16Noda Institute for Scientific Research GRANT
2014 Research Grant
Outline of Research Result

Genome analysis of unculturable, fermentative symbiotic bacteria
in termite guts
Yuichi Hongoh
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology

Research aims
Termites feed only on dead plant matter and contribute
greatly to the global carbon cycle on Earth. Their ability toefficiently digest lignocellulose is mostly attributable to thesymbiotic gut microbiota that is unique to termites and has
attracted interest in both basic and applied scientific research. However, because most of the diverse gut microbes(protists, bacteria, and archaea) are as-yet uncultivable, theexact details of the symbiotic mechanisms remain unclear.
This study aims to elucidate the contribution of an uncultivable, dominant bacterium, Candidatus Symbiothrix dinenymphae (order Bacteroidales) (Fig. 1), to the symbiotic
lignocellulolytic system.
Methods
Bacteria in the gut of the termite Reticulitermes speratus
were sorted to single cells on a fluorescently activated cellsorting (FACS) system, each of which was subjected to isothermal whole-genome amplification. The single-cell amplicons were screened by PCR amplification and Sanger sequencing to identify samples with Ca. S. dinenymphae
cells. Each identified single-cell amplicon was subjected topaired-end and mate-pair nucleotide sequence analysis on
an Illumina MiSeq platform. The generated sequence reads
were assembled to contigs and scaffolds, which wereFig. 1 The oxymonad protist Dinenympha porteri type II in the gut of
the termite Reticulitermes speratus and its ectosymbionts, Candidatus
Symbiothrix dinenymphae (6-FAM-labeled, green) and spirochetes (Texas
red-labeled, red), detected by FISH analysis using specific probes. Images
are from phase-contrast (left) and epifluorescence (right) microscopy analyses. Bar: 20 μm. See details in Ref. (1).

screened on the basis of sequence homology and GC content to remove contaminating fragments. In addition to thegenome sequence analysis, transmission electron microscopy (TEM) was conducted for detailed observation of thecell structure and the attachment site (Fig. 1) (1).
Results
The total length of the draft genome of Ca. S. dinenymphae obtained was 3.5 Mb, and the estimated genome coverage and size were approximately 82% and 4.3 Mb, respectively, on the basis of the conserved single-copy gene
repertoires. This bacterium is a permanently associatedectosymbiont of a protist cell (1), but its genome size has
not been reduced, unlike that of the intracellular symbiont
Candidatus Azobacteroides pseudotrichonymphae (orderBacteroidales) obligately associated with a termite-gut protist (2). The Ca. S. dinenymphae genome shares several
functional characteristics with Ca. A. pseudotrichonymphae: it ferments monosaccharides to acetate via glycolysis
and also undergoes fumarate respiration to generate ATPs,and it retains biosynthetic pathways for most amino acids.
On the other hand, Ca. S. dinenymphae lacks the genes required for nitrogen fixation, a process that is the prominent
feature of Ca. A. pseudotrichonymphae.
Strikingly, the Ca. S. dinenymphae genome contains
many genes for various glycoside hydrolases (GHs) involved in the degradation of the plant cell wall. These havenever been found in the genomes of intracellular symbionts

Fig. 2. Transmission electron micrographs of a Candidatus Symbiothrix
dinenymphae cell attached to the cell surface of the protist Dinenympha sp.
A magnified image (right) shows a socket-like structure, probably derived
from the host, and filamentous substances holding a tip of the ectosymbiont cell. Bars: 500 nm (left) and 200 nm (right). See details in Ref. (3).

		17of lignocellulose or on wood particles to digest their surface, remains unidentified (Fig. 3).
Interestingly, this ectosymbiotic bacterium possesses agene cluster responsible for gliding motility, which is occasionally observed in certain Bacteroidetes bacteria. This unexpected finding indicates that Ca. S. dinenymphae can detach from the host protist surface or possibly that the genes
are just reminiscent of their ancestral free-living lifestyle.
Conclusions

Fig. 3. Outline of the functions of the uncultivable bacterium Candidatus Symbiothrix dinenymphae, based on single-cell genome analysis.

such as Ca. A. pseudotrichonymphae (2). The GHs wereclassified into 16 families and included endo-β-1,4endoglucanase and β-glucosidase, which are required forcellulose degradation. A gene for cellobiohydrolase was notfound; this bacterium can degrade the amorphous site ofcellulose but not its crystalline structure. In addition to thecellulases, diverse hemicellulases such as endo-β-1,4xylanase, endo-1,5-α-l-arabinosidase, and α-mannosidase
were identified. This bacterium secretes these hydrolytic
enzymes from its cell while attached to the host protist cellsurface; however, the localization of the enzymes, whether
within the bacterial cell wall to capture soluble fragments

Candidatus Symbiothrix dinenymphae, an uncultivable,
dominant bacterium accounting for 2.5% of the total bacterial cells in the gut of Reticulitermes speratus, is an ectosymbiont of the cellulolytic protist Dinenympha. Singlecell genome analysis revealed that this bacterium also has
the potential to digest lignocellulose and contribute to thenutrition of the termite host. On the other hand, the benefit
to the host protist remains to be clarified.
References
Hongoh Y, Sato T, Noda S, Ui S, Kudo T, Ohkuma M (2007) Candidatus Symbiothrix dinenymphae: bristle-like Bacteroidales ectosymbionts of termite gut protists. Environ. Microbiol. 9, 2631–2635.
2) Hongoh Y, Sharma VK, Prakash T, Noda S, Toh H, Taylor TD, KudoT, Sakaki Y, Toyoda A, Hattori M, Ohkuma M (2008) Genome of anendosymbiont coupling N2 fixation to cellulolysis within protist cells
in termite gut. Science 322, 1108–1109.
3) Yuki M, Kuwahara H, Shintani M, Izawa K, Sato T, Starns D, Hongoh Y, Ohkuma M (2015) Dominant ectosymbiotic bacteria of cellulolytic protists in the termite gut also have the potential to digest lignocellulose. Environ. Microbiol. 17, 4942–4953.
1)