Up to now, it has been believed that the hindgut flagellates produce nutrients using their own cellulolytic enzymes for the benefit of their termite host. The glycolytic activities found in separated cells of Mixotricha paradoxa are compiled in Table 5.3 (Berchtold and König, unpubl. res.). Surprisingly, not all of these activities seemed to be produced by the flagellates themselves, but rather taken up with the gut contents. Two endoglucanases, Cel I and Cel II,
Monotrichomonas sp. Monotrichomonas carabina Drtrichomonas honigbergii Pseudotrichomonas keilini
Pentatrichomonas hominis Pseudotrypanosoma giganteum
Tetratrichomonas gailinanjm Thchomitus trypanoides Trichomonas lenax Trichomonas vaginalis Hypotrichomonas acosta Trichamäus batrachorum strain R105 Tnchomitus batrachorum strain BUS Monocercomonas Sp. Tritrichomonas foetus
Coronympha octonaria Metacoronympha senta Metadevescovina polyspira Devescovina sp, Foaina sp. clone Cf5
Trie ho monad inae
P. adamsoni symbiont clone gp 2-2 (Joenina) Metadevescovina extranea Dettotrichonympha nanalD. opercuiata
Koruga bonita Mixotricha paradoxa
Snyderella tabogae clone 3 Caionympha grassii clone 1 Dientamoeba fragilis
-- Holomastigotoides mirahile
nympha leidyi ■ Spirotrichonympha sp Eucomonympha sp Pseudotrichonympha grassii
Trichonympha cf. coliaris Trichonympha agilis
De ves covin ida e and
Fig. 5.4. Unrooted phylogenetic tree of parabasalids. Neighbor-joining analysis of SSU rDNA sequences. Bar represents five substitutions per 100 nucleotides. The bootstrap values are computed by three different reconstruction methods: distance matrix, maximum parsony and maximum likelihood. Asterisks designate nodes with bootstrap values below 40% (Li 2003).
with the molecular mass of approximately 48 kD, were isolated from the not yet culturable symbiotic flagellates living in the hindgut of the most primitive Australian termite Mastotermes darwiniensis (Li et al. 2003). The N-terminal sequences of these cellulases exhibited significant homo-logy to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates, but in the salivary glands of Mastotermes darwiniensis. A protein with the molecular mass of approximately 48 kD was also detected in crude extract of these flagellates, by western blot analysis using a polyclonal antiserum against the cellulase of the termite Mastotermes darwiniensis. The results gave evidence that cellulases isolated from the nutritive vacuole of the flagellates originated from the termite host. Probably, the cellulases are secreted from the salivary glands of Mastotermes darwiniensis. During the mechanical grinding of the wood particles by the termites, the cellulases are attached to wood particles or mixed with them, then the attached cellulases or the mixture move to the hindgut where they are most probably endocytosed by the flagellates.
Table 5.3. Determination of glycolytic and laccase activities of Mixotricha paradoxa
Enzyme Activity [|U/cell]
Laccase (ABTS)1' 0.001
It has been shown for Coptotermes formosanus that the endoglucanases of this termite are restricted to the salivary glands, the foregut, and the midgut (Nakashima et al. 2002). According to our work, the main endoglucanase activity found in cells of the hindgut flagellates of Mastotermes darwiniensis is likely to originate from the termite's cellulases. It has also been found that 40% of the endoglucanase activity of Mastotermes darwiniensis is present in the hindgut and most (ca. 84%) of the cellulase activity of the whole hindgut is present in the flagellate extract (Veivers et al. 1982). This implies that a certain amount of termite cellulases, secreted from the salivary glands, moves into the hindgut and enters the flagellate cells. They may be involved in the digestion of cellulose in the flagellate cells.
Using a PCR-based approach, DNA encoding cellulases belonging to the glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita, Deltotrichonympha nana and D. operculata. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 84% amino acid identity with each other. The deduced cellulase sequences of termite origin and flagellate origin consist of a single catalytic domain, lacking a cellulose-binding domain (CBD) and a spacer sequence found in most microbial cellulases (Li et al. 2003).
Although flagellate endoglucanase genes were even expressed in vivo, significant cellulase activity of flagellate origin was not found in the nutritive vacuole by SDS-PAGE. The flagellate cellulase proteins were not detected by western blot analysis. This implied that the native endoglucanase of flagellate origin has very low or even no CMC activity and the translation efficiency could also be very low. In the case of Mixotricha paradoxa, no cellulase gene could be detected.
It is conceivable that in the course of 200~300 million years, the symbiosis between termite and initially free-living intestinal flagellates mutually affected the enzymatic equipment of the other, such that the excess of termite cellulases led to a disuse of the flagellates' own enzymes. The lack of a selection pressure in the hindgut possibly directed low level translation, mutation, and inactivation of cellulolytic enzymes, from which the corresponding genes are still expressed. The production of an inactive enzyme may result in a complete loss of the corresponding genes. This means that the symbiotic flagellates are progressing to a state void of their own cellulolytic activities as was probably the case before the existence of cellulose-containing plants (though some cellulose-producing microorganisms, e.g., Acetobacter, appeared earlier than plants, the amount of the microbial cellulose should be much less than that of the plants). Presently, the symbiotic gut flagellates of the primitive Australian termite Mastotermes darwiniensis owe their endocellulolytic activity to their host.
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