| Budget Amount *help |
¥15,600,000 (Direct Cost: ¥15,600,000)
Fiscal Year 2006: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 2004: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Research Abstract |
The cellulolytic symbiotic protists belonging to the phylum Parabasalia in the gut of termites harbor hydrogenosomes, anaerobic energy and molecular hydrogen producing organelles, and play an important role for hydrogen emission from termites. In the termite Coptotermes formosanus that carries only three parabasalian species as gut symbiotic protists, a cDNA library of mixed-population of the gut protists was constructed and used for identification and characterization of a gene encoding cellulase. Expressed sequence tags (EST) analysis of the cDNA library enabled to infer their primary metabolic ability to identify such enzymes as involve in anaerobic energy production in hydrogenosomes. Protist origins of certain genes were identified using protist cells isolated manually, which allowed molecular phylogenetic analyses of the genes. Putative hydrogenases (enzymes evolving molecular hydrogen) were also identified, and among them, two genes from Pseudotrichonympha grassii that play critical roles in cellulose decomposition were hererologously expressed in Escherichia coli. Both of the purified recombinant enzymes preferentially catalyzed hydrogen evolution rather than hydrogen uptake, but the two were distinct in optimal pH for the reaction and in domain structures, suggesting their different physiological roles in the cells. Cellular fractionation experiments revealed that the hydrogenosome fraction exhibited high hydrogen evolving activity but endosymbiotic bacterial (identified as a novel member of Bacteroidales) fraction showed strong hydrogen uptake activity, suggesting that the symbiotic relationship involved inter-species hydrogen transfer. There are various endosymbiotic (intra-cellular) and ectosymbiotic (attached on to cell surface) associations of prokaryotes with gut protist cells, and the microbial community structures in termite guts and coevolution of termites, protists, and their associated bacteria were investigated and elucidated.
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