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Keywords:

  • Termite;
  • Symbiosis;
  • 16S rRNA;
  • Methanogen;
  • Bacteroides;
  • Spirochete

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The phylogeny of microorganisms of the symbiotic community in the gut of a lower termite, Cryptotermes domesticus (order Isoptera, family Kalotermitidae), was investigated without culturing the resident microorganisms. Portions of the small-subunit rRNA genes (16S rRNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial sequences of 16S rRNA showed the existence of prokaryotic species related to the genera Methanobrevibacter, Leuconostoc, Bacteroides and Treponema, but most of the sequences were those of yet unknown species. Unique sequences showing very low sequence similarity to known 16S rRNA sequences were also found although they were significantly clustered with the High G+C Gram-positive bacteria. Comparisons of these sequences with those from the symbiotic microorganisms in other termite species revealed the existence of termite-specific groups of organisms.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The mutualistic relationship between xylophagous termites and microorganisms inhabiting their gut is one of the most fascinating examples of symbiosis, a relationship that enables termites to live by xylophagy [1, 2]. Despite the isolation and cultivation of several bacteria and protists from within this community [3–7], our understanding of the biology and the physiology of intestinal microbiota is poor because many of the predominant species within the community, such as the spirochete-like bacteria, have not yet been cultured and characterized.

The application of molecular phylogenetic analysis to ecological studies has enhanced our ability to assess naturally occurring biodiversity in mixed microbial assemblages ([8, 9] and reviewed in [10]). In this approach, genes encoding small subunit ribosomal RNA (16S-like rRNA) derived from the extracted nucleic acids of mixed microbial populations are cloned and sequenced. These sequences can then be compared with each other as well as against databases of rRNA sequences from well-characterized microorganisms in order to determine the identity of organisms present in the natural microbial communities. This approach has already been applied to investigate the biodiversity of the symbiotic microbial community in the gut of a lower termite, Reticulitermes speratus[11–13]. These studies have shown that the termite symbiotic system includes many species yet-uncultured in the laboratory.

Termites (order Isoptera) are divided into seven families, showing considerable variation in life-style, ecology and types of symbiosis. A comparison of the constituents of the microbial communities between termite species may be beneficial to understand the nature of the termite symbiotic systems. In this work, on the basis of PCR-amplified 16S rRNA sequences, we report the phylogeny of members of the intestinal microbial community of the lower termite Cryptotermes domesticus. C. domesticus belongs to the family Kalotermitidae, while R. speratus, for which the phylogenetic diversity of the symbiotic system has already reported [11–13], belongs to the family Rhinotermitidae.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Termites

Wood-eating termites, C. domesticus (Isoptera, Kalotermitidae), were collected in the Iriomote island, Japan, in July 1996. Termite-infested wood moistened with distilled water was kept in plastic boxes at ambient temperature.

2.2DNA extraction, PCR amplification, and cloning

Approximately 100 termites were collected and, after their exterior surfaces were washed with distilled water, their entire guts were removed with forceps. The intestinal contents were gently squeezed and the gut debris was removed. DNA from the intestinal mixed population were extracted as described previously [11, 12]. Since many termite gut microbes tightly adhere to the gut wall [14], these populations may not have been included in the analysis described here. Ribosomal RNA genes were amplified from the purified DNA by PCR using an ExTaq DNA polymerase (TAKARA) according to manufacturer's directions. The PCR primers used were described previously [11] and corresponded to nucleotide positions 519–533 and 1392–1405 of E. coli 16S rRNA. The reaction conditions were for 35 cycles at 94°C for 30 s, 45°C for 45 s, 72°C for 2 min. PCR-products corresponding to the expected size of the prokaryotic rRNA gene (0.9 kb) were purified on a low melting agarose gel using a Wizard PCR preps DNA purification system (Promega). The purified PCR-products were ligated into a pGEM-T vector (Promega) according to manufacturer's directions and then introduced into E. coli. The insertion of the appropriated size was determined by PCR-amplification with the universal and reverse primers (TAKARA) which corresponded to the both sides of the cloning site on the vector.

2.3Nucleotide sequencing and phylogenetic analysis

Plasmid DNA was purified from each clone with a Wizard mini preps DNA purification system (Promega) and used as a template for sequencing with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA Polymerase, FS (Perkin Elmer). Sequencing reactions were determined on an automatic sequence analyzer (ABI model 373). Sequencing primers, EUB750F (5′-CRAACAGGATTAGATACCC-3′), EUB900F (5′-ACTCAAAKGAATTGACGG-3′), EUB1050F (5′-GGYTGTCGTCAGCTCGTG-3′), EUB1100R (5′-GGGTTGCGCTCGTTRYGG-3′), EUB900R (5′-CGTCAATTCMTTTGAGTT-3′), EUB750R (5′-TACCAGGGTATCTAATCC-3′), where R represents A or G, K represents G or T, Y represents C or T and M represents A or C, were used. These primers were deduced from consensus regions of eubacterial 16S rRNA. For archaeal 16S rRNA, sequencing primers described previously [12] were also used. The sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the accession numbers AB008898–AB008906.

The previously determined rRNA sequences used for comparisons in this study were retrieved from the GenBank, EMBL and DDBJ nucleotide sequence databases. Sequences were submitted to the CHECK-CHIMERA program of the Ribosomal Database Project [15] to detect the presence of possible chimeric artifacts. The chimeric probability was also examined by the predicted secondary structure of each sequence. Sequence data were aligned using the CLUSTAL W package [16], then corrected by manual inspection, and nucleotide positions of ambiguous alignment were omitted from subsequent phylogenetic analyses. Programs used to infer phylogenetic trees are contained in the PHYLIP package (version 3.5c) [17]. DNADIST was used to calculate evolutionary distances with the Kimura two-parameter model for nucleotide change. The Jukes–Cantor model for nucleotide change was also used and the results for both models were compatible. Thus, in this study, we only present data for the former model. Phylogenetic trees were reconstructed from evolutionary distance data by the neighbor-joining method [18], implemented through the program NEIGHBOR. A total of 100 bootstrapped replicate resampling data sets for DNADIST were generated with the program SEQBOOT, to provide confidence estimates for tree topologies [19]. Parsimony analysis was conducted with the program DNAPARS with random sequence addition and global rearrangement.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Among the clones isolated from the mixed community in of the termite gut, we analyzed a total of 37 clones. We first sequenced approximately 300 bases of the 5′-portion of each clonal segment. On the basis of sequence similarity, nine clone clusters in which less than two bases were different were identified. Then, we determined complete nucleotide sequences of a representative of each clone cluster (approximately 900 bases corresponding to E. coli position 534–1391). Chimeric rRNA gene clones can arise during PCR amplification of mixed-population DNAs [20]. Evaluation by the CHECK-CHIMERA program of the Ribosomal Database Project [15] and inspection of the predicted secondary structures indicated that the sequences reported in this study showed no obvious evidence of chimeric artifacts.

The sequences of the clones Cd10 and Cd45 shared 96.6% nucleotide identity with each other, but were distantly related to those of any known organisms in the databases (less than 82% identities). Phylogenetic analysis using representatives of several major groups of the bacteria (Fig. 1) indicated that these sequences showed slight relationships with the members of the high G+C Gram-positive bacteria. The bootstrap value of 91% significantly supported the clustering among Cd10, Cd45 and the members of the high G+C Gram-positive bacteria. However, these two sequences shared only low sequence similarities with any of the members and were deeply branched in the phylogenetic tree.

image

Figure 1. Phylogenetic tree showing the relationship of the clones Cd10 and Cd45 to the representatives of the bacteria. A total of 777 unambiguously aligned positions were included in the analysis. The bar represents 0.10 nucleotide substitution per position. Bootstrap values above 70 from 100 resamplings are shown for each node. Accession numbers for the reference sequences are as follows: M83548, X07998, J01859, M61006, X68176, M57740, S83624, S83623, L11306, X60514 and X80738.

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The branching order of the representatives of the major bacterial groups in the tree was not in agreement with the generally accepted evolutionary relationships [21]. However, the internal nodes were very short and were not supported by bootstrap analysis. We analyzed more than ten different data sets for representatives of the major bacterial groups and the high G+C Gram-positive bacteria. The branching order of the representatives of the major bacterial domains was unstable and different in each data set. The bootstrap analysis did not support these branching order in each case. However, in every case, the Cd10 and Cd45 sequences were grouped together with the members of the high G+C Gram-positive bacteria, showing the bootstrap values more than 80%. The parsimony analysis also supported this grouping. These results confirmed the significant evolutionary relationship of the Cd10 and Cd45 sequences with the high G+C Gram-positive bacteria.

We analyzed the predicted secondary structures for Cd10 and Cd45 and they share complementarity with those of other bacteria, indicating that the sequences encode functional rRNA. The sequence variations between Cd10 and Cd45 were located primarily in a stem region corresponding to the variable region 4 [22]. Another two clones within 37 clones analyzed here had identical nucleotide sequence to the Cd45, but Cd10 was unique.

The sequence of the clone Cd30 was related to the methanogenic archaea. Phylogenetic analysis indicated that the Cd30 sequence belongs to the genus Methanobrevibacter (Fig. 2). The Cd30 sequence shared the highest nucleotide identity 97.7% with the sequence from the symbiotic methanogen of the termite R. speratus[12], and had 94.6, 93.9 and 93.8% sequence identities with Methanobrevibacter arboriphilicus, M. curbatus and M. cuticularis, respectively. The latter two Methanobrevibacter have been isolated from the termite Reticulitermes flavipes[23]. The bootstrap value of 100% supported the monophyly of these five methanogens. These results indicated that members of the symbiotic methanogens reported so far were phylogenetically close relatives. However, from other termite species, we found sequences of symbiotic methanogens which were distantly related to Methanobrevibacter (unpublished result). Since many species of Methanobrevibacter, including strains isolated from termites, are virtually restricted to using H2 plus CO2 as the energy sources, it was suggested that the symbiont of C. domesticus also utilize them and may play a role as one of ‘H2 sink organisms’[2]. The clones represented by Cd30 were most abundant among our clones analyzed by the partial sequence (23 among 37 clones).

image

Figure 2. Phylogenetic tree showing the relationship of the clone Cd30 to the members of the order Methanobacteriales. A total of 798 unambiguously aligned positions were included in the analysis. The bar represents 0.05 nucleotide substitution per position. Bootstrap values above 70 from 100 resamplings are shown for each node. Mbr.=Methanobrevibacter, Msp.=Methanospaera, Mba.=Methanobacterium and Mth.=Methanothermus.

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The clone cluster represented by the clone Cd4 consisted of three identical partial sequences. The Cd4 sequence was closely related to the genus Leuconostoc belonging to the Low G+C Gram-positive bacteria, and shared high nucleotide identities 98.4, 98.4 and 96.1% with L. mesenteroides, L. cremoris and L. lactis, respectively. Species of Leuconostoc are lactic acid bacteria and it has been reported that lactic acid bacteria are one of the major isolates from termite guts [3–5].

The sequences of two clones, Cd22 and Cd39, were related to the genus Bacteroides. Phylogenetic analysis (Fig. 3) indicated that the Cd22 sequence was closely related to Bacteroides forsythus, sharing 92.0% nucleotide identity. The bootstrap value 100% supported their monophyly. The Cd39 sequence was clustered with the sequences from the symbionts of the termite R. speratus, UN71 and UN78 [11]. The Cd39 sequence showed 89.1 and 88.7% nucleotide identities with UN71 and UN78, respectively. The clustering of them was tenuously supported by the bootstrap value 85%. This cluster located phylogenetically different position from the three genera belonging to the Bacteroides group, Bacteroides, Porphyromonas and Prevotella, and seemed to be a specific cluster consisting of symbionts of termites. Both Cd22 and Cd39 were unique sequences among 37 clones analyzed.

image

Figure 3. Phylogenetic tree showing the relationship of the clones Cd22 and Cd39 to the representatives of the Bacteroides group in the CFB-phylum. A total of 720 unambiguously aligned positions were included in the analysis. The bar represents 0.05 nucleotide substitution per position. Bootstrap values above 70 from 100 resamplings are shown for each node. The clones obtained from the termite R. speratus are indicated by (Rs) after the clone names. B.=Bacteroides, Por.=Porphyromonas, Pre.=Prevotella and C.=Cytophaga.

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The remaining three clones, Cd3, Cd48 and Cd46 were related to spirochetes. Phylogenetic analysis including the sequences reported in the termites R. speratus[11], Mastotermes darwiniensis[24, 25] and Nasutitermes lujae[26], indicated that three sequences from C. domesticus and all the spirochetes sequences from termites were related to the genus Treponema. The bootstrap value 96% at the node supported this relationship. Two sequences from R. speratus (UN2 and UN100) and one from M. darwiniensis (sp40-12) clustered together with many members of the Treponema cluster, but the bootstrap values at the nodes for the clustering were low. All other sequences from the termites constituted another cluster which also contained the two cultivated spirochetes Treponema sp. H1 and Spirochaete stenostrepta, although the bootstrap value for this clustering was low. Within this cluster, two sequences from C. domesticus (Cd3 and Cd48) and three sequences from R. speratus (UN1, UN21 and UN96) were clustered together (more than 92%), and the bootstrap value 95% supported this clustering. A parsimony tree was constructed using the same data set and these clusterings were also obtained in this tree. In both methods, the Cd46 sequence seemed to be grouped with five sequences from M. darwiniensis, but the grouping was not supported by bootstrap analyses. The phylogenetic positions of NL1, UN90, UN114, sp40-2 and mpsp15 were unstable and changed in the parsimony tree.

In the termite phylogeny ([27], and references therein), M. darwiniensis is most basal species, and N. lujae and R. speratus are rather evolved species. In the symbiotic spirochetes phylogeny described here, a congruity with the termite phylogeny has not been obtained, rather the branching order seemed to be unrelated to the termite phylogeny. Furthermore, it was obscure in this study whether the termite species-specific clades of spirochetes were present or not. Further analysis of more spirochete sequences from more termite species is necessary to clarify the evolution of termite spirochetes. The Cd3 sequence was a representative of three clones among the 37 clones, within only one base difference in the partial sequences analyzed, and the Cd46 and Cd48 were unique sequences, respectively.

Phylogenetic analysis of the cloned 16S rRNA genes demonstrated that the symbiotic microbial community in the gut of the termite C. domesticus consisted of many yet-uncultured organisms. The similar results are obtained in the case of R. speratus, whose microbial community in the gut consists of numerous new species yet-uncultivated [11–13]. Although the two termite species belong to the different families, both are wood-eating termites. The results described in this study showed that both termite species harbored some phylogenetically related species, such as methanogens, members of the Bacteroides group and spirochetes (see Figs. 2–4). In these three cases, there are clusters specific for the termite sequences in the phylogenetic trees. One of the probable explanations is that the common ancestors of the clustering symbionts have been acquired by the earlier termites, which continued to evolve within the termite guts to the present diversity. Alternatively, diverse microorganisms, which are phylogenetically related due to some conserved functions necessary for the symbiosis, have been acquired independently in the course of diversification of termite species.

image

Figure 4. Phylogenetic tree showing the relationship of the clones Cd3, Cd46 and Cd48 to the members of the spirochetes group. A total of 743 unambiguously aligned positions were included in the analysis. The bar represents 0.05 nucleotide substitution per position. Bootstrap values above 70 from 100 resamplings are shown for each node. The termite hosts of the clones are indicated in the parentheses as follows: Rs=Reticulitermes speratus, Nl=Nasutitermes lujae and Md=Mastotermes darwiniensis. S.=Spirochaeta, T.=Treponema and B.=Borrelia.

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Differences between C. domesticus and R. speratus were also found. The unique sequences which were somewhat related to the high G+C Gram-positive bacteria were found from C. domesticus (clones Cd45 and Cd10, see Fig. 1), while the sequences related to them were not isolated in R. speratus. However, from R. speratus, its own unique sequences have been isolated (designated Termite group I in [11]). Sequences related to proteobacteria and the genus Clostridium have been obtained from R. speratus but not from C. domesticus. Further analyses are necessary to discover whether the microorganisms found in one termite species are really absent in another.

The numbers of clones of high similarity within the 16S rRNA gene clone library constructed here may reflect their numerical abundance in the termite gut. Especially, the methanogen clones represented by Cd30 were almost two-thirds of our clone library, suggesting the methanogen might be one of the major populations in the termite gut. However, since the DNA isolation, PCR and cloning may bias the representation of clones, a quantitative analysis of abundance using nucleic acid probes is necessary.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The authors thank to F. Aoki for assistance. This work was partially supported by grants for the Biodesign Research Program and the Genome Research Program from RIKEN.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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