Termite Gut Symbiotic Archaezoa Are Becoming Living Metabolic Fossils

Abstract

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology 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 M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.Cellulose is the major polysaccharide component of plant cell walls and the most abundant renewable energy source on earth. In the biological conversion of cellulose to glucose, at least three distinct types of glycolytic enzymes are involved. Endoglucanases (endo-1,4-␤-glucanase, EC 3.2.1.4) randomly hydrolyze 1,4-␤ bonds of the cellulose chains. Cellobiohydrolases (exo-1,4-␤-glucanase, EC 3.2.1.91) cleave cellobiosyl units from nonreducing ends of the cellullose chains. ␤-Glucosidases (EC 3.2.1.21) cleave glucosyl units from nonreducing ends of cello-oligosaccharides. The diverse spectra of cellulases are classified into 12 of the 57 glycosyl hydrolase families based on amino acid sequence similarities (5).Termites are among the most important lignocellulose-digesting insects and possess a great variety of symbiotic microorganisms in their hindguts, including Bacteria, Archaea and Eukarya, i.e., protozoa and yeasts (7). In the digestive tracts of lower termites, cellulose seems to be synergistically degraded by flagellates, bacteria, and yeasts (3, 7, 15) as well as by the termiteЈs own cellulases (14, 17). Cellulases of termite origin belong to glycosyl hydrolase family 9 (GHF9). Flagellated protozoa belonging to the Archaezoa are unique symbionts in the phylogenetically lower termites. They are cellulolytic and produce acetate from cellulose for the benefit of their hosts (10). The termite Mastotermes darwiniensis is the only species of the most primitive termite family, M...

Symbiotic “Archaezoa” of the Primitive TermiteMastotermes darwiniensisStill Play a Role in Cellulase Production

“…Thus, it is possible that Li et al (9) overlooked the presence of protozoan cellulases. To test this possibility, we attempted to fractionate the hindgut cellulases by gel filtration chromatography, which separates proteins based on molecular sizes.…”

“…Termite-derived cellulases belong to glycosyl hydrolase family 9 (GHF9), while those from protozoa (so far characterized from the termite species Coptotermes formosanus and Reticulitermes speratus) belong to GHFs 5,7, and 45 (4,5,12,16,26). It should be noted that GHFs 5,7,9, and 45 do not share any sequence similarity and are thus assumed to have evolved independently.…”

“…In the 1980s, both termite endogenous and protozoan cellulases were shown to function in the gut of M. darwiniensis on the basis of cellulase activities detected individually from the midguts (termite origin) and hindguts (protozoa origin) by gel filtration chromatography (25). Recently, Li et al (9) attempted to purify the protozoa-derived cellulase of M. darwiniensis using ion exchange chromatography. (The term "Archaezoa" was used by Li et al [9] to describe the cellulolytic symbionts; however, this term is no longer considered valid to refer those single-celled eukaryotes that lack mitochondria [2,18].)…”

“…(The term "Archaezoa" was used by Li et al [9] to describe the cellulolytic symbionts; however, this term is no longer considered valid to refer those single-celled eukaryotes that lack mitochondria [2,18].) Surprisingly, the N-terminal amino acid sequence of the cellulase purified from the hindgut extract was identical to that of a cellulase from salivary glands of the host termites (GHF9) (9). From the hindgut protozoan fauna, Li and colleagues also found mRNAs that encode cellulases of GHF45, similar to those of protozoa from other termites.…”

“…Recently, Li et al (9) attempted to purify the protozoa-derived cellulase of M. darwiniensis using ion exchange chromatography. (The term "Archaezoa" was used by Li et al [9] to describe the cellulolytic symbionts; however, this term is no longer considered valid to refer those single-celled eukaryotes that lack mitochondria [2,18].) Surprisingly, the N-terminal amino acid sequence of the cellulase purified from the hindgut extract was identical to that of a cellulase from salivary glands of the host termites (GHF9) (9).…”