction mixtures in
0.5-ml microcentrifuge
tubes. Reactions were performed using the reagents supplied with Gibco Taq
polymerase at a magnesium ion concentration of 1.5
mM, with 10 ng template DNA, and 100 pmol each
of primers A 189 (GGNGACTGGGACTTCTGG)
and
A682 (GAASGCNGAGAAGAASGC).
Deoxynucleotide triphosphates
were supplied at 200 PM
(final concentration). Reactions were carried out in a
Perkin-Elmer
model 360 thermal cycler with a hot
start. Enzyme was added after an initial denaturation
step of 96°C for 4 min. Twenty-eight cycles of 92%
1 min; 56”C, 1 min; 72°C 45 s were then performed
followed by a final extension of 5 min at 72°C. A
positive control was performed for each template
using the bacterial 16s rRNA-specific
primers f27
and r1492 [9]. Reaction products were checked for
size and purity on I% agarose gels visualised by
staining with ethidium bromide.
A.J. Holmes et al, / FEMS Microhiolog!
One organism, Nitrosospira sp., yielded two amplified products of 525 and 550 bp. Mb. albus failed to
give a product with the primer pair A 189/A682. A
1.8-kb product comprising the 3’ portion of pmoA
and the 5’ end of pmoB was amplified from this
strain
with
the primers
Al89
and
B 1983
(GAASCGRCTGTCSGGGTC)
using similar PCR
amplification conditions.
PCR primers were also tested against DNA from
a range of bacteria which do not oxidize methane or
ammonia, including
Meth_vlobacterium extorquens,
Escherichia coli. M~xococcus xanthus, Paracoccus
denitr@zms,
and strain M2, a novel methane sulfonic acid utilizing organism [ 111. No products of the
predicted size were obtained from any of these organisms. This specificity of the PCR for methanotrophs and nitrifiers was confirmed by Southern
hybridization of the products to a probe corresponding to the same portion of pmoA generated from
Mc. capsulatus (data not shown).
The identity of the pmoA and amoA products was
confirmed by cloning and sequencing. Overall amino
acid sequence conservation
between AmoA and
PmoA was approximately 40% identity (65% similarity). The most notable feature of sequence comparisons was that the level of conservation showed a
stronger correlation with the phylogenetic
relatedness of the organisms than with the function of the
gene products. Three identity groups representing the
. .
a, /3 and y subdlvlslons of the Proteobacteria were
2.3. Cloning and sequencing
The putative pmoA and amoA PCR products
were cloned into the pCRI1 vector using the TA
cloning kit (Invitrogen) according to the manufacturers instructions.
Plasmids were purified from selected clones using the method of Saunders and
Burke [lo] and DNA sequenced by both cycle sequencing using the dye-terminator
kit of Applied
Biosystems and conventional chain termination using
the Sequenase kit (United States Biochemicals). Sequences have been deposited in GenBank under the
accession numbers U3 1649-U3 1655.
3. Results and discussion
The high level of identity between AM0 and
pMM0 allowed the construction of oligonucleotides
targeting regions of shared amino acid sequence.
Degenerate primers were synthesized,
taking into
account codon usage in methanotrophs. Suitability of
the template DNAs for PCR was established by
performing a positive control reaction with all samples using universally
conserved primers targeting
16s rRNA genes.
The pMMO/AMO-specific
primers amplified a single DNA fragment of the
predicted size (525 bp) from all methanotroph DNAs
tested, except Mb. albus BG8, at an annealing temperature of 56°C and from all four nittifier DNAs.
Ms.tri
Fig. I. Identity/similarity
Mcy.par
NC.OCE
205
Letters 132 C19051 203-208
Mb.alb
Mm.met
MC.CapS
Nm.eur
NhlUl
Nspwa
matrix derived from comparison of 169 amino acids of methanotroph PmoA and nitrifier AmoA
sequences.
Values in the upper triangle of the matrix are percent identity and values in the lower triangle, percent similarity. Identity/similarity
consisting of: (A) a-Proteobacteria
Mm. mrthanicu, and Mr. capsulutus) and nitrifiers (Nc. occurmy): and (C) P-Proteobacteria nitrifiers
Nitrosospirc~
sp.)
are boxed.
groups
methanotrophs (Ms. trichosporiwn and MC!. purrus); (B) y-Proteobacteria methanotrophs (Mb. albus,
( Nm.ruropcwcc.
NI. n~ulti@-mis, and
206
A.J. Holmes et ~11./ FEMS Microbiology
seen rather than two identity groups representing
AmoA and PmoA (Fig. 1). Significantly, NC. oceanus
AmoA showed higher identity (> 75%) to all y-Proteobacteria methanotroph PmoA sequences than to
any of the P-Proteobacteria
nitrifier AmoA sequences (< 50%). It is interesting to note in this
context that NC. occunus has been reported to metabolize methane and may not fit the strict definition
of an ammonia-oxidizing
bacterium [ 121.
Fig. 2 shows an alignment of the predicted protein
sequences for PmoA and AmoA obtained in this
study, highlighting residues conserved within each of
the identity groups and those which are universally
conserved. Highly conserved residues are distributed
throughout the alignment, suggesting these proteins
will also show strong structural conservation.
This
hypothesis is supported by protein structure predic-
Letters
132 f I9951 203-2VK
tions and hydropathy plots for these peptides, which
are similar for all three groups (Fig. 3).
Neither pMM0 nor AM0 has been purified in
active form. Consequently, evidence for the identity
of the pmoA and amoA gene products as components of these enzymes is indirect. The degenerate
primers reported here allow specific amplification of
homologous
genes from nitrifiers
and methanotrophs, including representatives of the cy-, p- and
y-Proteobacteria.
Homologues of these genes could
not be detected by PCR in a range of bacteria which
do not oxidize either methane or ammonia. This
provides further evidence that the pmoA and amoA
gene products are components of the pMM0 and
AMO, respectively.
Both pMM0 and AM0 are key enzymes in major
biogeochemical
cycles and of potential significance
52
106
Ms.tri
Mc.par
NC. ace
Mb.alb
Mm.met
MC. cap
Nrn. cur
Nl.mul
Nspir
Ms.tri
r-lc.par
NC.OCe
Mb.alb
Mm.met
l.Ic.cap
Nm.eur
Nl.lnUl
Nspir
Ms.tri
kfc.par
NC.ax
W.alb
Mm.met
kfc.cap
Nnl.eur
Nl.mul
Nspir
Fig. 2. Alignment
Mstri,
of predicted amino acid sequencea of pmoA
Ms. trichosporiunz;
ctipsulatus:
Nm.eur,
Mc.par,
Nm. europaea;
and nmoA genes from methanotrophs and ammonia-oxidizing bacteria:
Mccaps. MC
NC. ocranus; Mb.alb, Mb. alhu.r: Mm.met, Mm. mrthaniw:
Nl. multiformis;
Nspira, Nitrosospirc~ sp. ReGdues boxed in grey are conserved within all
MC!. pcrnus;
NLmul,
Nc.oce,
members of a subdivision of the Proteobacteria. Residues boxed in black are universally conserved in the sequences included in this study.
Amino acids are numbered according to the published sequence for MC,. crrpsukrtus
PmoA [5].
in bioremediation programs. Further studies on these
enzymes are crucial to understanding
the role and
potential applications of methanotrophs and nitrifiers
in the environment. The universally conserved amino
acid residues identified in this study may be important in the activity of the enzyme and represent
potential targets for site-directed mutagenesis experiments to explore the biochemistry of these important
enzymes. Residues which show conservation at the
level of identity groups may reflect adaptation to
either the AM0 or pMM0 function. With the exception of Nc. oceLlI1us mnoA. sequence motifs capable
of discriminating
between AM0 and pMM0 could
be identified (amino acids 107-I IS, 210-215) and
may constitute
useful target sites for functional
group-specific probes to detect these groups in natural samples. Distinction between y-Proteobacteria nitrifiers (e.g. Nitrosoc,occ,u.s sp.) [ 131 and y-Proteobacteria methanotrophs
(e.g. Methylococcus sp.,
Methylohucter sp. and kfPth$onzanas sp.) [14] may
not be possible using pn~oA gene probes.
The enzymes AM0 and pMM0 appear to share
many functional similarities although their role in
cell metabolism of methanotrophs (pMM0) and nitrifiers (AMO) is very different. Both enzymes can
oxidize methane and ammonia. although their K,
values for these substrates differ. Methanotrophs and
nitrifiers appear to be mutually exclusive groups
despite their apparent capacity to oxidize both
methane
and ammonia.
Methanotrophs
oxidize
methane (their only carbon source) to carbon dioxide
via the intermediates
methanol, formaldehyde
and
formate. They assimilate carbon at the level of
formaldehyde and do not fix CO,. Nitrifiers oxidize
ammonia to nitrite via hydroxylamine
to obtain energy (and reductant) for the fixation of CO,. their
sole carbon source, using ribulose bisphosphate carboxylase/oxygenase.
Specialization to one of these
physiological
groups appears to be due mainly to
subsequent enzymes in the pathway of methane oxidation (e.g. methanol dehydrogenase)
and ammonia
oxidation (e.g. hydroxylamine
oxidoreductase).
In
this study, we have confirmed the presence of pmoA
or cmoA genes in all methanotrophs
and nitrifiers
examined and greatly extended the database of’ available sequences. The strong conservation of primary
and secondary structures of PmoA and AmoA from a
diverse collection of organisms suggests that these
genes share a common ancestry. Even more compelling evidence is the correlation of PmoA or AmoA
sequence identity clusters with the phylogenetic affiliations of these organisms. These genes may there-
HPhobic
IiPhilic
HPhobic
HPhilic
HPhobic
HPhilic
Fig. 3. Predicted hydropathy plot3 for the pmoA/mwA
were determined using the Kyte-Doolittle
HPhilic.
PmoA.
gene product\ ot (A)
Mq.
prrrr US: (B) Nm. ruropoecr; and (C) Mh. trlhus. Plots
algorithm with a window of 9 frclm a sequence of 169 amino acids. HPhobic. hydrophobic:
hydrophilic. The scale indicates the number of residue!, where ammo acid
I
i\ equivalent to amino acid 52 of MC,. urpsu/atus
208
A.J. Holmes et al. / FEMS Microbiology
fore be considered evolutionary homologues despite
their different roles in methanotrophs and the ammonia-oxidizing nitrifiers.
Letters 132 (I9951 203-208
[51 Semrau, J.D..
Chistoserdov,
A.,
Lebron,
J.. Costello,
A.,
Davagnino, J., Kenna, E.. Holmes, A.J., Finch, R., Murrell,
J.C.
and
Lidstrom,
M.E.
(1995)
Particulate
methane
monooxygenase &enes in methanotrophs. J. Bacterial.
177.
307 I-3079.
[6] McTavish,
Acknowledgements
H.,
Fuchs. J.A. and Hooper,
Nitrosomonns
Work on methanotrophs in J.C.M.‘s laboratory is
funded by grants from the BBSRC, NERC and EU.
A.J.H. is supported by the EU. This work was also
supported by a University Research Initiative grant
from ARPA (NO00 14-92-J- 1901) to M.E.L. We thank
J. Prosser, University of Aberdeen, for the cultures
of ammonia-oxidizing
bacteria.
ruropuerr.
J. Bacterial.
175. 2436-2444.
Mrth,dococ~cus
cqwuIutusBath.
FEMS Microbial. Lett.
29, lO5- 109.
[81 Oakley,
C.J. and Murrell, J.C. (1988)
methane oxidizing bacteria. FEMS
nrj‘?f genes in obligate
Microbial.
Lett. 49, S3-
57.
[91 Giovannoni, S.J. (1991) The polymerase chain reaction. In:
Acid
Techniques
in
E. and Goodfellow.
Bacterial
Systematic&
M., Eds.), pp. 177-203.
John Wiley and Sons, Chichester.
References
[lOI Saunders, S.E. and Burke, J.F. (1990)
Oremland, R.S. and Culbertson, C.W. (1992)
methane-oxidizing
Importance of
bacteria in the methane budget as re-
vealed by use of a specific inhibitor. Nature 356, 421-423.
121DiSpirito.
A.A..
Gulledge, J., Murrell.
Lidstrom.
M.E.
and Krema. C.L.
J.C., Shiemke. A.K.,
(1992)
Trichloroethylene
oxidation by the membrane associated methane monooxygenase in type I, type II. and type X methanotrophs. Biodegradation 2, 151-164.
Stanley,
(1983)
Se-
substrate and active site probe for methane monooxygenase
from
Nucleic
Dl
(1993)
[71 Prior. S.D. and Dalton, H.D. (1985) Acetylene as a suicide
(Stackebrandt.
Ill
A.B.
quence of the gene coding for ammonia monooxygenase in
S.H..
miniprep DNA
S.D.,
Leak,
D.J.
and Dalton,
Copper stress underlies the fundamental
H.D.
change in
intracellular location of methane monooxygenase in methane
Reh. 18. 4948.
J.C. (1994)
ylotrophic
Methanesulfonate
Microbiology
140, l419-
iI31 Head, I.M..
Hiorns,
M.E.,
A.K.,
Jacobs, S.J.. Hales,
and Chan. S.I. (1994)
The nature of the
copper ions in the membranes
containing
methane
Mrthylococcu.s
monooxygenase
from
(Bath). J. Biol. Chem. 269, 14995-15005.
B.J..
the particulate
cupsulntus
W.D..
and Saunders. J.R. (1993)
ammonia-oxidizing
Shiemke,
O~CLIWS. Arch. Microbial.
147.
Embley.
A.J.
126-133.
ribosomal
Lidstrom.
1426.
[I21 Ward, B.B. (1987) Kinetic studies on ammonia and methane
Biotechnol. Lett. 5, 487-492.
H.-H.,
utilization by a novel meth-
bacterium involves an unusual monooxygenase.
oxidizing organisms: studies in batch and continuous culture.
[41 Nguyen,
isolation of
[I II Kelly, D.P., Baker, S.C.. Trickett, J.. Davey, M. and Murrell.
oxidation by Nitrosococcus
Prior.
Rapid
for double strand sequencing. Nucleic Acids
1147-l
RNA
T.M.,
The phylogeny
McCarthy.
of autotrophic
bacteria as determined by analysis of IhS
gene sequences. J. Gen.
Microbial.
139.
153.
[I41 Tsuji. K.. Tsien. H.C., Hanson. R.S..
R. and LaRoche, S. (1990)
determination
of
phylogenetic
ylotropha. J. Gen. Microbial.
DePdlma.
16s rRNA
relationship
136, I-IO.
S.R.. Scholtz.
sequence analysis for
among
meth-