Phylogenetic Identification of the Intestinal Anaerobic Microbial Community in the Termite Hindgut without Cultivation
Toshiaki KUDO*, Moriya OHKUMA, Shigeharu MORIYA, Satoko NODA, and Kuniyo OHTOKO
The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama, 351-01, Japan
Life originated in the pre-oxic worlds and anoxic environments still persist in many places on Earth such as lake sediments, the gut of ruminants, and the deep waters of some marine basins. A termite maintains an anaerobic microbial community in its hindgut which seems the mimium size of an anaerobic habitat. This microbial community consists of bacteria and various anaerobic flagellates and it is established that termites are totally dependent on the microbes for the utilization of their food.
The phylogenetic diversity of the intestinal microflora of a lower termite Reticulitermes speratus was examined by a strategy which 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 termite gut by the polymerase chain reaction (PCR) and were clonally isolated. Nucleotide sequence analysis of partial 16S rRNA showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rRNA sequence data are available. Most of sequenced clones were phylogenetically affiliated with the four major groups of the domain Bacteria: the Proteobacteria group, the Spirochete group, the Bacteroides group, and the low G+C Gram-positive bacteria. The 16S rRNA sequence data shows that the majority of the intestinal microflora of the termite consists of new, yet-to-be-cultivable species previously unknown to microbiologists.
The diversity of nitrogen-fixing organisms was also investigated without culturing the resident microorganisms as one of the model genes. Fragments of nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite gut and were clonally isolated. The phylogenetic analysis of the nifH amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut.
We also present and discuss molecular phylogenetic identification of symbiotic methanogen and protists.
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