Biosci. Biotechnol. Biochem., 74 (6), 1145–1151, 2010
Award Review
Diversity and Genomes of Uncultured Microbial Symbionts in the Termite Gut Yuichi H ONGOH1;2 1
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Tokyo 152-8550, Japan 2 Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Saitama 351-0198, Japan Online Publication, June 7, 2010 [doi:10.1271/bbb.100094]
Termites play a key role in the global carbon cycle as decomposers. Their ability to thrive solely on dead plant matter is chiefly attributable to the activities of gut microbes, which comprise protists, bacteria, and archaea. Although the majority of the gut microbes are as yet unculturable, molecular analyses have gradually been revealing their diversity and symbiotic mechanisms. Culture-independent studies indicate that a single termite species harbors several hundred species of gut microbes unique to termites, and that the microbiota is consistent within a host termite species. To elucidate the functions of these unculturable symbionts, environmental genomics has recently been applied. Particularly, single-species-targeting metagenomics has provided a breakthrough in the understanding of symbiotic roles, such as the nitrogen fixation, of uncultured, individual microbial species. A combination of single-species-targeting metagenomics, conventional metagenomics, and metatranscriptomics should be a powerful tool to dissect this complex, multilayered symbiotic system. Key words:
whole-genome amplification; Phi29 DNA polymerase; uncultivable; symbiosis; cellulose
Termites are social insects inhabiting temperate to tropical regions. They thrive solely on dead plant matter and greatly contribute to the global carbon cycle. Their ability to degrade lignocellulose efficiently has made some species destructive pests of woody buildings and, on the other hand, has intrigued researchers who seek hints for the development of novel biofuels from woody biomass. Extensive physiological, biochemical, and morphological analyses of termites have been conducted for nearly a century, and have concluded that termites rely mostly on gut microbes for survival on their recalcitrant food materials. Phylogenetically lower termites harbor protists, bacteria, and archaea in their gut, while phylogenetically higher termites generally lack gut protists and harbor only bacteria and archaea. Most of the lower termites are wood-feeders, while the higher termites comprise wood-,
grass-, litter-, lichen-, and soil (humus)-feeders. Because the majority of gut microbes are as yet unculturable, culture-independent approaches are essential to ecological studies of these microbes. Clone analyses of small subunit rRNA genes have revealed the phylogenetic diversity of the gut microbes, and microscopic observations, including fluorescent in situ hybridization (FISH) analysis, have revealed their spatial distribution in the gut. The functions of this unculturable microbiota as a whole have been analyzed by metagenomics and metatranscriptomics. In addition, recent attempts to acquire the complete genome sequences of unculturable bacterial species in the termite gut have succeeded. In this article, I review recent progress in molecular ecological studies of termite-gut microbiota, with emphasis on the diversity of bacteria and their functions as predicted by genomic analysis.
I. Diversity of Termite Gut Symbionts In lower termites, 103 to 105 cells of protists are present in a single gut,1,2) and occupy 90% or more volume of the dilated portion of the hindgut (Fig. 1). The protists belong to either the phylum Parabasalia or the phylum Preaxostyla. Each termite species has a specific and consistent protistan microbiota that consists of 1 to 20 morphologically distinguishable species.3,4) These protists are unique to termites and their closest relatives, wood-feeding Cryptocercus cockroaches. It has been suggested that the ancestor of termites and Cryptocercus cockroaches possessed the ancestors of extant protistan lineages, which have diversified along with host divergence.5) Bacteria and archaea are found in the guts of both lower and higher termites. Approximately 106 to 108 prokaryotic cells are present in a single gut.6–9) The majority are bacteria, and archaea account only for 0 to 10%.10,11) The most frequently found archaea are methanogens. Among these, methanobrevibacters are found in various lower and higher termites,12–14) and three strains of Methanobrevibacter have been isolated from a lower termite.10,15) The isolates generate methane
This review was written in response to the author’s receipt of Japan Society for Bioscience, Biotechnology, and Agrochemistry Award for the Encouragement of Young Scientists in 2008. Correspondence: Fax: +81-3-5734-2946; E-mail:
[email protected] Abbreviations: FISH, fluorescent in situ hybridization; GHF, glycosyl hydrolase family; WGA, whole-genome amplification; CDS, protein-coding sequences
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Fig. 1. Termites and Their Gut Microbiota. A, Japanese subterranean termite Reticulitermes speratus. B, The entire gut of R. speratus. C, The gut microbiota of R. speratus. Bars ¼ 1 mm in panels A and B, 50 mm in panel C. Table 1. Termite Species Used in Clone Analysis of 16S rRNA Genes Amplified from Bacterial Gut Microbiota Termites Family Kalotermitidae (lower) Cryptotermes cavifrons Family Rhinotermitidae (lower) Coptotermes formosanus Reticulitermes flavipes Reticulitermes speratus Reticulitermes sp. RPK Family Termitidae (higher) Subfamily Macrotermitinaea Macrotermes gilvus Macrotermes michaelseni Odontotermes formosanus Subfamily Termitinae Cubitermes orthognathus Microcerotermes spp. Termes comis Subfamily Nasutitermitinae Nasutitermes ephratae Nasutitermes takasagoensis a
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30, 70) 21, 25) 7, 20, 22) 7)
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Termites in this subfamily are fungus-growers.
from H2 and CO2 , and probably play a role in lignocellulose fermentation in the gut by consuming H2 . They are tolerant of microoxic conditions, and methanobrevibacters are present on the gut epithelium10,16) as well as within the cells of gut protists such as Dinenympha parva12) and Spirotrichonympha leidyi.17) Studies of methanogenic archaea in the termite gut have been reviewed elsewhere by Purdy18) and by Hongoh and Ohkuma19) in detail. The taxonomic compositions of gut bacteria have been examined in various termite species by 16S rRNA
clone analysis (Table 1).7,8,20–32) More than 1,500 phylotypes in total (with a criterion of >97% sequence identity in near-full length 16S sequence) have been obtained to date, most of which have never been detected in other environments and show low sequence similarities to cultured bacterial strains. In both lower and higher termites, it has been estimated that a single termite species harbors several hundred or more gut bacterial phylotypes.7,8,22,29,33) The phylotypes have been classified into 24 phylum-level clusters (Fig. 2), and they form one or more monophyletic clusters in each bacterial phylum. Although these bacteria have not strictly co-speciated with their termite hosts, their phylogeny loosely reflects the host taxonomic positions, and the bacterial microbiota is consistent within a host species or genus.7,8) This suggests that the majority of termite gut bacteria are not allochthonous but autochthonous symbionts, vertically transmitted from parents to offspring via proctodeal trophallaxis (i.e., transmission of the gut contents from the anus of a donor to the mouth of a recipient).4) In the guts of termites examined thus far, one of three anaerobic bacterial groups, the genus Treponema in Spirochaetes, the order Bacteroidales in Bacteroidetes, and the class Clostridia in Firmicutes, predominated, followed by the other two.7,8,22,24,27,28) Occasionally, the candidate class ‘‘Endomicrobia’’ in the phylum Elusimicrobia (originally designated the candidate phylum Termite Group 1)20,34) was also found as a dominant group in the guts of lower termites,7,22) and the candidate phylum TG3 (Termite Group 3) and the phylum Fibrobacteres were the second most dominant groups in the guts of wood-feeding higher termites.7,27) In summary, distinct termite species harbor different
Diversity and Genomes of Termite Gut Microbes
Fig. 2. Phylogenetic Diversity of Bacteria in the Termite Gut. A phylogenetic tree showing the diversity of gut bacteria at the phylum level based on 16S rRNA sequences obtained from various termite species, the majority of which are shown in Table 1. The tree was constructed using ARB software (http://www.arb-home.de/) on the basis of a parsimonious criterion. The dimensions of the trapezoids indicate approximately the diversity of bacteria in each phylum-level cluster.
bacterial species with community structures specific to the host species, but most of those bacteria belong to phylogenetic clusters that are unique to termites and shared among diverse termite species.
II. In Situ Localization of Termite Gut Bacteria FISH analyses targeting 16S rRNA have revealed that bacterial species are not randomly distributed in the gut, but are localized to specific sites such as the gut epithelium, luminal fluid, and the surface or cytoplasm of protist cells. The cellular association of bacteria and protists is a prominent feature of gut microbiota in lower termites.35–37) The endo- and ectosymbiotic bacteria of gut protists comprise diverse taxonomic groups: Treponema,38–41) ‘‘Endomicrobia,’’42–44) Bacteroidales,28,40,45–51) Deltaproteobacteria,52) Mycoplasmatales,53) and Synergistetes.28) Among these, Treponema and Bacteroidales are the most frequently observed ectosymbionts of gut protists. They inhabit the cell surfaces of various protist species and occasionally coinhabit a single host cell (Fig. 3A, B).40) In the protist
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Fig. 3. Ecto- and Endosymbionts of Flagellated Protists in the Termite Gut. A, Phase contrast image of the oxymonad protist Dinenympha porteri from the gut of Reticulitermes speratus. Arrowheads indicate the four true flagella of the protist. B, FISH image of panel A. Cells of Treponema and Bacteroidales were detected using oligonucleotide probes targeting the 16S rRNA specific to each bacterial group. Red signals (Texas-red), Treponema; green signals (6FAM), Bacteroidales. Panels A and B were originally published in reference 40. C, Phase contrast image of the parabasalid protist Trichonympha agilis from the gut of R. speratus. D, FISH image of panel C. The cells of endomicrobial phylotype Rs-D17 were detected with red signals (Texas-red). The green signals indicate cells of other bacteria. E, Transmission electron micrograph of Rs-D17. Panels C–E were originally published in reference 69. F, Phase contrast image of the parabasalid protist Pseudotrichonympha grassii from the gut of Coptotermes formosanus. G, FISH image detecting specifically (green signals: 6FAM) the cells of Bacteroidales phylotype CfPt1-2 (‘‘Candidatus Azobacteroides pseudotrichonymphae’’) within a P. grassii cell. Panel G was originally published in the supporting online material of reference 70. Bars ¼ 10 mm in panel A, 25 mm in panel C, 0.5 mm in panel E, 50 mm in panel F, and 10 mm in panel G.
Mixotricha paradoxa, the ectosymbiotic treponemes reportedly confer motility on the host cell by their synchronized movement.41,54) ‘‘Candidatus Tammella caduceiae,’’ in the phylum Synergistetes, is another motility ectosymbiont, the flagella of which propel the cells of the host protist Caduceia versatilis.28,55) Bacteria belonging to the candidate class ‘‘Endomicrobia’’ in the phylum Elusimicrobia, or Termite Group 1, have been found to be intracellular symbionts of
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various protist species in the termite gut (Fig. 3C– E).42–44) Endomicrobial bacteria in many cases coexist in the host cytoplasm with one or more different groups of bacteria. Within the cells of protist Trichonympha agilis in the gut of the termite Reticulitermes speratus, the endomicrobial phylotype Rs-D17 always coexists with ‘‘Candidatus Desulfovibrio trichonymphae,’’52) which belongs to the class Deltaproteobacteria. A Bacteroidales bacterium, ‘‘Candidatus Azobacteroides pseudotrichonymphae,’’ is present within the cells of the protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus (Fig. 3F, G).46) The ‘‘Azobacteroides’’ bacteria and the Pseudotrichonympha protists have almost strictly co-speciated.49) Strict co-speciation has also been reported between ‘‘Candidatus Endomicrobium’’ in the class ‘‘Endomicrobia’’ and the Trichonympha protists,56) and between ‘‘Candidatus Armantifilum’’ (ectosymbiotic Bacteroidales bacteria) and the devescovinid protists.51) However, while co-speciation is observed on a lower taxonomic level, i.e., within a genus of the protist host, the topology between the host and symbiont does not coincide at a higher taxonomic level.43,50) This indicates that multiple, independent acquisitions of the symbiotic bacteria by the protist hosts have occurred, and that the symbiotic bacteria are vertically transmitted until they are replaced by other bacteria.
III. Metagenomics and Metatranscriptomics of Termite Gut Microbiota The protists in the termite gut are very difficult to cultivate and only a few studies have been successful. Yamin (1981) succeeded in cultivation of the gut protists Trichonympha sphaerica and Trichomitopsis termopsidis from the termite Zootermopsis angusticollis, and he demonstrated that these protists degrade cellulose: ðC6 H12 O6 þ 2H2 OÞn ! ð2CH3 COOH þ 2CO2 þ 4H2 Þn.57) Acetate is the major carbon and energy source for the termite host.58) However, to my knowledge, no cultured strains of termite-gut protists exist at present, and their detailed physiology and ecology remain unclear. To obtain comprehensive information, particularly on the lignocellulose-degrading system, metatranscriptomic searches have been conducted for protistan microbiota in the guts of several lower termite species, Mastotermes darwiniensis, Hodotermopsis sjoestedti, Neotermes koshunensis, and Reticulitermes speratus, and the wood-feeding cockroach Cryptocercus punctulatus.59,60) Approximately 1,000 cDNA clones per host species were sequenced, and it was found that about 10% of the clones in each sample belonged to the glycosyl hydrolase families (GHFs) such as GHF5, 7, 10, and 45. These included cellulases (endoglucanase and cellobiohydrolase) and hemicellulases. The predominant GHF varied among the host species, probably reflecting the host’s preference for a wood environment and the taxonomic compositions of the protistan microbiota.60) Tartar et al. (2009) performed both metatranscriptomic analysis of the gut protistan microbiota and transcriptomic analysis of the gut cells of the host termite Reticulitermes flavipes.61) Approximately 5,000 clones were sequenced for each cDNA library, and the
metatranscriptome of the protistan microbiota was found to be similar to that of R. speratus.59) The transcriptome of the host, R. flavipes, contained transcripts of a gene encoding a cellulase (endoglucanase) of GHF9, as previously reported for the transcriptomic analysis of the salivary gland and gut of R. speratus.59) The researchers also discovered transcripts of a laccase gene, and they claimed that it is perhaps involved in lignin degradation.61) This appears to parallel a recent report that lignin degradation was observed in the gut of the termite Z. angusticollis,62) but it has been found that homologous laccase genes in the genomes of various insects are involved in tanning cuticles.63) Since the sequencing efforts in these metatranscriptomic analyses were too weak to be useful to predict the entire functions of the protistan microbiota, more exhaustive analysis is needed. Because the majority of bacterial microbiota in the termite gut are as yet unculturable and are distantly related to the cultured species, their detailed functions are unclear. The metagenomics of the bacterial microbiota in the gut of a wood-feeding higher termite, Nasutitermes ephratae, has been reported.29) In that study, numerous genes of bacterial origin putatively involved in the degradation of cellulose and hemicellulose, H2 production, reductive acetogenesis, and nitrogen fixation were identified. These functions have been reported in biochemical and physiological analyses of the whole gut microbiota64–66) and also in studies of several cultured bacterial strains isolated from the termite gut.67,68) Since the merit of metagenomics is to provide a comprehensive view of the diversity and functions of the entire community, comparisons of metagenomes among host species with different taxonomic positions and feeding habits are most helpful for an understanding of this complicated symbiotic system.
IV. Single-Species-Targeting Metagenomics of Termite Gut Bacteria While metagenomics and metatranscriptomics can reveal the comprehensive functions of a whole microbiota, these analyses generally provide poor information on the functions of and interrelationships among the individual microbial species in a community. To obtain deeper insights into a microbial ecosystem, which consists of complex interrelationships among diverse species, it is essential to clarify the functions of the individual species, at least the dominant ones, in the microbiota. Recently, Hongoh et al. (2008) attempted to acquire the complete genome sequences of uncultured bacterial species in the termite gut from a small number of cells.69,70) Since a standard genome sequence analysis requires 1010 bacterial cells, they applied an isothermal whole-genome amplification (WGA) technique using Phi29 DNA polymerase, which enables 1010 fold amplification of entire genome regions within several hours.71) Two unculturable bacterial endosymbionts of unculturable cellulolytic protists were chosen for genome analysis. One was phylotype Rs-D17 (or ‘‘Candidatus Endomicrobium sp.’’), belonging to the phylum Elusimicrobia (Termite Group 1). Rs-D17 is found exclusively within the cells of the protist Trichonympha agilis
Diversity and Genomes of Termite Gut Microbes
and one of the predominant phylotypes in the gut of R. speratus (Fig. 3C–E).22,43) The other was phylotype CfPt1-2 (‘‘Candidatus Azobacteroides pseudotrichonymphae’’), belonging to the order Bacteroidales in the phylum Bacteroidetes. CfPt1-2 is a specific endosymbiont of the protist Pseudotrichonympha grassii, and accounts for two-thirds of the prokaryotic cells in the gut of Coptotermes formosanus (Fig. 3F, G).46,49) Almost no information was available on the functions of these unculturable endosymbionts of the protists despite their predominance in the gut. To minimize the intraspecific variation of the targeted bacterial genomes, only a single host protist cell was physically isolated and used for the collection of its endosymbiotic bacteria. The 102 to 103 bacterial cells collected were lysed in alkaline solution and subjected to WGA. From the amplified samples, the complete circular chromosomes (1.1 Mb) were successfully reconstructed without ambiguity for each bacterium.69,70) The chromosome of Rs-D17 contains 761 proteincoding sequences (CDSs) and additional 121 pseudogenes. The pseudogenes comprise genes involved in functions such as DNA replication/repair, lipopolysaccharide biosynthesis, transport, and defense mechanisms. For instance, the gene for the chromosome replication initiation factor DnaA was found to be a pseudogene. In contrast, the genes required for biosynthesis of amino acids and cofactors are abundantly retained. Because the genes encoding glutamine synthetase GlnA and ammonium transporter AmtB have been pseudogenized, it is likely that Rs-D17 requires glutamine from the host cytoplasm and upgrades it to various compounds. The predicted pathways suggest that glucose-6-phosphate is the major carbon and energy source for Rs-D17 (Fig. 4A). The chromosome of CfPt1-2 contains 758 CDSs and 21 pseudogenes. The most striking feature of this bacterium, predicted from the genome data, was its ability to fix dinitrogen. Although N2 -fixing activity has been demonstrated in diverse termites, including C. formosanus, this finding was unexpected, because there were no reports on N2 -fixing ability or the presence of nitrogenase genes from any known bacteria in the phylum Bacteroidetes. The predicted pathways suggest that the fixed nitrogen, in the form of NH3 , is assimilated initially by the activity of glutamine synthetase and is then used for the biosynthesis of diverse amino acids and cofactors. This bacterium also possesses a gene encoding an ammonium transporter and a gene cluster encoding urease and a urea transporter. The ability to import and assimilate ammonium and urea implies that CfPt1-2 not only fixes atmospheric nitrogen but also recycles the putative nitrogen waste products of the host protist. CfPt1-2 possesses genes for importing and utilizing monosaccharides derived from lignocellulose, i.e., glucose, xylose, and hexuronates, which are likely to be the major carbon and energy sources (Fig. 4B). The profile of the cluster of orthologous groups of proteins and the general features of the CfPt1-2 genome differ greatly from those of known Bacteroidales bacteria, but are strikingly similar to those of Rs-D17. The functional similarity of CfPt1-2 and Rs-D17 implies that the primary role of the endosymbionts of cellulolytic protists is efficient and stable biosynthesis of amino
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Fig. 4. Outline of the Symbiotic Roles of Endosymbiotic Bacteria within the Cells of Cellulolytic Protists in the Termite Gut as Predicted by Genome Analysis. A, Symbiotic roles of endomicrobial phylotype Rs-D17 within cells of the protist Trichonympha agilis in the gut of Reticulitermes speratus. The original figure was published in the supporting information of reference 69. B, Symbiotic roles of Bacteroidales phylotype CfPt1-2 (‘‘Candidatus Azobacteroides pseudotrichonymphae’’) within cells of the protist Pseudotrichonympha grassii in the gut of Coptotermes formosanus. The original figure was published in the supporting online material of reference 70.
acids and cofactors, which are critically deficient in woody materials. The processes of N2 fixation and biosynthesis of amino acids and cofactors conducted by the endosymbionts are considered to be much more stable and efficient than those conducted by freeswimming gut bacteria. The endosymbionts can utilize ample carbon and energy sources without competition, and their genomes have been reduced, streamlined, and specialized for nitrogen metabolism. Particularly in CfPt1-2, its ability to couple N2 fixation directly to cellulolysis makes possible highly efficient growth of the host cellulolytic protist, the termite, and the termite colony, without the limitation of nitrogen deficiency.
V. Perspectives Since the introduction of molecular biology into environmental microbiology in 1990s, it has been recognized that more than 99% of microbial species on Earth are as yet unculturable. The termite-gut microbiota also comprises unculturable microbes, and
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therefore detailed analysis has been hampered, but now researchers have tools to unveil and dissect this black box. Community analysis using a high-throughput sequencer can generate 104 clones of 16S rRNA genes, which should provide more a precise estimation of the species richness in the gut microbiota. Metagenomics and metatranscriptomics by means of a highthroughput sequencer are also very useful for researchers to form a comprehensive view of the functions and diversity of gut microbiota. Nevertheless, our knowledge of the functions of individual microbial species is still poor and difficult to obtain. Hence, single-speciestargeting metagenomics and the development of its ultimate form, single-cell genomics,72–74) are of great importance. I expect innovations in these genomic approaches to provide new insight into this complicated, multi-layered symbiotic system, which might also contain valuable information for pest control and biofuel production.
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Acknowledgments I am grateful to Dr. Moriya Ohkuma, Dr. Satoko Noda, Dr. Shigeharu Moriya, Dr. Tetsushi Inoue, Dr. Toru Johjima, Dr. Satoshi Hattori, Dr. Savitr Trakulnaleamsai, Dr. Napavarn Noparatnaraporn, and other cooperators for the research on termite-gut microbiota. I sincerely thank Dr. Toshiaki Kudo, who gave me the opportunity to study termites, and also the late Dr. Hajime Ishikawa, who gave me guidance for research on symbiosis. Parts of the figures and text are reproduced here with modifications from original articles with generous permission from John Wiley and Sons, the American Association for the Advancement of Science, and the US National Academy of Sciences.
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