35[Noda Institute for Scientific Research GRANT]2006 Research Grant
Outline of Research Result

Metabolic Symbiosis between Termite Gut Protists and Their
Intracellular Bacteria
Moriya OHKUMA
Environmental Molecular Biology Laboratory, RIKEN

Research aims

Results

Termites are one of the few arthropods that exclusively
thrive on dead plant matter and efficiently decompose lignocellulose. A dense and diverse microbial community inthe gut of termites, comprising of both flagellated protists(single cell eukaryotes) and prokaryotes, is essential for theefficient decomposition, and thus is expected to be an attractive model for efficient utilization of biomass resources1). Although most members of the community aredifficult to cultivate, molecular ecological studies reveal theenormous diversity and novelty of the resident microbial
species2,3). A unique and impressive character of the termite-gut community is its highly structured nature; the specific associations of various prokaryotes with the cells ofgut protists comprise predominant population in the gut
community3–8). These associations are considered as keys tounderstand the mechanisms of the efficient decomposition.
In this study, the primary metabolism of the host protists
and symbiotic interactions with their associated prokaryotes
were investigated.

In the EST analysis, various genes for cellulases wereidentified, which were categorized to glycosyl hydrolase
families 5, 7, and 45 as shown in the case of gut protists inthe termite Reticulitermes speratus 11). Genes for most glycolytic enzymes and hydrogenosomal functions, such as
pyruvate:ferredoxin oxidoreductase and hydrogenase werealso identified. It is noted that genes encoding phosphoenolpyruvate (PEP) carboxykinase (PCK) and malate dehydrogenase (MDH) in the cytoplasm as well as hydrogenosomal malic enzyme (ME) were highly expressed when compared to the gene involving in direct transformation of PEP
to pyruvate. The results suggest that malate is produced in
the cytoplasm and transported into hydrogenosomes to ferment to acetate, CO2, and H2 (Fig. 1).
Two genes encoding hydrogenosomal iron-only (Fe-) hydrogenases, which were phylogenetically distinct and carried different N-terminal accessory domains, were identified in the largest cellulolytic protist Pseudotrichonympha
grassii. The active recombinant enzymes of these two Fehydrogenases were successfully obtained and exhibited different specific activity and optimal pH, suggesting the different physiological roles (Table 1). Both Fe-hydrogenases
preferentially catalyzed H2 evolution rather than H2 uptake.
H2 evolution of at least the short-form enzyme was still active even under high hydrogen partial pressure. The results
suggest that termite-gut symbionts are a rich reservoir ofnovel Fe-hydrogenases whose property is adapted to the gut

Methods
Culture-independent molecular approaches were mainlyapplied. Expressed sequence tags (EST) analysis of acDNA library constructed for mixed-population of gut protists of the termite Coptotermes formosanus 9) and the following comparisons with the public databases were conducted to reconstruct the primary metabolism of gut protists (manuscript in preparation). This termite harbors only
the three species of protist belonging to the phylum
Parabasalia that carry anaerobic energy-producing organelles, hydrogenosomes10). Genes encoding hydrogenase
homologs were heterologously expressed, and the recombinant enzymes were purified and characterized. The protist
origins of the hydrogenase genes were also identified by sequence-specific diagnostic PCR with manually isolated
cells of each protist species and whole-cell in situ hybridizations using specific probes. After enrichment of theprotist cells, the cellular components were fractionated toevaluate hydrogenase activity11).

Fig. 1. Schematic showing the reconstructed primary metabolism ofparabasalian protists in the gut of termites, with the emphasis on the flowof reducing equivalents (e).

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Table 1.

Characteristics of two Fe-hydrogenases in P. grassii
Activity

Long-form
Short-form

H2 evolution

H2 uptake1217.8213154

ND43112OptimalpH
6.0
8.0

Hydrogenase activity (standard deviation) measured using methylviologen as an electron carrier is expressed as mmol H2 min1 mg-protein1.
ND, not detected.

tists and prokaryotes as well as the metabolic functions of
the protists greatly contribute to the efficiency of cellulose
utilization of termites.
References
1.
2.

3.

environment.
Fractionation of cellular components of P. grassii demonstrated significant H2 evolution activity in the hydrogenosomal fraction. The vigorous H2 uptake activity was detectedin the fraction enriched in its endosymbiotic bacteria thatare affiliated to a novel lineage of the order Bacteroidales8),
and as a result, the strong H2 evolution activity of the hostprotists was compensated. Indeed, living termites emit H2 atthe considerably lower level (0.75 mol H2 emitted per molar
glucose derived from cellulose) than that predicted stoichiometrically (4 mol H2 per molar glucose). The potentialof H2 production in termite guts had been largely underestimated.

4.

5.

6.

7.

Conclusion
It is generally believed that gut protists phagocytose
wood particles and degrade cellulose within their cells. The
reconstruction of their primary metabolic pathway supports
the cellulolytic property of gut protists. The merit to produce malate in the cytoplasm is that most of the reducing
equivalents produced during the cellulose fermentation as aform of NADH were regenerated in the cytoplasm, andtransferred as a form of malate to hydrogenosomes where
the strong Fe-hydrogenases dispose the reducing power as aform of H2 (Fig. 1). The endosymbiotic bacteria effectively
eliminate the produced H2. Further studies are necessary toclarify the primary functions of the endosymbionts, particularly the electron acceptor of the endosymbionts that utilizehydrogen. The genome analysis of the endosymbiont is now
in progress. Nevertheless, the efficient elimination of reducing equivalents, an important rate-limiting step for anaerobic fermentation, leads to the enhanced cellulose fermentation. The symbiotic associations between termite-gut pro-

8.

9.

10.

11.

12.

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Hongoh, Y., Sato, T., Dolan, M. F., Noda, S., Ui, S., Kudo, T.,Ohkuma, M. The motility symbiont of the termite gut flagellate Caduceia versatilis is a member of the “Synergistes” group. Appl. Environ. Microbiol. 73: 6270–6276 (2007)
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Vongkaluang, C., Kudo, T., Ohkuma, M. Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach. Environ. Microbiol. 8: 11–20 (2006)
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Endosymbiotic Bacteroidales bacteria of the flagellated protist
Pseudotrichonympha grassii in the gut of the termite Coptotermes
formosanus. Appl. Environ. Microbiol. 71: 8811–8817 (2005)
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Inoue, J.-I., Saita, K., Kudo, T., Ui, S., Ohkuma, M. Hydrogen production by termite gut protists: characterization of iron hydrogenasesof parabasalian symbionts of the termite Coptotermes formosanus.
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cDNA analysis of the genes involved in lignocellulose digestion in
the symbiotic protist community of Reticulitermes speratus. FEMS
Microbiol. Ecol. 59: 592–599 (2007)