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 arch-
aea. Although the majority of the gut microbes are as
yet unculturable, molecular analyses have gradually
been revealing their diversity and symbiotic mecha-
nisms. 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 sym-
bionts, environmental genomics has recently been
applied. Particularly, single-species-targeting metage-
nomics has provided a breakthrough in the under-
standing 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, multi-
layered symbiotic system.
Key words: whole-genome amplification; Phi29 DNA
polymerase; uncultivable; symbiosis; cellu-
lose
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, bac-
teria, 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-,
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: yhongo@bio.titech.ac.jp
Abbreviations: FISH, fluorescent in situ hybridization; GHF, glycosyl hydrolase family; WGA, whole-genome amplification; CDS, protein-coding
sequences
grass-, litter-, lichen-, and soil (humus)-feeders. Because
the majority of gut microbes are as yet unculturable,
culture-independent approaches are essential to eco-
logical studies of these microbes. Clone analyses of
small subunit rRNA genes have revealed the phyloge-
netic 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
1146 Y. HONGOH

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 clone analysis (Table 1).7,8,20–32) More than 1,500 phy-
Genes Amplified from Bacterial Gut Microbiota lotypes in total (with a criterion of >97% sequence
identity in near-full length 16S sequence) have been
Termites Food Refs
obtained to date, most of which have never been
Family Kalotermitidae (lower) detected in other environments and show low sequence
Cryptotermes cavifrons Wood 28)
similarities to cultured bacterial strains. In both lower
Family Rhinotermitidae (lower)
Coptotermes formosanus Wood 30, 70)
and higher termites, it has been estimated that a single
Reticulitermes flavipes Wood 21, 25) termite species harbors several hundred or more gut
Reticulitermes speratus Wood 7, 20, 22) bacterial phylotypes.7,8,22,29,33) The phylotypes have been
Reticulitermes sp. RPK Wood 7) classified into 24 phylum-level clusters (Fig. 2), and
Family Termitidae (higher) they form one or more monophyletic clusters in each
Subfamily Macrotermitinaea bacterial phylum. Although these bacteria have not
Macrotermes gilvus Litter 8)
strictly co-speciated with their termite hosts, their
Macrotermes michaelseni Litter 32)
Odontotermes formosanus Wood 31) phylogeny loosely reflects the host taxonomic positions,
Subfamily Termitinae and the bacterial microbiota is consistent within a host
Cubitermes orthognathus Soil 26) species or genus.7,8) This suggests that the majority of
Microcerotermes spp. Wood 7) termite gut bacteria are not allochthonous but autoch-
Termes comis Interface 24) thonous symbionts, vertically transmitted from parents
Subfamily Nasutitermitinae to offspring via proctodeal trophallaxis (i.e., transmis-
Nasutitermes ephratae Wood 29)
Nasutitermes takasagoensis Wood 27)
sion of the gut contents from the anus of a donor to the
mouth of a recipient).4)
a
Termites in this subfamily are fungus-growers. In the guts of termites examined thus far, one of three
anaerobic bacterial groups, the genus Treponema in
from H2 and CO2 , and probably play a role in Spirochaetes, the order Bacteroidales in Bacteroidetes,
lignocellulose fermentation in the gut by consuming and the class Clostridia in Firmicutes, predominated,
H2 . They are tolerant of microoxic conditions, and followed by the other two.7,8,22,24,27,28) Occasionally, the
methanobrevibacters are present on the gut epithe- candidate class ‘‘Endomicrobia’’ in the phylum Elusi-
lium10,16) as well as within the cells of gut protists such microbia (originally designated the candidate phylum
as Dinenympha parva12) and Spirotrichonympha lei- Termite Group 1)20,34) was also found as a dominant
dyi.17) Studies of methanogenic archaea in the termite group in the guts of lower termites,7,22) and the candidate
gut have been reviewed elsewhere by Purdy18) and by phylum TG3 (Termite Group 3) and the phylum
Hongoh and Ohkuma19) in detail. Fibrobacteres were the second most dominant groups
The taxonomic compositions of gut bacteria have in the guts of wood-feeding higher termites.7,27) In
been examined in various termite species by 16S rRNA 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) Bacter-
oidales,28,40,45–51) Deltaproteobacteria,52) Mycoplasma-
tales,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 co-
inhabit a single host cell (Fig. 3A, B).40) In the protist
1147
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 oligonucleo-
tide 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 ‘‘Endomi-
crobia’’ in the phylum Elusimicrobia, or Termite Group
1, have been found to be intracellular symbionts of
1148 Y. HONGOH
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 Bacteroi-
dales bacterium, ‘‘Candidatus Azobacteroides pseudo-
trichonymphae,’’ 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 Pseudotricho-
nympha protists have almost strictly co-speciated.49)
Strict co-speciation has also been reported between
‘‘Candidatus Endomicrobium’’ in the class ‘‘Endomi-
crobia’’ and the Trichonympha protists,56) and between
‘‘Candidatus Armantifilum’’ (ectosymbiotic Bacteroi-
dales bacteria) and the devescovinid protists.51) How-
ever, 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 cellu-
lose: 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 hydro-
lase families (GHFs) such as GHF5, 7, 10, and 45. These
included cellulases (endoglucanase and cellobiohydro-
lase) 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 metatranscrip-
tomic 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 micro-
biota 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 hemi-
cellulose, 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 taxo-
nomic 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 micro-
biota, 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 isother-
mal 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 uncul-
turable cellulolytic protists were chosen for genome
analysis. One was phylotype Rs-D17 (or ‘‘Candidatus
Endomicrobium sp.’’), belonging to the phylum Elusi-
microbia (Termite Group 1). Rs-D17 is found exclu-
sively within the cells of the protist Trichonympha agilis
 Diversity and Genomes of Termite Gut Microbes 1149

and one of the predominant phylotypes in the gut of

R. speratus (Fig. 3C–E).22,43) The other was phylotype
A
CfPt1-2 (‘‘Candidatus Azobacteroides pseudotricho-
nymphae’’), belonging to the order Bacteroidales in the
phylum Bacteroidetes. CfPt1-2 is a specific endosym-
biont 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 B
to WGA. From the amplified samples, the complete
circular chromosomes (1.1 Mb) were successfully re-
constructed without ambiguity for each bacterium.69,70)
The chromosome of Rs-D17 contains 761 protein-
coding sequences (CDSs) and additional 121 pseudo-
genes. The pseudogenes comprise genes involved in
functions such as DNA replication/repair, lipopolysac-
charide biosynthesis, transport, and defense mecha-
nisms. For instance, the gene for the chromosome
replication initiation factor DnaA was found to be a
pseudogene. In contrast, the genes required for biosyn-
thesis of amino acids and cofactors are abundantly
retained. Because the genes encoding glutamine synthe-
tase 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 Fig. 4. Outline of the Symbiotic Roles of Endosymbiotic Bacteria
that glucose-6-phosphate is the major carbon and energy within the Cells of Cellulolytic Protists in the Termite Gut as
Predicted by Genome Analysis.
source for Rs-D17 (Fig. 4A). A, Symbiotic roles of endomicrobial phylotype Rs-D17 within
The chromosome of CfPt1-2 contains 758 CDSs and cells of the protist Trichonympha agilis in the gut of Reticulitermes
21 pseudogenes. The most striking feature of this speratus. The original figure was published in the supporting
bacterium, predicted from the genome data, was its information of reference 69. B, Symbiotic roles of Bacteroidales
ability to fix dinitrogen. Although N2 -fixing activity has phylotype CfPt1-2 (‘‘Candidatus Azobacteroides pseudotrichonym-
phae’’) within cells of the protist Pseudotrichonympha grassii in the
been demonstrated in diverse termites, including C. gut of Coptotermes formosanus. The original figure was published in
formosanus, this finding was unexpected, because there the supporting online material of reference 70.
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 acids and cofactors, which are critically deficient in
that the fixed nitrogen, in the form of NH3 , is assimilated woody materials. The processes of N2 fixation and
initially by the activity of glutamine synthetase and is biosynthesis of amino acids and cofactors conducted by
then used for the biosynthesis of diverse amino acids and the endosymbionts are considered to be much more
cofactors. This bacterium also possesses a gene encod- stable and efficient than those conducted by free-
ing an ammonium transporter and a gene cluster swimming gut bacteria. The endosymbionts can utilize
encoding urease and a urea transporter. The ability to ample carbon and energy sources without competition,
import and assimilate ammonium and urea implies that and their genomes have been reduced, streamlined, and
CfPt1-2 not only fixes atmospheric nitrogen but also specialized for nitrogen metabolism. Particularly in
recycles the putative nitrogen waste products of the host CfPt1-2, its ability to couple N2 fixation directly to
protist. CfPt1-2 possesses genes for importing and cellulolysis makes possible highly efficient growth of
utilizing monosaccharides derived from lignocellulose, the host cellulolytic protist, the termite, and the termite
i.e., glucose, xylose, and hexuronates, which are likely colony, without the limitation of nitrogen deficiency.
to be the major carbon and energy sources (Fig. 4B).
The profile of the cluster of orthologous groups of V. Perspectives
proteins and the general features of the CfPt1-2 genome
differ greatly from those of known Bacteroidales Since the introduction of molecular biology into
bacteria, but are strikingly similar to those of Rs-D17. environmental microbiology in 1990s, it has been
The functional similarity of CfPt1-2 and Rs-D17 implies recognized that more than 99% of microbial species
that the primary role of the endosymbionts of cellulo- on Earth are as yet unculturable. The termite-gut
lytic protists is efficient and stable biosynthesis of amino microbiota also comprises unculturable microbes, and
1150 Y. HONGOH
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. Metage-
nomics and metatranscriptomics by means of a high-
throughput sequencer are also very useful for research-
ers to form a comprehensive view of the functions and
diversity of gut microbiota. Nevertheless, our knowl-
edge of the functions of individual microbial species is
still poor and difficult to obtain. Hence, single-species-
targeting 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.
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 micro-
biota. 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.
References
1) Yoshimura T, Wood Res., 82, 68–129 (1995).
2) Inoue T, Murashima K, Azuma J-I, Sugimoto A, and Slaytor M,
J. Insect Physiol., 43, 235–242 (1997).
3) Yamin MA, Sociobiology, 4, 1–120 (1979).
4) Kitade O, Maeyama T, and Matsumoto T, Sociobiology, 30,
161–167 (1997).
5) Ohkuma M, Noda S, Hongoh Y, Nalepa CA, and Inoue T, Proc.
R. Soc. Lond. B, 276, 239–245 (2009).
6) Schultz JE and Breznak JA, Appl. Environ. Microbiol., 35, 930–
936 (1978).
7) Hongoh Y, Deevong P, Inoue T, Moriya S, Trakulnaleamsai S,
Ohkuma M, Vongkaluang C, Noparatnaraporn N, and Kudo T,
Appl. Environ. Microbiol., 71, 6590–6599 (2005).
8) Hongoh Y, Ekpornprasit L, Inoue T, Moriya S, Trakulnaleamsai
S, Ohkuma M, Noparatnaraporn N, and Kudo T, Mol. Ecol., 15,
505–516 (2006).
9) Tholen A, Schink B, and Brune A, FEMS Microbiol. Ecol., 24,
137–149 (1997).
10) Leadbetter JR and Breznak JA, Appl. Environ. Microbiol., 62,
3620–3631 (1996).
11) Brauman A, Dore J, Eggleton P, Bignell DE, Breznak JA, and
Kane MD, FEMS Microbiol. Ecol., 35, 27–36 (2001).
12) Tokura M, Ohkuma M, and Kudo T, FEMS Microbiol. Ecol.,
33, 233–240 (2000).
13) Friedrich MW, Schmitt-Wagner D, Lueders T, and Brune A,
Appl. Environ. Microbiol., 67, 4880–4890 (2001).
14) Donovan SE, Purdy KJ, Kane MD, and Eggleton P, Appl.
Environ. Microbiol., 70, 3884–3892 (2004).
15) Leadbetter JR, Crosby LD, and Breznak JA, Arch. Microbiol.,
169, 287–292 (1998).
16) Pester M and Brune A, ISME J., 1, 551–565 (2007).
17) Inoue J, Noda S, Hongoh Y, Ui S, and Ohkuma M, Microbes
Environ., 23, 94–97 (2008).
18) Purdy KJ, Adv. Appl. Microbiol., 62, 63–80 (2007).
19) Hongoh Y and Ohkuma M, ‘‘Microbiology Monographs:
(Endo)symbiotic Methanogenic Archaea,’’ ed. Hackstein JHP,
Springer-Verlag, Berlin and Heidelberg, in press.
20) Ohkuma M and Kudo T, Appl. Environ. Microbiol., 62, 461–
468 (1996).
21) Lilburn TG, Schmidt TM, and Breznak JA, Environ. Microbiol.,
1, 331–345 (1999).
22) Hongoh Y, Ohkuma M, and Kudo T, FEMS Microbiol. Ecol.,
44, 231–242 (2003).
23) Nakajima H, Hongoh Y, Usami R, Kudo T, and Ohkuma M,
FEMS Microbiol. Ecol., 54, 247–255 (2005).
24) Thongaram T, Hongoh Y, Kosono S, Ohkuma M,
Trakulnaleamsai S, Noparatnaraporn N, and Kudo T, Extrem-
ophiles, 9, 229–238 (2005).
25) Yang H, Schmitt-Wagner D, Stingl U, and Brune A, Environ.
Microbiol., 7, 916–932 (2005).
26) Schmitt-Wagner D, Friedrich MW, Wagner B, and Brune A,
Appl. Environ. Microbiol., 69, 6007–6017 (2003).
27) Hongoh Y, Deevong P, Hattori S, Inoue T, Noda S,
Noparatnaraporn N, Kudo T, and Ohkuma M, Appl. Environ.
Microbiol., 72, 6780–6788 (2006).
28) Hongoh Y, Sato T, Dolan MF, Noda S, Ui S, Kudo T, and
Ohkuma M, Appl. Environ. Microbiol., 73, 6270–6276 (2007).
29) Warnecke F, Luginbühl P, Ivanova N, Ghassemian M,
Richardson TH, Stege JT, Cayouette M, McHardy AC,
Djordjevic G, Aboushadi N, Sorek R, Tringe SG, Podar M,
Martin HG, Kunin V, Dalevi D, Madejska J, Kirton E, Platt D,
Szeto E, Salamov A, Barry K, Mikhailova N, Kyrpides NC,
Matson EG, Ottesen EA, Zhang X, Hernandez M, Murillo C,
Acosta LG, Rigoutsos I, Tamayo G, Green BD, Chang C, Rubin
EM, Mathur EJ, Robertson DE, Hugenholtz P, and Leadbetter
JR, Nature, 450, 560–565 (2007).
30) Shinzato N, Muramatsu M, Matsui T, and Watanabe Y, Biosci.
Biotechnol. Biochem., 69, 1145–1155 (2005).
31) Shinzato N, Muramatsu M, Matsui T, and Watanabe Y, Biosci.
Biotechnol. Biochem., 71, 906–915 (2007).
32) Mackenzie LM, Muigai AT, Osir EO, Lwande W, Keller M,
Toledo G, and Boga HI, Afr. J. Biotechnol., 6, 658–667 (2007).
33) Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N,
Chen F, Ochman H, and Hugenholtz P, ISME J., 4, 642–647
(2010).
34) Hugenholtz P, Goebel BM, and Pace NR, J. Bacteriol., 180,
4765–4774 (1998).
35) Berchtold M and König H, Syst. Appl. Microbiol., 19, 66–73
(1996).
36) Brune A and Stingl U, Prog. Mol. Subcell. Biol., 41, 39–60
(2006).
37) Ohkuma M, Trends Microbiol., 16, 345–352 (2008).
38) Iida T, Ohkuma M, Ohtoko K, and Kudo T, FEMS Microbiol.
Ecol., 34, 17–26 (2000).
39) Noda S, Ohkuma M, Yamada A, Hongoh Y, and Kudo T, Appl.
Environ. Microbiol., 69, 625–633 (2003).
40) Hongoh Y, Sato T, Noda S, Ui S, Kudo T, and Ohkuma M,
Environ. Microbiol., 9, 2631–2635 (2007).
41) Wenzel M, Radek R, Brugerolle G, and König H, Eur. J.
Protistol., 39, 11–23 (2003).
42) Stingl U, Radek R, Yang H, and Brune A, Appl. Environ.
Microbiol., 71, 1473–1479 (2005).
43) Ohkuma M, Sato T, Noda S, Ui S, Kudo T, and Hongoh Y,
FEMS Microbiol. Ecol., 60, 467–476 (2007).
44) Ikeda-Ohtsubo W, Desai M, Stingl U, and Brune A, Micro-
biology, 153, 3458–3465 (2007).
45) Stingl U, Maass A, Radek R, and Brune A, Microbiology, 150,
2229–2235 (2004).
46) Noda S, Iida T, Kitade O, Nakajima H, Kudo T, and Ohkuma
M, Appl. Environ. Microbiol., 71, 8811–8817 (2005).
47) Noda S, Inoue T, Hongoh Y, Kawai M, Nalepa CA,
Vongkaluang C, Kudo T, and Ohkuma M, Environ. Microbiol.,
8, 11–20 (2006).
 Diversity and Genomes of Termite Gut Microbes
48) Noda S, Kawai M, Nakajima H, Kudo T, and Ohkuma M,
Microbes Environ., 21, 16–22 (2006).
49) Noda S, Kitade O, Inoue T, Kawai M, Kanuka M, Hiroshima K,
Hongoh Y, Constantino R, Uys V, Zhong J-H, Kudo T, and
Ohkuma M, Mol. Ecol., 16, 1257–1266 (2007).
50) Noda S, Hongoh Y, Sato T, and Ohkuma M, BMC Evol. Biol., 9,
e158 (2009).
51) Desai MS, Strassert JFH, Meuser K, Hertel H, Ikeda-Ohtsubo
W, Radek R, and Brune A, Environ. Microbiol., doi: 10.1111/
j.1462-2920.2009.02080.x.
52) Sato T, Hongoh Y, Noda S, Hattori S, Ui S, and Ohkuma M,
Environ. Microbiol., 11, 1007–1015 (2009).
53) Fröhlich J and König H, Syst. Appl. Microbiol., 22, 249–257
(1999).
54) Cleveland LR and Grimstone AV, Proc. R. Soc. Lond. B, 159,
668–686 (1964).
55) Tamm SL, J. Cell Biol., 94, 697–709 (1982).
56) Ikeda-Ohtsubo W and Brune A, Mol. Ecol., 18, 332–342 (2009).
57) Yamin MA, Science, 211, 58–59 (1981).
58) Slaytor M, ‘‘Termites: Evolution, Sociality, Symbioses, Ecol-
ogy,’’ eds. Abe T, Bignell DE, and Higashi M, Kluwer
Academic Publishers, Dordrecht, pp. 307–332 (2000).
59) Todaka N, Moriya S, Saita K, Hondo T, Kiuchi I, Takasu H,
Ohkuma M, Piero C, Hayashizaki Y, and Kudo T, FEMS
Microbiol. Ecol., 59, 592–599 (2007).
60) Todaka N, Inoue T, Saita K, Ohkuma M, Nalepa CA, Lenz M,
Kudo T, and Moriya S, PLoS One, 5, e8636 (2010).
61) Tartar A, Wheeler MM, Zhou X, Coy MR, Boucias DG, and
Scharf ME, Biotechnol. Biofuels, 2, 25 (2009).
62) Geib SM, Filley TR, Hatcher PG, Hoover K, Carlson JE,
1151
Jimenez-Gasco MD, Nakagawa-Izumi A, Sleighter RL, and
Tien M, Proc. Natl. Acad. Sci. USA, 105, 12932–12937 (2008).
63) Arakane Y, Muthukrishnan S, Beeman RW, Kanost MR, and
Kramer KJ, Proc. Natl. Acad. Sci. USA, 102, 11337–11342
(2005).
64) Breznak JA, Brill WJ, Mertins JW, and Coppel HC, Nature,
244, 577–580 (1973).
65) Breznak JA and Switzer JM, Appl. Environ. Microbiol., 52,
623–630 (1986).
66) Tokuda G and Watanabe H, Biol. Lett., 3, 336–339 (2007).
67) Leadbetter JR, Schmidt TM, Graber JR, and Breznak JA,
Science, 283, 686–689 (1999).
68) Lilburn TG, Kim KS, Ostrom NE, Byzek KR, Leadbetter JR,
and Breznak JA, Science, 292, 2495–2498 (2001).
69) Hongoh Y, Sharma VK, Prakash T, Noda S, Taylor TD, Kudo
T, Sakaki Y, Toyoda A, Hattori M, and Ohkuma M, Proc. Natl.
Acad. Sci. USA, 105, 5555–5560 (2008).
70) Hongoh Y, Sharma VK, Prakash T, Noda S, Toh H, Taylor TD,
Kudo T, Sakaki Y, Toyoda A, Hattori M, and Ohkuma M,
Science, 322, 1108–1109 (2008).
71) Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P,
Sun Z, Zong Q, Du Y, Du J, Driscoll M, Song W, Kingsmore
SF, Egholm M, and Lasken RS, Proc. Natl. Acad. Sci. USA, 99,
5261–5266 (2002).
72) Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J,
Chisholm SW, and Church GM, Nat. Biotechnol., 24, 680–686
(2006).
73) Lasken RS, Curr. Opin. Microbiol., 10, 510–516 (2007).
74) Walker A and Parkhill J, Nat. Rev. Microbiol., 6, 176–177
(2008).