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Applied and Environmental Microbiology, December 1999, p. 5198-5206, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Soluble Methane Monooxygenase Gene Clusters from
Trichloroethylene-Degrading Methylomonas sp. Strains and
Detection of Methanotrophs during In Situ Bioremediation
Toru
Shigematsu,1,*
Satoshi
Hanada,1
Masahiro
Eguchi,2
Yoichi
Kamagata,1
Takahiro
Kanagawa,1 and
Ryuichiro
Kurane1
National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology, Tsukuba,
Ibaraki 305-8566,1 and Central Research
Laboratories, Organo Corporation, Saitama
335-0015,2 Japan
Received 19 April 1999/Accepted 10 September 1999
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ABSTRACT |
The soluble MMO (sMMO) gene clusters from group I methanotrophs
were characterized. An 8.1-kb KpnI fragment from
Methylomonas sp. strain KSWIII and a 7.5-kb
SalI fragment from Methylomonas sp. strain
KSPIII which contained the sMMO gene clusters were cloned and
sequenced. The sequences of these two fragments were almost identical.
The sMMO gene clusters in the fragment consisted of six open reading
frames which were 52 to 79% similar to the corresponding genes of
previously described sMMO gene clusters of the group II and group X
methanotrophs. The phylogenetic analysis of the predicted amino acid
sequences of sMMO demonstrated that the sMMOs from these strains were
closer to that from M. capsulatus Bath in the group X
methanotrophs than to those from Methylosinus trichosporium
OB3b and Methylocystis sp. strain M in the group II
methanotrophs. Based on the sequence data of sMMO genes of our strains
and other methanotrophs, we designed a new PCR primer to amplify sMMO
gene fragments of all the known methanotrophs harboring the
mmoX gene. The primer set was successfully used for
detecting methanotrophs in the groundwater of
trichloroethylene-contaminated sites during in situ-biostimulation treatments.
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INTRODUCTION |
Trichloroethylene (TCE) is one of
the most common contaminants in the soil environment and groundwater in
many countries. Several methane-oxidizing bacteria efficiently degrade
the contaminant, and a number of investigations of biological removal
of TCE from contaminated soil by using methanotrophs have been reported
(15, 22). The enzyme responsible for the biodegradation of
TCE is methane monooxygenase (MMO), which catalyzes the oxidation of methane to methanol. Two distinct types of MMOs are known:
membrane-bound particulate MMO, present in all methanotrophs (43,
54), and soluble MMO (sMMO), which has been found in only several
species of methanotrophs (12, 30, 36, 47). Both types of MMO
can degrade TCE, but sMMO degrades it at a very high rate compared with
particulate MMO (50). Therefore, sMMO has received special attention in the bioremediation of TCE.
Methanotrophs are taxonomically classified into three groups: group I,
group II, and group X (22). The extensively characterized sMMO proteins are those purified from group II methanotrophs, Methylosinus trichosporium OB3b (3, 17, 18) and
Methylocystis sp. strain M (37), and a group X
methanotroph, Methylococcus capsulatus Bath (8, 20,
53). The enzyme complexes of these three strains have very
similar properties and consist of three components: a hydroxylase
component (MMOH), a reductase component (MMOR), and a regulatory
protein B (MMOB) (7). The X-ray crystal structures of the
hydroxylase components from M. trichosporium OB3b and
M. capsulatus Bath have also been reported (16,
40). The DNA sequence of the gene cluster that codes for the sMMO
proteins has been determined for the three methanotrophs (5, 6,
34, 45, 46). In each strain, six genes, mmoX,
mmoY, mmoB, mmoZ, orfY, and
mmoC, were detected (5, 6, 34, 45, 46). MMOH is
encoded by mmoX, mmoY, and mmoZ genes.
MMOR and MMOB are encoded by mmoC and mmoB, respectively.
On the other hand, there is little published information on the
properties of sMMO in group I methanotrophs. The presence of sMMO
protein has been demonstrated in two strains, Methylomonas methanica 68-1 and Methylomonas sp. strain GYJ3
(26, 44). From strain GYJ3, the hydroxylase component and
the reductase component of sMMO were purified, and the regulatory
protein B was partially purified (44). Fuse et al. reported
the partial sequence of mmoX from
Methylomicrobium sp., which belongs to group I
(19). However, the complete DNA sequence of an sMMO gene
cluster from the group I methanotrophs has not been reported.
Recently, two TCE-degrading methanotrophic strains, KSWIII and KSPIII,
from a TCE-contaminated field were isolated in our laboratory
(21). Phylogenetic analysis based on 16S rRNA sequences suggested that they were affiliated with the genus
Methylomonas of group I methanotrophs (21). In
this report, we characterize the strains in terms of morphological and
chemotaxonomic aspects and analyze the sMMO genes from the strains.
This is the first report on characterization of an sMMO gene cluster
from the group I methanotrophs. We also designed a gene probe for
sMMO genes based on the sequence data and tested its validity for
detection of methanotrophs in an aquifer during in situ-biostimulation treatments.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The methanotrophic
strains KSWIII and KSPIII were collected from the site at Kururi
(Kimitsu, Chiba Prefecture, Japan), which is contaminated with TCE, and
isolated in the previous study (21). The reference strains
M. trichosporium OB3b (ATCC 35070) and M. capsulatus Bath (ATCC 33009) were obtained from the American Type Culture Collection (Manassas, Va.). Methlocystis sp. strain
M was a kind gift from H. Uchiyama, National Institute for
Environmental Studies, Tsukuba, Japan. The strains were grown on NMS
medium (51) with gentle agitation (100 rpm) at 30°C
(strains KSWIII, KSPIII, OB3b, and M) and at 37°C (strain Bath) under
a methane-containing air atmosphere (air methane ratio, 8:2).
Taxonomic studies.
Cell morphology was examined by
phase-contrast microscopy and transmission electron microscopy. For
transmission electron microscopy, a centrifuged cell pellet was fixed
with 5% (vol/vol) glutaraldehyde and 1% (vol/vol) osmium tetroxide.
Ultrathin sections of the sample embedded in epoxy resin
(28) were prepared with a Reinchert ultramicrotome. Samples
were stained with uranyl acetate and lead citrate and examined with a
Hitachi H-7000 transmission electron microscope. In vivo absorption
spectra were measured in cell extracts with a Beckman DU 640 spectrophotometer after disruption by sonication (100 W; 3 min).
Quinones were extracted from the cells with chloroform-methanol (2:1
[vol/vol]) and analyzed by reverse-phase high-performance liquid
chromatography (Beckman System Gold), as previously described
(48), with ubiquinone standards, including
18-methylene-ubiquinone-8 extracted from methanotrophically grown cells
of M. capsulatus Bath (9). The G+C content of the
total DNA was measured according to the method described previously
(24). The extracted total DNAs were digested with P1
nuclease and alkaline phosphatase with a Yamasa GC Kit (Yamasa Shoyu
Co., Choshi, Japan) and analyzed by high-performance liquid
chromatography (Shimadzu LC-9A with a CLC-ODS column).
Probes for the sMMO gene.
Two probes for detecting
mmoX and mmoC genes were generated by PCR with
the primer sets described by Miguez et al. (35). A 370-bp
fragment of the mmoX gene amplified from
Methylomonas sp. strain KSWIII with the
mmoX-specific primers mmoX1 and mmoX2 (35) was used as the mmoX-specific probe. A
890-bp fragment of the mmoC gene amplified from M. capsulatus Bath with primers mmoC1 and mmoC2
(35) was used as the mmoC-specific probe.
Amplification reactions were performed with the reagents supplied with
AmpliTaq Gold (Perkin-Elmer Applied Biosystems, Foster City,
Calif.) at a magnesium ion concentration of 2 mM, with 500 ng to 1 µg
of DNA and 40 pmol of each primer. The reactions were carried out with
a TaKaRa PCR Thermal Cycler MP (Takara Shuzo, Kyoto, Japan) with
preincubation at 95°C for 9 min and 35 cycles of 95°C for 1 min,
50°C for 1 min, and 72°C for 2 min. The PCR products were purified
with Microspin S-200 HR columns (Amersham Pharmacia Biotech, Uppsala, Sweden).
Cloning and sequencing of sMMO gene clusters.
The total DNAs
of methanotrophic strains were isolated with the Qiagen (Hilden,
Germany) genomic DNA buffer set and Qiagen Genomic-Tip 20/G. The
KpnI-digested total DNA of strain KSWIII was analyzed by
Southern hybridization with the digoxigenin (DIG)-labeled mmoX- or mmoC-specific probe. DNA labeling was
carried out with a DIG DNA labeling and detection kit (Boehringer
Mannheim GmbH, Mannheim, Germany). Hybridizations were carried out by
the standard methods (42) with DIG Easy Hyb (Boehringer
Mannheim) at 42°C. Washing of membrane filters after hybridization
was carried out at 68°C. An 8.1-kb fragment was detected with both
the mmoX- and mmoC-specific probes. Restriction
fragments were ligated into ZAP Express vector (Stratagene, La Jolla,
Calif.). The ligated DNA was packaged in bacteriophage particles with a
Gigapack III Gold (Stratagene) and transfected into Escherichia
coli XL1-Blue MRF'. The clone library was then probed with the
DIG-labeled mmoX-specific probe to identify the 8.1-kb
KpnI fragment. The fragment was sequenced by the
primer-walking method with an ABI 377 automated DNA sequencer (Perkin-Elmer Applied Biosystems) by cycle sequencing with a dRhodamine terminator cycle sequencing FS ready-reaction kit (Perkin-Elmer Applied
Biosystems). The 7.5-kb SalI fragment containing sMMO genes
of strain KSPIII was also cloned and sequenced as described above.
Sequence analysis.
DNA and deduced amino acid sequences were
analyzed with the GENETYX-WIN (version 3.1.0) software package
(Software Development Co. Ltd., Tokyo, Japan). Searching for homologous
proteins was done with the BLAST (version 2.0) program (1).
Multiple alignments were run under the Clustal W (version 1.6) program
(49). Secondary-structure predictions were made with the
SIMPA96 program (29) based on the nearest-neighbor method.
Phylogenetic trees were constructed by the neighbor-joining method
(41) with the MEGA program (27). The reference
DNA sequences used in the comparison were retrieved from the DDBJ,
EMBL, and GenBank nucleotide sequence databases, and the reference
protein sequences were retrieved from the SWISS-PROT database.
Field treatments of the in situ-biostimulation experiment.
The field treatments for in situ biostimulation were carried out at the
Kururi test site. The TCE concentration in groundwater at the test site
was approximately 200 µg/liter. The groundwater was continuously
pumped from the extraction well at an extraction rate of 1.5 liters/min
and injected into the injection well (for the locations of the
injection, sampling, and extraction wells at the test site, see Fig.
5A). At first, to survey the movement of the groundwater during the
treatments, bromide was continuously added to the extracted water and
injected. The concentration of bromide was measured after 10 days of
circulation of the groundwater. Then the biostimulation was started on
25 September 1998 by the addition of (i) methane to a dissolved
concentration of 10 mg/liter, (ii) oxygen to a dissolved concentration
of 30 mg/liter, and (iii) KNO3 (18 mg/liter) and
KH2PO4 (15 mg/liter) to serve as nutrient salts. The addition was repeatedly carried out in the following cycle:
addition of methane, 190 min; no addition, 50 min; addition of oxygen
and nutrient salts, 190 min; and no addition, 50 min.
PCR amplification of mmoX gene from total DNA
extracted from groundwater samples.
Groundwater samples were
collected 1 day before the start of the biostimulation treatments (24 September) and 10 days after the start (5 October) at the five sampling
wells (S1, S2, S3, S4, and S5) and at the extraction well (E).
Groundwater samples (200 ml) were then filtered with a
0.2-µm-pore-size Isopore membrane filter (Millipore Corporation,
Bedford, Mass.) to collect bacteria. The DNA from bacteria collected
with the filter was extracted with the FastDNA SPIN kit (for soil)
(Bio101 Inc., Vista, Calif.). The DNA sample was then dissolved in 100 µl of Tris-EDTA, and 1 µl was used for PCR. The amounts of DNA
samples which were used for amplification were 23.4, 12.4, 23.8, 11.7, and 31.7 ng for the DNA samples collected from wells S1, S2, S3, S4, E,
and S5, respectively, on 24 September, and 26.2, 14.4, 15.0, 11.1, 12.4, and 14.9 ng for the samples collected from wells S1, S2, S3, S4, E, and S5, respectively on 5 October. The PCR was carried out with a
combination of primer mmoXr901
(5'-TGGGTSAARACSTGGAACCGCTGGGT-3'; nucleotide positions 926 to 901 from the beginning of mmoX), which was newly designed
based on the sequence data including the mmoX genes from
strains KSWIII and KSPIII, and primer mmoX1 (35). Amplification reactions were performed as described above with one
modification: the annealing temperature was altered to 60°C.
Nucleotide sequence accession numbers.
The complete sequence
of the sMMO gene cluster of Methylomonas sp. strains KSWIII
and KSPIII will appear in the DDBJ, EMBL, and GenBank databases with
accession no. AB025022 and AB025021.
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RESULTS AND DISCUSSION |
Classification of strains KSWIII and KSPIII.
The phylogenetic
analysis based on 16S rRNA sequences revealed that strains KSWIII and
KSPIII were almost identical, with a sequence similarity of 99.2%, and
that both strains were closely related to M. methanica, with
sequence similarities of 97.8 and 98.1%, respectively (21).
It suggested that the isolates belonged to the genus
Methylomonas.
Several findings in this study support the suggestion. The cells of the
strains were short rods that were 0.8 to 1.1 µm wide and 1.3 to 2.0 µm long. The cultures of the strains were pink. Ultrasonically
disrupted cells of both strains had three absorption maxima, at 474, 504, and 539 nm, which reflected the presence of carotenoids. The
possession of carotenoids is a distinctive feature of species in the
genus Methylomonas and is rarely observed in other species
of methanotrophs (2). Ultrathin-section electron microscopy
showed that the cells grown on NMS medium in the presence of methane
and 1 mM CuSO4 possessed bundle-like intracytoplasmic membrane structure, which is typical of the group I methanotrophs (52). Strain KSWIII predominantly contained an
18-methylene-ubiquinone-8, which has been found in several members of
the group I methanotrophs, e.g., M. methanica, M. capsulatus, and Methylocaldum gracile (9). Strain KSWIII had 52.0 mol% G+C content, which was within the range of
the previously described DNA base composition in M. methanica (51 to 53%) (2). On the basis of these data,
we decided to designate the strains KSWIII and KSPIII
Methylomonas sp.
Cloning and sequencing of sMMO gene clusters in strains KSWIII and
KSPIII.
An 8,107-bp KpnI fragment from strain KSWIII
and a 7,531-bp SalI fragment from strain KSPIII were cloned
and sequenced. Six open reading frames which showed high homology to
mmoX, mmoY, mmoB, mmoZ,
orfY, and mmoC (partial in the fragment from
strain KSPIII) were found in each fragment (Fig.
1). The deduced amino acid sequences of
mmo gene products from the two strains were almost identical
(only one amino acid mismatch was found in MmoC) (Table
1).
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FIG. 1.
Restriction maps of the mmo gene clusters.
The upper line shows the cloned 8.1-kb KpnI fragment from
strain KSWIII. The lower line shows the cloned 7.5-kb SalI
fragment from strain KSPIII. The open boxes represent the
mmoX, -Y, -Z, -B,
orfY, and mmoC genes. The locations of
restriction sites are marked as follows: S1, SalI; Kp,
KpnI; Sp, SphI; Ec, EcoRI; Xb,
XbaI.
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The deduced amino acid sequences of MmoX, MmoY, MmoZ, and MmoC proteins
from the two strains were aligned with those of Mmo
proteins from
M. capsulatus Bath (
45,
46),
M. trichosporium OB3b (
5,
6),
Methylocystis sp.
strain M (
34), and the
partial sequences of MmoX from
acidophilic methane-oxidizing strains
K, M131, and S6 (
14)
and an uncultured bacterium (
13) (see
Fig.
3 and
4).
The two consensus regions for putative iron binding sites, which were
found in the three known MmoXs (
5,
34,
46), and
other iron
binding proteins (
4,
38,
39) were well-conserved
in the
MmoXs of the strains KSWIII and KSPIII, located at amino
acid positions
143 to 147 and 242 to 246 (Fig.
2A). The
six amino
acids,
114E,
144E,
147H,
209E,
243E, and
246H, which were found by X-ray crystal structure analysis
to form
the diiron center on the hydroxylase component of sMMOs (MMOH)
from
M. capsulatus Bath and
M. trichosporium OB3b
(
16,
40),
were also conserved in the MmoXs from the two
strains (Fig.
2A).
The secondary-structure prediction analysis of the
MmoXs of the
strains KSWIII and KSPIII demonstrated that most of the
-helical
and
-strand structure elements from the X-ray structure
analysis
of strain Bath and strain OB3b were present in the MmoXs of
the
strains KSWIII and KSPIII, with only the one exception of
-helical
element 5 (
5) (Fig.
2A).
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FIG. 2.
Alignments of deduced amino acid sequences of the
mmoX (A), mmoY (B), and mmoZ (C) genes
from strain KSWIII, M. capsulatus Bath (accession no. M90050
and M32314) (45, 46), M. trichosporium OB3b
(X55394 and S81887) (5, 6), and Methylocystis sp.
strain M (U81594) (34). The partial sequences of the
mmoX genes from strains K, M131, and S6 (K, M131, S6_MmoX;
AF004554) (14) and an uncultured bacterium (PeatBog24_MmoX;
AF004555) (13) are also aligned (A). Identical residues are
in solid boxes, and similar residues are in shaded boxes. Underneath
the aligned sequences, the secondary structural features of the MMOH
from strain OB3b obtained from the crystal structure analysis are
marked with bars (2nd structures); the helical () and sheet ()
regions are assigned as described previously (16). The
predicted -strand () and -helical () regions for MmoX, -Y,
and -Z from strain KSWIII (KSWIII_pred.) are also marked. Two conserved
regions containing amino acids which are believed to serve as potential
iron ligands are enclosed by open boxes. The asterisks above the
aligned sequences indicate the amino acids which form the diiron center
revealed by X-ray crystal analysis of the MMOHs from strain OB3b and
strain Bath.
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The multiple alignments of the deduced amino acids sequences of MmoY
and MmoZ showed that there is relatively low conservation
among the
C-terminal portions of MmoYs (amino acid positions between
300 and 393 in strain KSWIII) and the N-terminal portions of MmoZs
(amino acid
positions between 1 and 100 in strain KSWIII). However,
the locations
of most putative secondary-structure elements of
MmoY and MmoZ from
strains KSWIII and KSPIII agreed with those
from the X-ray structure
analysis of the MMOHs of strains Bath
and OB3b, with only one exception
for MmoY (
2 was not predicted)
and only one exception for MmoZ (
4
was not predicted) (Fig.
2B
and C). These findings suggest that the
MMOHs from the
Methylomonas sp. strains KSWIII and KSPIII
have structures similar to those
from
M. capsulatus Bath and
M. trichosporium OB3b.
The sequences of MmoCs of strains KSWIII and KSPIII agreed with the
previous assignment of the reductase components (MMOR)
to the extended
flavoprotein reductase (FNR) family (
32). The
consensus
sequences, which were observed in the members of the
extended FNR
family (
10,
11), including the known MMORs (
6,
46), conserved cysteine residues for the N-terminal [2Fe-2S]
domain, RXYS and GXXS for the central [FAD] domain, and GTGIXP,
YXCGP, and EXF for the C-terminal [NAD] domain (
10), were
observed
in the MmoCs of strains KSWIII and KSPIII (Fig.
3). The deduced
amino acid sequences of
MmoCs from strains KSWIII and KSPIII enabled
improved accuracy of the
secondary-structure prediction analysis
of the multiple alignment of
MmoCs, which showed that most of
the locations of the putative
-helix and
-strand structures
were identical to those of the
X-ray crystal structure of FNR
(
25) and phthalate
dioxygenase reductase (PDR) (
10), although
the sequence
similarities between MmoCs and FNR or PDR were not
so high (Fig.
3).
The three domains of MmoCs, including those
from
Methylomonas sp. strains KSWIII and KSPIII were, therefore,
likely to form structures similar to those of the corresponding
domains
of other proteins in the extended FNR family.
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FIG. 3.
Alignments of the [2Fe-2S] domain (A), the [FAD]
domain (B), and the [NAD] domain (C) of the deduced amino acid
sequences of the mmoC genes from strain KSWIII, M. capsulatus Bath, M. trichosporium OB3b, and
Methylocystis sp. strain M. The primary structures of the
[2Fe-2S] domain (A) and the [NAD] domain (C) of PDR from
Burkholderia cepacia (accession no. P33164) (10)
and the [FAD] domain (B) of FNR from spinach (P00455) (25)
are also aligned. Identical residues are in solid boxes, and similar
residues are in shaded boxes. Underneath the aligned sequences, the
secondary-structural features of PDR (A and C) or FNR (B) obtained in
the crystal structure analyses are shown (2nd structures) as solid bars
(-helical regions []) and shaded bars (-strand regions
[]), together with the consensus features of predicted -strand
() and -helical regions () for the MmoCs (consensus pred.).
The asterisks above the aligned sequences indicate the consensus
sequences for FNR family proteins.
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In the regulatory protein B of sMMO (MMOB), the
29Q-
30V cleavage site, which was responsible
for forming a truncated inactive
form called B' in all the known MmoBs
(
31), was found in amino
acid sequences from strains KSWIII
and KSPIII. However, the
12M-
13G site, which
was responsible for forming another inactive form
called B" found only
in MmoB of
M. capsulatus Bath (
31), was
not found
in amino acid sequences of strains KSWIII and KSPIII.
The open reading
frame
orfY has been identified in the sequences
of all sMMO
genes, including our
Methylomonas sp. strains. There
was
significant conservation, especially in their central regions,
among
these deduced amino acid sequences. However, the function
of this
orf is not clear, since it differs from any sequences
in the
database.
A putative promoter sequence upstream of
mmoX from strains
KSWIII and KSPIII
(C
2083TGGCACN
5TTGCA
2099
in strain KSWIII) was similar to the consensus sequence
recognized
by
E. coli RNA polymerase containing
54 (
23). Similar
54 promoters
were also found upstream of the
mmoX genes from
M. capsulatus Bath (
46),
M. trichosporium OB3b
(
5), and
Methylocystis sp. strain M
(
34).
Phylogenetic analysis of sMMO gene clusters.
DNA and amino
acid sequence similarities are shown in Table 1. The sMMO genes from
the strains KSWIII and KSPIII were more closely related to those from
M. capsulatus Bath than to those from M. trichosporium OB3b or those from Methylocystis sp.
strain M (Table 1). The partial amino acid sequence of MmoX from
Methylomicrobium sp. strain NI (group I) (19)
showed high homology with those from our Methylomonas sp.
strains, with 96.6% identity (data not shown).
The phylogenetic analysis of the deduced amino acid sequences of MmoX
proteins demonstrated that the sequences form two clusters
(Fig.
4A).
One cluster consisted of three strains in the
Proteobacteria (
M. capsulatus Bath [group X]
and
Methylomonas sp. strains KSWIII
and KSPIII [group I]),
and the other cluster consisted of two
strains in the
Proteobacteria (
M. trichosporium OB3b [group
II] and
Methylocystis sp. strain M [group II]) and the
partial
sequence of a PCR product amplified from environmental samples
(Peatbog 24). The partial sequence of the acidophilic methane-oxidizing
strains K, M131, and S6, which were affiliated with
Beijerinckia indica subsp.
indica in the
Proteobacteria (
14), showed almost
equal
distances to the two clusters. The phylogenetic relationship
determined
from MmoXs was significantly correlated with the relationship
determined from 16S rRNA sequences (Fig.
4B). The phylogenetic
analysis of the
deduced amino acid sequences of the other Mmo
proteins also showed
similar results (data not shown).
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FIG. 4.
Phylogenetic relationship of the deduced amino acid
sequences of the mmoX genes (A) and of the 16S ribosomal DNA
(rDNA) sequences (B) among methanotrophs. The trees were constructed
from evolutionary distances obtained by the neighbor-joining method
(41). The bars represent 2 amino acid substitutions per 100 amino acids in MmoXs (A) and 2 nucleotide substitutions per 100 nucleotides in 16S rDNA sequences (B). The 16S rDNA sequence of
Desulfovibrio desulfuricans that belongs to the  subclass
of Proteobacteria was used to root the tree (B). Bootstrap
probabilities (27) are indicated at the branching points.
The accession number of each reference sequence is shown in
parentheses.
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PCR primer design for detection of sMMO gene.
There have been
several reports of the detection of methanotrophs possessing sMMO genes
in environmental samples by amplification with PCR primers for sMMO
genes (13, 33, 35). Miguez et al. used a primer set
(mmoX1 and mmoX2) to detect mmoX genes
in pure cultures and in environmental samples (35). However,
the primer mmoX2 had several mismatches with the gene of our
Methylomonas sp. strains, so the primer set did not work to
amplify the mmoX gene from a small quantity (below 1 ng) of
DNA from the strains.
We therefore designed a new PCR primer (
mmoXr901) based on
the sequence data including strains KSWIII and KSPIII to cover
all of
the known methanotrophs harboring the
mmoX gene. By using
a
combination of this primer and primer
mmoX1, the
corresponding
DNA fragment was amplified from a minimum of 100 pg of
purified
DNA from
M. capsulatus Bath,
M. trichosporium OB3b,
Methylocystis sp. strain M, and
strains KSWIII and KSPIII (data not
shown).
Detection of sMMO gene at in situ-biostimulation site.
The
primer set was applied to detect the methanotrophs in the groundwater
samples at the in situ-biostimulation site. There were no amplified
products from total DNAs from the groundwater samples 1 day before the
start of the biostimulation treatment (Fig.
5B, lanes 2 to 7). However, a distinct
amplified product of 396 bp was obtained from the groundwater sample
extracted at well S1 10 days after the start (Fig. 5B, lane 8).
Significant amplified products were observed from the groundwater
sample extracted from wells S2, S3, and S4 (Fig. 5B, lanes 9, 10, and
11). No significant amplification was observed in the samples from
wells S4 and S5 (Fig. 5B, lanes 12 and 13). Very weak PCR product
signals (less than 10 ng per PCR) were detected in the samples shown in
Fig. 5B, lanes 3 and 12. Some of the amplified products were cloned and
sequenced and were found to consist of mmoX genes similar to
those of Methylocystis sp. strain M or our
Methylomonas sp. strains (data not shown). This indicated
that our new PCR primer can detect not only the mmoX genes
of the group II and group X methanotrophs but also those of the group I
methanotrophs in environmental samples. In the tracer experiment with
bromide carried out prior to the biostimulation treatments, high
concentrations of bromide (23 to 27 mg/liter) were detected at wells
S1, S2, S3, and S4. However, 7 mg of bromide/liter was detected at the
extraction well, and trace levels of bromide were detected at well S5.
These results show that the chemical added to the groundwater spread into the aquifer near the injection well at a high concentration but
did not reach well S5 and that the extraction well contained an inflow
of external groundwater. The results of PCR amplification showed good
agreement with the results of the tracer experiment.
View larger version (44K):
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|
FIG. 5.
(A) Location of the injection (I), sampling (S1 to S5),
and extraction (E) wells at Kururi test site. The direction of
groundwater flow is indicated by the arrow. (B) Amplification of
mmoX from total DNA extracted from groundwater samples.
Lanes: 1, size marker (HaeIII-digested  X174 DNA); 2 to 7, PCR product from the groundwater at the S1, S2, S3, S4, E, and S5
wells, respectively, collected on 24 September 1998; 8 to 13, PCR
product from the groundwater at the S1, S2, S3, S4, E, and S5 wells,
respectively, collected on 5 October 1998; 14, PCR product from 100 pg
of purified total DNA of M. trichosporium OB3b.
|
|
These results could be valuable for further studies on monitoring the
population of methanotrophs during in situ-bioremediation
treatments
and on examining the diversity of methanotrophs present
in
environmental
samples.
| |
ACKNOWLEDGMENTS |
We thank Xian-Ying Meng for transmission electron microscopy. We
thank Kimitsu City Hall in Chiba Prefecture for their kind cooperation.
This work was conducted as one of the research and development
activities for the bioremediation project which is conducted by the
Research Institute of Innovative Technology for the Earth (RITE),
Japan, and funded by the Ministry of International Trade and Industry
(MITI) through the New Energy and Industrial Technology Development
Organization (NEDO).
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, Kurokami 2-39-1, Kumamoto-City, Kumamoto 860-8555, Japan. Phone: 81-96-342-3668. Fax: 81-96-342-3679. E-mail:
shige{at}kumamoto-u.ac.jp.
| |
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Abstract
Introduction
