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Applied and Environmental Microbiology, February 1999, p. 837-840, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Phylogenetic Diversity of Symbiotic Methanogens
Living in the Hindgut of the Lower Termite Reticulitermes
speratus Analyzed by PCR and In Situ Hybridization
Naoya
Shinzato,1,2,*
Tadao
Matsumoto,1
Ikuo
Yamaoka,3
Tairo
Oshima,2 and
Akihiko
Yamagishi2
Department of Biology, School of Arts and
Science, University of Tokyo, Komaba, Meguro-ku, Tokyo
153-0041,1
Department of Molecular
Biology, School of Life Science, Tokyo University of Pharmacy & Life
Science, Horinouchi, Hachiouji, Tokyo
192-0392,2 and
Biological Institute,
Faculty of Science, Yamaguchi University, Yoshida, Yamaguchi
753-0841,3 Japan
Received 24 August 1998/Accepted 9 November 1998
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ABSTRACT |
A phylogenetic analysis of the sequences of 60 clones of archaeal
small-subunit rRNA genes amplified from the termite
Reticulitermes speratus revealed that most of them (56 clones) clustered in the genus Methanobrevibacter. Three
clones were classified in the order Thermoplasmales. The
Methanobrevibacter-related symbionts were detected by in
situ hybridization analysis.
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TEXT |
Termites, both lower and higher
ones, harbor methanogenic Archaea in their hindguts (7,
13). The H2 consumption of methanogens is
expected to promote anaerobic cellulose decomposition in the termite
hindgut. Methanogenic symbionts of termites are present in two
different niches: endosymbiotic methanogens are present in flagellates,
and nonendosymbiotic ones adhere to hindgut surfaces (6, 7).
Recently, Leadbetter and Breznak (6) reported on the
isolation and characterization of two novel species of methanogenic
bacteria from Reticulitermes flavipes which belong to the
genus Methanobrevibacter. These species were related to the
cells colonizing the hindgut epithelium but not to the cells in the
symbiotic flagellates, as determined by morphological observation. In
the present investigation we surveyed (by PCR) the phylogenetic diversity of methanogens in Reticulitermes speratus termites
collected in the Japan archipelago.
Termites.
Six colonies of the termite R. speratus
(Kolbe, order Isoptera, family Rhinotermitidae) were collected from
forests in Japan (Table 1). One colony
was collected from each sampling point except for the Tokyo area, where
three colonies were sampled.
DNA extraction.
Ten individual workers from each colony
were selected for the DNA extraction. After surface sterilization with
70% ethanol, the digestive tracts of the termites were pulled out with
a pair of fine-tipped forceps. The gut tissues and their contents were crushed with a Teflon homogenizer in 1 ml of extraction buffer consisting of 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 0.1% sodium dodecyl sulfate. The mixture was frozen in liquid nitrogen and thawed
at 57°C five times and then treated with 0.5 mg of proteinase K per
ml at 57°C for 16 h. The solution was extracted with phenol and
chloroform, and the DNA was precipitated with ethanol (11).
PCR amplification and sequence analysis.
PCR primers were
designed to amplify a part of the archaeal small-subunit (SSU) rRNA
gene. The primers used were ME855F
(5'-TTAA AGGAATTGGCGGGGGA-3') and ME1354R
(5'-TGACGGGCGGTGTGTGCAAG-3'). The PCR
reaction was performed with a thermal program which comprised 40 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 90 sec.
The amplified DNA fragments were ligated with a ddT-tailed vector
as described by Holton and Graham (5) and cloned. The nucleotide sequences were determined by the dideoxynucleotide chain
termination method (12) on an A. L. F. model II
DNA sequencer (Pharmacia Biotech).
Phylogenetic analysis.
Phylogenetic trees were constructed
by neighbor-joining distance matrix methods (10) with
the programs in the software package PHYLIP, version 3.572 (J. Felsenstein and the University of Washington). The
aligned sequences were also analyzed by maximum-parsimony and
maximum-likelihood methods to check the tree topology.
Phylogeny of symbiotic methanogens of R. speratus.
Ten
clones were isolated for each termite colony, and the sequences of 60 clones were determined in total. Clones which had evolutionary
distances within 0.02 substitutions (sequence identity, >98%)
were grouped together. As a result, the clones were classified into six
types: 1A, 1B, 1C, 1D, 2, and 3 (Table 1).
The phylogenetic tree shown in Fig. 1 was
constructed by the neighbor-joining method. Types 1, 2, and 3 were
separated from each other by more than 0.1 evolutionary distances. The
clones belonging to types 1A to 1D were located in the order
Methanobacteriales. Ohkuma et al. (9) have reported on the
amplification and cloning of SSU ribosomal DNA sequences of methanogens
from R. speratus. The sequence of the clone M4 reported by
Ohkuma et al. was found to be type 1B.
Methanobrevibacter-related methanogens were detected from
all colonies used in this study. Methanobrevibacter-related methanogens seem to be most abundant in R. speratus.
Leadbetter and Breznak (6) have recently isolated
Methanobrevibacter cuticularis and Methanobrevibacter
curvatus from R. flavipes. These previously isolated
methanogens are also in the genus Methanobrevibacter. Methanobrevibacter-related methanogens may be the major phylum of
symbiotic methanogens in termites. However, it should be kept in mind
that PCR may have some biases in the amplification of sequences.
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FIG. 1.
Phylogenetic positions of the PCR clones obtained from
R. speratus hindgut contents within the members of
Euryarchaeota. Type clones of respective groups of the
clones were used to construct the tree and are indicated in
parentheses. The tree was inferred from 499 unambiguously aligned
nucleotide sequences of SSU rRNA by using the neighbor-joining method.
Thermococcus celer was used as the out-group. Bar, 0.1 substitutions per nucleotide position. Numbers are bootstrap values
(3). The bootstrap confidence interval was calculated from
1,000 repetitions of resampling.
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The type 2 clone fell in the order Methanomicrobiales (Fig.
1). This
clone was closely related to
Methanocorpusculum parvum with
95.3% sequence identity. The clustering of the clone and
M. parvum was strongly supported by a bootstrap value of 100%.
In the present study, three clones of type 3 were obtained from
R. speratus hindgut microflora. The closest neighbors of
these
clones were
Thermoplasma relatives
(
Picrophilus oshimae, with
82.5% identity), and the
second closest one was
Thermoplasma acidophilum,
showing a similarity of 81.3%. Type 3 clones formed a
monophyletic
clade with species of Thermoplasmales, which was supported
by
a relatively high bootstrap value (90%). Many sequences which
are
related to those of
Thermoplasma have been amplified from
various environmental samples and reported (
1,
2,
4).
However, these sequences could not be used in the phylogenetic
analysis
because of the differences in the region determined.
Judging from the
sequence analysis, the type 3 symbiont seemed
to be a
Thermoplasma-related novel archaeon. Known
Thermoplasma species grow in acidic and hot
environments. The type 3 symbiont
must be a neutrophilic and
mesophilic archaeon, because the physical
conditions of the
Reticulitermes hindgut are at a nearly neutral
pH
(
8) and of course at an ambient
temperature.
In situ hybridization.
We also performed whole-cell
hybridizations with fluorescent oligonucleotide probes. Oligonucleotide
probes were designed to bind to the SSU rRNAs of the methanogens
detected in this study (Fig. 2). Two
archaeon-specific probes, ARC344
(5'-GCGCCTGCTGCGCCCGT-3') and ARC915 (5'-GTGCTCCCCGCCAATTCCT-3'), de-scribed
by Stahl et al. (14), were also used as positive controls.
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FIG. 2.
Alignment of the oligonucleotide probe sequences and the
corresponding SSU rRNA sequences of termite symbiont clones,
methanogens, and Escherichia coli. Only nucleotides which
are different from those of the type 1 sequence are shown.
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A worker from colony RS1, sampled in the Tokyo area, was used in this
analysis. Samples were prepared basically according
to the work of
Stahl et al. (
14). The hindgut contents were
collected by
centrifugation at 16,000 rpm (15,000 ×
g) for 10
min and
fixed in 1% paraformaldehyde in phosphate-buffered saline
at 4°C for
30 min. The hindgut contents of the termite were hybridized
with
the archaeon-specific probes labeled with fluorescein and
with those
specific to cloned sequences that had been labeled
with rhodamine. They
were inspected with a laser scanning confocal
imaging system (MRC-600;
Bio-Rad) with a krypton-argon mixed-gas
laser
generator.
Though wood particles present in the sample emitted fluorescence with a
wide spectrum, prokaryotic cells could be morphologically
distinguished
from the particles (Fig.
3). The cells
that hybridized
with the archaeon-specific probes were all
straight rods, and
they frequently existed as two cells attached
to each other. The
morphological features of the cells are
consistent with those
of some observed on the epithelial surface of the
hindgut. The
cells that hybridized with the archaeon-specific
probes also hybridized
with type 1-specific probes (Fig.
3).
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FIG. 3.
Whole-cell in situ hybridization of hindgut contents
from R. speratus. (A) In this image taken with
fluorescein-labeled archaea-specific probes, ARC344 and ARC915,
hybridized cells are indicated by arrows. (B) The same field as in
panel A, showing the rhodamine fluorescence of type 1-specific probes
1109TY1R and 1174TY1R. (C) Merged field of panels A and B. The cells
hybridized with both sets of probes appear yellow on the real merged
field. Wood particles (W) which emitted fluorescence in scans for both
fluorescein and rhodamine were also yellow on the real merged field.
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|
In summary, we report that the methanogen community in the hindgut of
R. speratus consists mainly of
Methanobrevibacter-related
methanogens, which had
some divergence. The type 1 symbiotic methanogens
were detected by in
situ hybridization
analysis.
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ACKNOWLEDGMENTS |
This work was supported by a grant-in-aid for scientific
research from the Ministry of Education, Science, and Culture of Japan (07265209 and 08228221).
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FOOTNOTES |
*
Corresponding author. Mailing address: c/o A. Yamagishi, Department of Molecular Biology, School of Life Science,
Tokyo University of Pharmacy & Life Science, 1432-1, Horinouchi,
Hachiouji, Tokyo 192-0392, Japan. Phone: 81-426-76-7141. Fax:
81-426-76-7145. E-mail: shinzato{at}nibh.go.jp.
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Applied and Environmental Microbiology, February 1999, p. 837-840, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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