Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

Abstract

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.Symbiotic microorganisms that inhabit the guts of termites enable the termites to feed on lignocelluloses (4, 13, 21). The gut microbiota forms a dense and complex symbiotic community consisting of both flagellated protists (single-cell eukaryotes) and prokaryotes. Recent culture-independent studies based on molecular sequences have enabled us to classify the gut symbionts phylogenetically as both protists (22,26) and prokaryotes (9, 16-18, 23-25, 28, 29). These studies revealed that a great majority of the prokaryotes in the gut are novel organisms that have not been cultivated, which has limited our knowledge of termite gut symbionts.Associations of prokaryotes with gut protists are frequently observed, and gut protists themselves are the hosts of prokaryotic symbionts (21). In fact, the protist-associated prokaryotes comprise a significant portion of the gut microbial community (3). Previous observations revealed the presence of methanogens within cells (as endosymbionts) and spirochetes attached to cell surfaces (as ectosymbionts) of the gut protists, and these groups of prokaryotes have been identified in situ and classified phylogenetically by using molecular sequences (references 8, 12, 20, 33, and 35 and references therein). Recently, bacteria belonging to the order Bacteroidales were identified as ectosymbionts of a number of protist species in termite guts (18,30,35), but they belong to at least three polyphyletic lineages in this order (18). These studies and other studies have gradually disclosed bacterial species associated with gut protists of termites and the spatial distribution of t...

The gut microbiota of the wood-feeding termite Reticulitermes lucifugus (Isoptera; Rhinotermitidae)

“…Members of the phylum Bacteroidetes have been reported to reside in the gut as ecto-or endosymbionts of flagellated protozoa (Noda et al 2005;Nakajima et al 2006), and cellularlevel symbioses between Bacteroidales and gut protists are as common as those between spirochetes and gut protists. The Bacteroidales bacteria in termite guts, including symbionts of protists, constitute several monophyletic clusters specific to termites and wood-feeding cockroaches.…”

Acetogenesis from H2plus CO2and nitrogen fixation by an endosymbiotic spirochete of a termite-gut cellulolytic protist

“…The Eucomonympha-Teranympha lineage is phylogenetically a sister to Pseudotrichonympha (39), but their endosymbionts are completely different. The Pseudotrichonympha endosymbiont belongs to the order Bacteroidales (40). It is possible that, after the two lineages separated, they acquired the endosymbionts independently or that the endosymbiont of one lineage was replaced by another.…”

“…Confidence of tree topology was estimated with bootstrap analyses of 500 resamplings. FISH and transmission electron microscopy (TEM) were conducted as described previously (27,40).…”

Quantitative detection of culturable methanogenic archaea abundance in anaerobic treatment systems using the sequence-specific rRNA cleavage method

“…This method has been successfully applied to quantify targeted microbial populations in different environments such as termite gut (Noda et al, 2005), biological treatment processes (Uyeno et al, 2004;Sekiguchi et al, 2005), daily cattle rumen (Uyeno et al, 2007), cow feces (Uyeno et al, 2004) and human feces (Uyeno et al, 2008). In this study, this method is further extended to quantify different methanogens in anaerobic treatment processes through the use of a comprehensive set of 40 16S rRNA-based scissor probes targeting almost all known methanogens at different phylogeny specificities.…”