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Applied and Environmental Microbiology, September 2001, p. 4177-4185, Vol. 67, No. 9
Max-Planck-Institut für terrestrische
Mikrobiologie, D-35043 Marburg, Germany
Received 22 December 2000/Accepted 26 June 2001
The diversity of methanotrophic bacteria associated with roots of
submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of
pmoA, which encodes the The atmospheric trace gas
methane (CH4) is a prominent "greenhouse"
gas. Its atmospheric concentration has been increasing until recently
at a rate of about 1% a year (8). Up to 70 to 80% of
atmospheric CH4 is biogenic (55).
Flooded rice fields are one of the major sources of biogenic
CH4 (34, 50). Estimations of the
annual emission rate from flooded rice fields range between 60 and 110 Tg (8, 21, 45). The upper limit of this emission rate accounts for approximately 25% of the total annual
CH4 emission into the atmosphere (8,
21).
Approximately 90% of the CH4 that is emitted
from rice paddies escapes through the aerenchyma of the rice plants,
whereas only 10% escapes through the floodwater (19, 52).
However, the aerenchyma does not merely function as a gas transport
system but rather constitutes a dynamic, oxygenated biofilter. The
diffusive input of oxygen into the below-ground plant surface area
enables aerobic methanotrophs to oxidize CH4.
Gilbert and Frenzel (22) showed that the activities of
methanotrophs were directly dependent on the oxygen availability in the
rice root environment. It was shown that up to 30% of the
CH4 produced in rice paddy soil is oxidized by
root-associated methanotrophs (5, 9, 15).
Based on phylogenetic, physiological, morphological, and biochemical
characteristics, methanotrophs are divided into two major subgroups
(27). The The methanotrophic diversity in rice field soil has been assessed in
detail (28, 30), but knowledge about the diversity of
methanotrophic populations associated with rice roots is still limited.
Type II strains were isolated from the terminal positive-dilution steps
of a most-probable-number dilution series (23).
However, whether these results reflect the natural situation on rice
roots, i.e., predominance of type II methanotrophs, or instead were the consequence of cultivation bias, is unclear.
We assessed the methanotrophic diversity associated with roots of
submerged rice plants using various cultivation-independent techniques.
This assessment was carried out in relation to the methanotrophic
diversity detectable in rice paddy bulk soil. Despite the phylogenetic
distance between type I and type II methanotrophs, almost all known
methanotrophs possess a pmoA gene, which encodes the To examine the meaningfulness of the pmoA-based results, we
also assessed the methanotrophic diversity detectable by retrieval of mmoX (25) and mxaF
(42). The mmoX gene encodes the Rice microcosms.
Rice (Oryza sativa var. Roma,
type japonica) was grown in three flooded, unfertilized microcosms for
70, 84, or 90 days using conditions described previously (20,
26).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4177-4185.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Methanotroph Diversity on Roots of
Submerged Rice Plants by Molecular Retrieval of
pmoA, mmoX, mxaF, and
16S rRNA and Ribosomal DNA, Including pmoA-Based
Terminal Restriction Fragment Length Polymorphism Profiling
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
subunit of the particulate
methane monooxygenase. A novel methanotroph-specific
community-profiling method was established using the terminal
restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP
profiles clearly revealed a more complex root-associated methanotrophic
community than did banding patterns obtained by
pmoA-based denaturing gradient gel electrophoresis. The
comparison of pmoA-based T-RFLP profiles obtained from
rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on
rice roots than in the bulk soil. These were affiliated to the genera
Methylomonas, Methylobacter,
Methylococcus, and to a novel type I methanotroph
sublineage. By contrast, type II methanotrophs of the
Methylocystis-Methylosinus group
could be detected with high relative signal intensity in both soil and
root compartments. Phylogenetic treeing analyses and a set of
substrate-diagnostic amino acid residues provided evidence that a novel
pmoA lineage was detected. This branched distinctly from
all currently known methanotrophs. To examine whether the retrieval of
pmoA provided a complete view of root-associated
methanotroph diversity, we also assessed the diversity detectable by
recovery of genes coding for subunits of soluble methane monooxygenase
(mmoX) and methanol dehydrogenase (mxaF).
In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved
using a PCR primer set specific to type I methanotrophs. The overall
methanotroph diversity detected by recovery of mmoX,
mxaF, and 16S rRNA and 16S rDNA corresponded well
to the diversity detectable by retrieval of pmoA.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-proteobacterial type I methanotroph
group comprises the genera Methylomonas,
Methylocaldum, Methylomicrobium, Methylobacter, Methylosarcina,
Methylosphaera, and Methylococcus (also
classified as type X) (4, 6, 27, 58), while the -proteobacterial type II methanotroph group consists of the genera Methylocystis and Methylosinus (27)
and one more distant species, Methylocella palustris
(14).
subunit (PmoA) of the particulate methane monooxygenase (pMMO). The
only exception is Methylocella palustris (13,
14). Consequently, this study focused mainly on the retrieval of
pmoA using PCR primers described previously
(32). These primers also target amoA,
which encodes the subunit (AmoA) of the ammonia monooxygenase in
autotrophic ammonia oxidizers. Based on the pmoA sequence
database created in this study, we established a novel methanotroph-specific community-profiling method using the
terminal restriction fragment length polymorphism (T-RFLP)
technique (37, 39). The methanotroph diversity
detectable by pmoA-based T-RFLP profiling was compared
with those detectable by comparative sequence analysis of
cloned pmoA and by pmoA-based denaturing
gradient gel electrophoresis (DGGE).
subunit (MmoX) of the hydroxylase component of the soluble methane
monooxygenase (sMMO). This monooxygenase is present in most
type II methanotrophs, in members of the genus
Methylococcus, and in some Methylomonas strains
(53) but not in most of the other type I methanotrophs (27). The mxaF gene codes for the subunit
of the methanol dehydrogenase, which is present in all
methylotrophs. In addition, both 16S ribosomal DNA (rDNA) and 16S rRNA
were retrieved using PCR primers specific to type I methanotrophs
(57).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Extraction of total DNA. Total DNA from the bulk soil of the flooded rice microcosms was extracted and purified using a protocol reported previously (38). In this protocol, microbial cells were lysed directly in the soil matrix by bead beating.
For extraction of total DNA from rice roots, the samples M84 and M90 were lyophilized, and the dried root material was subsequently pulverized with a mortar under liquid N2. The pulverized root material was resuspended in 1 ml of extraction buffer. Enzymatic lysis of microbial cells, as well as isolation and purification of total DNA, was performed using an extraction protocol described previously (26).Simultaneous extraction of total DNA and RNA.
Rice roots of
sample M70 were placed in a 2-ml reaction tube together with 1 g
of glass beads (diameter, 0.17 to 0.18 mm) and 700 µl of precooled
TPM buffer (50 mM Tris-HCl [pH 7.5], 1.7% [wt/vol]
polyvinylpyrrolidone, 10 mM MgCl2)
(18). This suspension was shaken for 60 s at maximum
speed in a bead beater (Dismembrator-S; B. Braun Biotech GmbH,
Melsungen, Germany). Glass beads, root particles, and cell debris were
pelleted by centrifugation for 5 min at 4°C, and the supernatant was
transferred to a new reaction tube. Seven hundred microliters of a
phenol-based lysis buffer was added to the pellet, and the bead-beating
procedure was repeated. After centrifugation, both supernatants were
pooled and extracted three times with cold phenol-chloroform (1:1,
vol/vol) followed by precipitation of total nucleic acids with 0.1 volume of sodium acetate (3 M; pH 5.2) and 2.5 volumes of ethanol. The
pellet was dried and suspended in 100 µl of Tris-EDTA buffer. For
subsequent DNA-based analyses a 50-µl aliquot was stored at 
20°C.
For preparation of total RNA, the other 50-µl aliquot was mixed with
1 volume of TMC buffer (10 mM Tris-HCl [pH 7.5], 5 mM
MgCl2, 0.1 mM CsCl2) (18) and 5 U of RNase-free DNase (Promega, Madison, Wis.)
and incubated for 1 h at 37°C to remove the DNA. The reaction
was stopped by extraction with 1 volume of chloroform. Precipitation and resuspension of total RNA were performed as described above.
PCR amplification.
The primer sets used in this study are
listed in Table 1. For
pmoA-based T-RFLP analysis, the 5' primer A189 was labeled with the dye carboxyfluorescein. The reaction mixture contained 1 to 5 ng of DNA, 50 µl of MasterAmp PCR premix F (Epicentre Technologies, Madison, Wis.), 0.3 µM concentrations of each primer (MWG-Biotech, Ebersberg, Germany), and 2.5 U of Taq DNA polymerase
(AmpliTaq; PE Applied Biosystems, Foster City, Calif.). Amplification
was performed in a total volume of 100 µl in 0.2-ml reaction tubes, using a DNA thermal cycler (model 2400; PE Applied Biosystems). The
thermal PCR profile was as follows: initial denaturation for 2 min at
94°C and 30 cycles consisting of denaturation at 94°C for 45 s, primer annealing for 60 s (annealing temperature specific to
each target gene [Table 1]), and elongation at 72°C for 120 s.
The final elongation step was 6 min. Aliquots of the amplicons (10 µl) were checked by electrophoresis on a 1% agarose gel.
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RT-PCR of 16S rRNA. Ribosomal copy DNA (rcDNA) of type I methanotrophs was synthesized from total RNA using primer MethT1cR (Table 1), Moloney murine leukemia virus reverse transcriptase (RT) (RNase H minus; Promega, Mannheim, Germany), and a previously described protocol (38). PCR of 16S rcDNA was carried out as described above.
pmoA-based T-RFLP analysis. T-RFLP analysis was performed for each total DNA extract in triplicate using a protocol reported previously (38, 39). The pmoA was amplified by PCR as described above. After purification with Qiaquick spin columns (Qiagen, Hilden, Germany), approximately 100 ng of the amplicons were digested with 10 U of the restriction endonuclease MspI (Promega). The digestions were carried out in a total volume of 10 µl for 3 h at 37°C. Aliquots (2.5 µl) of the digested amplicons were mixed with 2.0 µl of formamide and 0.5 µl of an internal lane standard (GeneScan-1000 ROX; PE Applied Biosystems). The mixtures were denatured at 100°C for 3 min and then chilled on ice. Electrophoresis on a polyacrylamide gel (6%) was performed using an automated DNA sequencer (model 373; PE Applied Biosystems) for 6 h at the following settings: 2,500 V, 40 mA, and 27 W (24-cm gel length). After electrophoresis, the sizes of the 5'-terminal restriction fragments (T-RFs) and the intensities of their fluorescence emission signals (i.e., signal intensities) were automatically calculated by the GeneScan Analysis software, version 2.1 (PE Applied Biosystems). The accuracy of size estimation between replicates was ±1 bp. The relative signal intensity of each T-RF was calculated based on the signal intensity of the individual T-RF in relation to the total signal intensity of all T-RFs (including the 531-bp fragment) detected in the respective T-RFLP community profile. The 531-bp fragment corresponds to pmoA amplicons without any restriction site for MspI.
pmoA-based DGGE. PCR amplification of pmoA and DGGE in the Dcode System (Bio-Rad, Munich, Germany) were carried out as described by Henckel et al. (28). In brief, PCR products were separated in 1-mm-thick polyacrylamide gels (6.5% [wt/vol] acrylamide-bisacrylamide [37.5:1]) using a linear denaturing gradient that ranged from 35 to 80%. A denaturing gradient of 80% corresponded to 6.5% acrylamide, 5.6 M urea, and 32% deionized formamide. The electrophoresis was performed in 0.5× TAE buffer (0.04 M Tris-base, 0.02 M sodium acetate, 1 mM EDTA [pH 7.4]) for 15 h at a constant voltage of 74 V at 60°C. Gels were stained with 1:10,000 (vol/vol) SYBR-Green I (Biozym, Hessisch-Oldendorf, Germany) for 45 min and scanned with a Storm 860 Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.).
Cloning and sequencing. PCR products of pmoA, mmoX, mxaF, 16S rDNA, and 16S rcDNA were cloned using the TOPO TA cloning kit (Invitrogen Corp., San Diego, Calif.) as recommended by the manufacturer. The preparation of plasmid DNA of randomly selected clones, PCR amplification of cloned inserts, and nonradioactive sequencing were carried out as described previously (48). In addition, oligonucleotide primers targeting internal regions of the cloned inserts were used for sequencing of mmoX and 16S rDNA and rcDNA.
Phylogenetic analysis. Based on sequence information deposited either in public-domain databases or generated in the course of this study, we established sequence databases for pmoA, mxaF, and mmoX. Each of these sequence databases was integrated into the ARB program package (developed by O. Strunk and W. Ludwig; Technische Universität München [http://www.arb-home.de]) and was manually put into an aligned format. The 16S rDNA and rcDNA clone sequences were added to a database of about 14,000 complete or partial bacterial 16S rRNA sequences. Evolutionary distances (ARB and PHYLIP [17]) between pairs of inferred amino acid sequences (pmoA, mmoX, and mxaF) were calculated using various models (11, 36). Evolutionary-distance values between pairs of 16S rDNA and rcDNA clone sequences were calculated by applying the Jukes-Cantor correction (35). The trees were constructed using the neighbor-joining method (49). The statistical significance levels of interior nodes were determined by performing bootstrap analyses by the neighbor-joining method (500 data resamplings). To exclude obvious chimeric primary structures from the pmoA, mmoX, mxaF, and 16S rDNA and rRNA sequence databases, separate treeing analyses of the 5' and 3' halves of the respective sequence data sets were carried out.
Nucleotide sequence accession numbers. The environmental pmoA (plus amoA and sequence types of uncertain affiliation), mmoX, mxaF, 16S rDNA, and 16S rcDNA clone sequences recovered in this study from rice roots of flooded rice microcosms have been deposited in the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession no. AJ299946 to AJ299968, AJ299515 to AJ29953, AJ299504 to AJ299514, AJ299969 to AJ299983, and AJ299984 to AJ299989, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The use of PCR primer sets with intended-target specificity for partial stretches of pmoA, mmoX, mxaF, and 16S rDNA resulted in amplicons of the predicted sizes (Table 1). Clone libraries were generated from the PCR products, and subsequently individual clones were randomly selected for comparative sequence analysis. In addition, methanotroph diversity was assessed by pmoA-based T-RFLP analysis.
pmoA-based cloning approach. One clone library (each) was generated from samples M84 and M90. In total, 47 pmoA clones were randomly selected for comparative sequence analysis (28 and 19 clones from samples M84 and M90, respectively).
Thirty-six clones formed three distinct clusters within the phylogenetic radiation of type I methanotrophs (Fig. 1A). Cluster I (16 clones) was affiliated with Methylomonas methanica. Cluster II (10 clones) formed a separate lineage without any clear affiliation to any of the known genera. Cluster III (10 clones) exhibited a moderate relationship to Methylococcus capsulatus.
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50 are shown. Scale bar, 0.1 substitution per amino acid site. Database accession numbers of
reference organisms are as follows: Methylocystis sp.
strain M, 
-proteobacterial
Nitrosomonas-Nitrosospira group. Five sequences
formed the lineages A, B, and C (Fig. 1A), which branched
distinctly from all currently known methanotrophs or autotrophic
ammonia oxidizers.
Separate treeing analysis of the 5' and 3' halves of the
respective sequence types suggested that the clone sequences assigned to the lineages A, B, and C were of natural origin, i.e., separate treeing analysis did not provide evidence that any of these clones were
chimeric. We therefore determined amino acid signature residues for the inferred peptide sequences of the lineages A, B, and
C (Table 2) (33). These are
either universal to PmoA (methanotrophs) and AmoA (autotrophic ammonia
oxidizers) or specific for either PmoA or AmoA
(substrate-diagnostic residues). Based on this approach, Holmes et al. (33) assigned a newly detected
cluster of sequence types (forest clones) (Table 2) to an
uncharacterized group of methanotrophs. The percentage distribution of
substrate-diagnostic residues suggests that lineage A corresponds to a
novel pmoA cluster rather than to an amoA
cluster. By contrast, due to the relatively low number of conserved
substrate-diagnostic residues, sequence types of the lineages B and C
could not be assigned to either PmoA or AmoA.
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pmoA-based T-RFLP analysis. Based on our pmoA sequence database derived from cultured methanotrophs and environmental samples, we predicted that the tetrameric restriction enzyme MspI would be the restriction enzyme most appropriate to analyze the genetic diversity of methanotrophic communities in a single electrophoretic profile. Defined mixtures of genomic DNA from cultured methanotrophs subjected to MspI-based T-RFLP analysis exactly produced those T-RFs predicted based on our pmoA sequence database (data not shown). Consequently, MspI was used for pmoA-based T-RFLP analysis of methanotrophic communities. Extraction of total DNA from the same root sample in triplicate followed by PCR amplification of pmoA and T-RFLP analysis produced highly similar T-RFLP community profiles. The coefficients of variation of the relative signal intensities between these profiles were between 5.4 and 12.3% for the major peaks, i.e., those with sizes of 80, 245, 350, 440, 505, and 531 bp (Fig. 1B; see also Fig. 2). The coefficients of variation for the major peaks between T-RFLP community profiles generated in triplicate from individual DNA extracts ranged from 1.7 to 7.6%. This analysis showed that the T-RFLP technique was reliable for a rapid PCR-based fingerprinting of methanotrophic communities. High reproducibility of the T-RFLP technique has been reported previously (e.g., for 16S rDNA-based T-RFLP analysis [39, 44]).
The comparison of T-RFLP community profiles obtained from rice root sample M84 with T-RFs of individual pmoA and amoA sequence types allowed the differentiation among type I methanotrophs, type II methanotrophs, and autotrophic ammonia oxidizers (Fig. 1). Type I methanotrophs were further differentiated into five distinct T-RFs, which correspond to phylogenetically distinct sublineages affiliated with the following: (i) pmoA cluster III plus members of the genera Methylococcus and Methylocaldum (80-bp T-RF); (ii) Methylomicrobium album (350-bp T-RF); (iii) pmoA cluster I plus Methylomonas methanica (440-bp T-RF); (iv) Methylobacter sp. strain BB5.1 (505-bp T-RF); and (v) pmoA cluster II (531-bp peak; no recognition site for MspI). The type II methanotrophs of the Methylocystis-Methylosinus group were characterized by the 245-bp T-RF, while the 47-bp T-RF was indicative of amoA (Nitrosomonas-Nitrosospira group). Three further minor peaks could be assigned to the lineages A (280-bp T-RF), B (228-bp T-RF), and C (113-bp T-RF). Several peaks of mainly lower relative signal intensity could be assigned to none of the pmoA or amoA sequence types retrieved from rice roots or deposited in public-domain databases. These T-RFs may correspond to novel pmoA or amoA sequence types. Root-associated methanotroph diversity detectable by T-RFLP analysis was compared to that detectable by DGGE using the same extract of total DNA as the starting material. DGGE revealed the presence of only pmoA sequence types affiliated with type II methanotrophs and pmoA cluster III (Fig. 1C). The community profiles obtained by pmoA-based T-RFLP analysis from root samples M70, M84, and M90 showed similar relative abundances of the major T-RFs, i.e., of T-RFs with sizes of 80, 245, 350, 440, 505, and 531 bp. The T-RFLP profiles generated from the anoxic bulk soil were characterized mainly by the 245-bp T-RF of type II methanotrophs, but minor peaks indicative of type I methanotrophs (80- and 350-bp T-RFs) and ammonia oxidizers (47-bp T-RF) were also present. The comparison of T-RFLP profiles obtained from bulk soil versus rice roots is shown for rice microcosm M84 (Fig. 2).
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mmoX.
Five of 15 clones analyzed were closely
related to the mmoX of Methylocystis sp.
strain LR1 (Fig. 3). This type II
methanotroph was isolated in Canada (16). Three
mmoX clones were assigned to Methylocystis sp.
strain M. The treeing analysis suggested that clone M84-S38 was
affiliated to the mmoX of the acidophile Methylocella palustris (13, 14). However, the
dissimilarity values of the predicted peptide sequence of clone
M84-S38 with MmoX of Methylocella palustris
(15.7%), the Methylocystis-Methylosinus group
(17.1 to 18.5%), and Methylococcus capsulatus Bath (17.5%) were all similar. The MmoX sequence types of the phylogenetically distinct type I methanotrophs Methylomonas sp. strain KSWIII
and Methylococcus capsulatus Bath differ by 11.3%
(53). Taking into account these dissimilarity values, it
can only be speculated that clone M84-S38 corresponds to a novel
methanotrophic bacterium which harbors sMMO. The remaining six clones
were false positives containing non-mmoX sequence types.
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mxaF.
Twenty-four of 50 clones analyzed formed
a coherent mxaF sequence cluster related to
Methylomonas methanica and Methylomicrobium album. One sequence type each could be assigned to
Methylocystis sp. strain LR1 (16), strain LK6
(Fig. 4), and
Hyphomicrobium sp. strain CM2 (data not shown). The
remaining 23 clones were false positives containing non-mxaF
sequence types.
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16S rDNA and 16S rRNA analysis of type I methanotrophs.
16S
rDNA clone libraries were generated from samples M70, M84, and M90. An
rcDNA clone library was created only from freshly prepared roots of
sample M70. Because RT-PCR was unsuccessful with the primer set
MethT1dF and MethT1bR, we replaced MethT1bR with primer MethT1cR (Table
1). The use of MethT1cR resulted in an amplicon of the predicted size
(556 bp). The retrieval of both 16S rDNA and 16S rRNA led to the
identification of a diverse community of type I methanotrophs (Fig.
5). No false-positive clone sequences
were detected in a set of 23 16S rDNA and 6 16S rcDNA clones randomly
selected for analysis. This underlines the target specificity of these
PCR primers for type I methanotrophs (57).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Root-associated methanotroph diversity assessed by comparative analysis of pmoA, mmoX, mxaF, and 16S rRNA and rDNA sequences. The branching pattern of the pmoA tree showed a remarkable congruence with that of the 16S rDNA and rcDNA tree (Fig. 1A and 5). Therefore, it is highly likely for example that pmoA cluster III corresponds to the 16S rDNA branch characterized by the clones M90-D37, M84-D38, and M84-D36. Although it is difficult to deduce a close phylogenetic correspondence between distinct clusters of environmental sequence types from two different gene markers, the similarity in the tree topologies suggests that members of the same type I sublineages were detected by both the pmoA and 16S rDNA approaches. This finding agrees well with the results of a cultivation-independent characterization of methanotrophic populations in the sediment of Lake Washington (Seattle, Wash.) (10). The comparison of environmental libraries constructed for methanotroph 16S rRNA and pmoA genes also showed that the two different genes cover very similar ranges of diversity.
The mxaF gene represents a universal marker for methylotrophs. Its retrieval should allow detection of a range of methanotroph diversity similar to that detected by recovery of pmoA and 16S rRNA and rDNA. However, almost all mxaF clones formed only one distinct cluster which might phylogenetically correspond to members of pmoA cluster I (Fig. 1A and 4). This finding suggests that the currently available mxaF PCR assay is only of limited value for the assessment of methanotroph and methylotroph diversity and should be applied only in conjunction with other gene markers. Both the pmoA-based T-RFLP profiles (Fig. 1 and 2) and the retrieval of mmoX confirmed the presence of type II methanotrophs on rice roots. The mmoX-targeted primers used in this study can be considered to be complementary to primer sets previously published for the specific detection of mmoX (2, 40, 43). Their applicability in environmental studies is demonstrated especially by the retrieval of clone M84-S38 (Fig. 3). This sequence type expands our current knowledge about mmoX-based methanotroph diversity. Overall, comparative sequence analysis of cloned pmoA, mmoX, mxaF, and 16S rDNA and rcDNA revealed a genetically highly diverse methanotrophic community associated with rice roots.Comparison of various pmoA-based approaches to assessing methanotroph diversity (cloning, DGGE, and T-RFLP analysis). The frequency distribution of methanotroph subgroups in the clone library generated from sample M84 was not consistent with the relative signal intensities of the corresponding T-RFs observed in the T-RFLP community profiles (Fig. 1). For instance, pmoA sequence types affiliated with Methylomicrobium album (350-bp T-RF) and Methylobacter sp. strain BB.1 (505-bp T-RF) were not detected by the cloning approach. This finding might be indicative of cloning bias and/or sampling error (i.e., analysis of too few pmoA clones). Similarly, the proportion of type II methanotroph pmoA sequence types in the clone library was lower (2 of 28 clones analyzed) than one would expect based on the high relative signal intensity of the 245-bp T-RF (35.4%). This observation indicates either some bias against cloning of pmoA genes from type II methanotrophs or errors in the strict correlation of the 245-bp T-RF with type II methanotrophs of the Methylocystis-Methylosinus group. However, both T-RFLP analysis of individual pmoA clones retrieved from rice roots and a thorough in silico analysis of our current database of approximately 130 pmoA sequences suggest such a strict correlation of the 245-bp T-RF with type II methanotrophs.
The disparity between the frequency distribution of methanotroph subgroups in the clone library and the relative signal intensities of the corresponding T-RFs stresses a major methodological advantage of MspI-based T-RFLP analysis
that it provides an exact
quantitative measure of the T-RF composition of pmoA PCR
products. Also, pmoA-based methanotroph diversity detectable
by T-RFLP analysis was clearly higher than that detectable by
DGGE (Fig. 1B versus C). Explanations for this finding might be
that sequence types are separated in T-RFLP analysis and DGGE by
different methodological principles and staining of DGGE gels is less
sensitive than fluorescence detection in T-RFLP analysis. Taking
these observations together, it can be concluded that for
cultivation-independent assessment of methanotroph diversity
pmoA-based T-RFLP analysis represents an important
tool to complement the pmoA-based cloning approach and DGGE.
Ecological significance of root-associated methanotroph diversity. The analysis of phospholipid ester-linked fatty acids (PLFA) recovered from rhizosphere soil of flooded rice microcosms indicated an increased abundance of type I methanotrophs after NH4+ fertilization (ninefold increase in the type-I-specific PLFA biomarker), while the type-II-specific PLFA biomarker increased only two- to threefold after NH4+ fertilization (3). These results led to the conclusion that a high ammonium concentration is essential for growth of type I methanotrophs. In a control experiment based on molecular retrieval of 16S rDNA and DGGE, type I methanotrophs were also detected in rhizosphere soil of unfertilized rice microcosms. However, type I methanotrophs were not detected in the bulk soil of these microcosms. Based on these preliminary data, it was concluded that the rice plant itself also favors growth of type I methanotrophs (3).
The latter conclusion is clearly supported by this study. The T-RFLP profiles suggest a substantially higher relative abundance of type I methanotrophs on rice roots than in the bulk soil (Fig. 2). The detection of rRNA from type I methanotrophs (Fig. 5) provides evidence that these species are metabolically active and thus further supports the idea that rice roots are an important habitat for type I methanotrophs. The promotion of methanotrophic activity by rice plants might be of twofold nature. The diffusional input of oxygen into the root environment directly affects the activities of the obligately aerobic methanotrophs. An indirect effect on NH4+ availability might be mediated by the escape of oxygen from the root tissue into the rhizosphere soil, which can increase the redox potential in the root vicinity and, as a consequence, lead to the desorption of fixed NH4+ ions from clay minerals (51). Increased availability of ammonium should especially favor proliferation of type I methanotrophs (3, 27). The T-RFLP profiles obtained from rice roots and bulk soil are characterized by high relative signal intensities of the type II methanotroph-specific 245-bp T-RF (35.4 and 67%, respectively). In the anoxic bulk soil, 16S rRNA genes extracted from desiccation-resistant exospores and lipid cysts formed by Methylosinus spp. and Methylocystis spp. (56), respectively, as well as from vegetative cells present in a stage of anaerobic dormancy (46, 47), might have contributed to the high relative signal intensity. The cultivation-independent characterization of type I methanotrophs in unfertilized rhizosphere soil by Bodelier et al. (3) resulted in the detection of only one distinct cluster of highly similar 16S rDNA sequence types related to Methylobacter spp. (Fig. 5, sequence type RRI1). By contrast, our results of comparative sequence analysis of cloned pmoA and T-RFLP community profiling revealed a more complex population structure encompassing type I methanotrophs affiliated with the genera Methylomonas, Methylobacter, Methylomicrobium, and Methylococcus and a novel type I methanotroph sublineage. The considerable number of distinct methanotrophic populations that colonized the root compartment is also illustrated by the recovery of various 16S rDNA sublineages of type I methanotrophs. The presence of such a highly diverse methanotrophic community might indicate that there are a large number of ecological microniches characterized by spatiotemporal variations in the mixing ratios of CH4 and O2 (1, 30, 31) and in the availability of nitrogen (3, 24). The hypothesis that the CH4/O2 mixing ratio is an important regulator of root-associated methanotroph diversity is supported by the close correspondence between mxaF sequence types obtained in a previous study (clones Mo1/Mo2 [31]) and the mxaF cluster detected on rice roots (Fig. 4). Mo1 and Mo2 were detected by pmoA-based DGGE in a methanotrophic consortium enriched from rice field soil under a high CH4/low O2 mixing ratio, while enrichment conditions using low CH4/high O2 and low CH4/low O2 mixing ratios did not favor growth of these type I methanotrophs.Final conclusions. The comparison of data obtained by retrieval of pmoA with those obtained by recovery of mmoX, mxaF, and 16S rDNA and rRNA clearly indicates that pmoA represents an excellent functional gene marker for cultivation-independent analysis of methanotrophic diversity. However, the data also indicate that a comprehensive view of methanotrophic diversity can be obtained only by a combined use of various molecular techniques, i.e., by cloning and sequencing and by cloning-independent fingerprinting. Within this framework, the pmoA-based T-RFLP analysis proved to be a suitable tool to rapidly assess methanotrophic diversity. Taking all molecular data together, a highly diverse community of type I and type II methanotrophs was detected. Except for Methylomicrobium album, type I methanotroph populations were detected in the root environment with clearly higher relative signal intensities than in the bulk soil. Thus, the data as a whole agree well with the hypothesis that type I methanotrophs are predominant in environments that allow rapid growth of methanotrophic bacteria, while type II methanotrophs are more abundant in environments where growth rates are periodically restricted (27, 54).
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ACKNOWLEDGMENTS |
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We are grateful to Sonja Fleissner for excellent technical assistance.
This work was supported by a grant from the European Community RTD Programme Biotechnology (contract BIO-CT96-0419). M.T.Y. thanks the Deutschen Akademischen Austauschdienst for financial support in the form of a PhD scholarship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany. Phone: 49 (6421) 178 720. Fax: 49 (6421) 178 809. E-mail address: liesack{at}mailer.uni-marburg.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 2001, p. 4177-4185, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4177-4185.2001
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