MOLECULAR PHYLOGENETIC IDENTIFICATION OF THE INTESTINAL ANAEROBIC MICROBIAL COMMUNITY IN THE HINDGUT OF THE TERMITE, RETICULITERMES-SPERATUS, WITHOUT CULTIVATION

A termite maintains an anaerobic microbial community in its hindgut, w hich seems to be the minimum size of an anaerobic habitat. This microb ial community consists of bacteria and various anaerobic flagellates, and it is established that termites are totally dependent on the micro bes for the utilization of their food. The molecular phylogenetic dive rsity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy that does not rely on cultivation of the resident microorganisms. Small subunit ribosomal RNA (ssrRNA) genes were directly amplified from the mixed-population DNA of the ter mite gut by polymerase chain reaction (PCR) and clonally isolated. Mos t sequenced clones were phylogenetically affiliated with the four majo r groups of the domain Bacteria: the Proteobacteria group, the Spiroch ete group, the Bacteroides group, and the Low G + C gram-positive bact eria. The 16S rRNA sequence data show that the majority of the intesti nal microflora of the termite consists of new species that are pet to be cultured. The phylogeny of a symbiotic methanogen inhabiting the gu t of a lower termite (R. speratus) was analyzed without cultivation. T he nucleotide sequence of the ssrDNA and the predicted amino acid sequ ence of the mcrA product were compared with those of the known methano gens, Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the kn own members of this order. The diversity of nitrogen-fixing organisms was also investigated without culturing the resident microorganisms. F ragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite g ut and were clonally isolated. The phylogenetic analysis of the nifH a mino acid sequences showed that there was a remarkable diversity of ni trogenase genes in the termite gut: The molecular phylogeny of a symbi otic hypermastigote Trichonympha agilis (class Parabasalia; order Hype rmastigida) in the hindgut of R. speratus was also examined by the sam e strategy. The whole-cell hybridization experiments indicated that th e sequence originated from a large hypermastigote in the termite hindg ut, Trichonympha agilis. According to the phylogenetic trees construct ed, the hypermastigote represented one of the deepest branches of euka ryotes. The hypermastigote along with members of the order Trichomonad ida formed a monophyletic lineage, indicating that the hypermastigote and trichomonads shared a recent common ancestry.