Yuichi Hongoh

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

ABSTRACT We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.

Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae)

The molecular diversity and community structure of bacteria from the gut of the termite Reticulitermes speratus were analyzed by the sequencing of near-full-length 16S rRNA genes, amplified by polymerase chain reaction. The results of the analysis of 1344 clones indicated a predominance of spirochetes in the gut. Spirochetal clones accounted for approximately half of the analyzed clones. The clones related to Bacteroides, Clostridia, and the candidate division Termite Group I each accounted for approximately 5-15% of the analyzed clones. The rest were comprised of Proteobacteria, Actinobacteria, Mycoplasma and others. Using the criterion of 97% sequence identity, the clones were sorted into 268 phylotypes, including 100 clostridial, 61 spirochetal and 31 Bacteroides-related phylotypes. More than 90% of the phylotypes were found for the first time, and some constituted monophyletic clusters with sequences recovered from the gut of other termite species.

Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment

We investigated biases occurring in the polymerase chain reaction (PCR) amplification of 16S rRNA genes from an environmental sample, by comparing the clone libraries that we had previously prepared from the gut homogenate of the termite Reticulitermes speratus. We detected a significant increase in the expected number of phylotypes by lowering the annealing temperature, and a significant decrease in the proportion of clones belonging to the predominant group by raising the number of PCR cycles. We also found that the Bacteria-universal primer, 63F, introduced a seriously biased amplification caused by primer mismatches, in contrast to a previous report. These results, together with suggestions from previous studies using simplified model samples, will help us to recognize the limitations of PCR-based analysis.

Complete genome of the uncultured Termite Group 1 bacteria in a single host protist cell

Termites harbor a symbiotic gut microbial community that is responsible for their ability to thrive on recalcitrant plant matter. The community comprises diverse microorganisms, most of which are as yet uncultivable; the detailed symbiotic mechanism remains unclear. Here, we present the first complete genome sequence of a termite gut symbiont—an uncultured bacterium named Rs-D17 belonging to the candidate phylum Termite Group 1 (TG1). TG1 is a dominant group in termite guts, found as intracellular symbionts of various cellulolytic protists, without any physiological information. To acquire the complete genome sequence, we collected Rs-D17 cells from only a single host protist cell to minimize their genomic variation and performed isothermal whole-genome amplification. This strategy enabled us to reconstruct a circular chromosome (1,125,857 bp) encoding 761 putative protein-coding genes. The genome additionally contains 121 pseudogenes assigned to categories, such as cell wall biosynthesis, regulators, transporters, and defense mechanisms. Despite its apparent reductive evolution, the ability to synthesize 15 amino acids and various cofactors is retained, some of these genes having been duplicated. Considering that diverse termite-gut protists harbor TG1 bacteria, we suggest that this bacterial group plays a key role in the gut symbiotic system by stably supplying essential nitrogenous compounds deficient in lignocelluloses to their host protists and the termites. Our results provide a breakthrough to clarify the functions of and the interactions among the individual members of this multilayered symbiotic complex.

Genome of an Endosymbiont Coupling N2 Fixation to Cellulolysis Within Protist Cells in Termite Gut

Termites harbor diverse symbiotic gut microorganisms, the majority of which are as yet uncultivable and their interrelationships unclear. Here, we present the complete genome sequence of the uncultured Bacteroidales endosymbiont of the cellulolytic protist Pseudotrichonympha grassii, which accounts for 70% of the bacterial cells in the gut of the termite Coptotermes formosanus. Functional annotation of the chromosome (1,114,206 base pairs) unveiled its ability to fix dinitrogen and recycle putative host nitrogen wastes for biosynthesis of diverse amino acids and cofactors, and import glucose and xylose as energy and carbon sources. Thus, nitrogen fixation and cellulolysis are coupled within the protists cells. This highly evolved symbiotic system probably underlies the ability of the worldwide pest termites Coptotermes to use wood as their sole food.

Toward the functional analysis of uncultivable, symbiotic microorganisms in the termite gut

Termites thrive on dead plant matters with the aid of microorganisms resident in their gut. The gut microbiota comprises protists (single-celled eukaryotes), bacteria, and archaea, most of which are unique to the termite gut ecosystem. Although this symbiosis has long been intriguing researchers of both basic and applied sciences, its detailed mechanism remains unclear due to the enormous complexity and the unculturability of the microbiota. In the effort to overcome the difficulty, recent advances in omics, such as metagenomics, metatranscriptomics, and metaproteomics have gradually unveiled the black box of this symbiotic system. Genomics targeting a single species of the unculturable microbial members has also provided a great progress in the understanding of the symbiotic interrelationships among the gut microorganisms. In this review, the symbiotic system organized by wood-feeding termites and their gut microorganisms is outlined, focusing on the recent achievement in omics studies of this multilayered symbiotic system.

Cospeciation in the triplex symbiosis of termite gut protists (Pseudotrichonympha spp.), their hosts, and their bacterial endosymbionts

A number of cophylogenetic relationships between two organisms namely a host and a symbiont or parasite have been studied to date; however, organismal interactions in nature usually involve multiple members. Here, we investigated the cospeciation of a triplex symbiotic system comprising a hierarchy of three organisms — termites of the family Rhinotermitidae, cellulolytic protists of the genus Pseudotrichonympha in the guts of these termites, and intracellular bacterial symbionts of the protists. The molecular phylogeny was inferred based on two mitochondrial genes for the termites and nuclear small‐subunit rRNA genes for the protists and their endosymbionts, and these were compared. Although intestinal microorganisms are generally considered to have looser associations with the host than intracellular symbionts, the Pseudotrichonympha protists showed almost complete codivergence with the host termites, probably due to strict transmissions by proctodeal trophallaxis or coprophagy based on the social behaviour of the termites. Except for one case, the endosymbiotic bacteria of the protists formed a monophyletic lineage in the order Bacteroidales, and the branching pattern was almost identical to those of the protists and the termites. However, some non‐codivergent evolutionary events were evident. The members of this triplex symbiotic system appear to have cospeciated during their evolution with minor exceptions; the evolutionary relationships were probably established by termite sociality and the complex microbial community in the gut.

Diversity and Genomes of Uncultured Microbial Symbionts in the Termite Gut

Termites play a key role in the global carbon cycle as decomposers. Their ability to thrive solely on dead plant matter is chiefly attributable to the activities of gut microbes, which comprise protists, bacteria, and archaea. Although the majority of the gut microbes are as yet unculturable, molecular analyses have gradually been revealing their diversity and symbiotic mechanisms. Culture-independent studies indicate that a single termite species harbors several hundred species of gut microbes unique to termites, and that the microbiota is consistent within a host termite species. To elucidate the functions of these unculturable symbionts, environmental genomics has recently been applied. Particularly, single-species-targeting metagenomics has provided a breakthrough in the understanding of symbiotic roles, such as the nitrogen fixation, of uncultured, individual microbial species. A combination of single-species-targeting metagenomics, conventional metagenomics, and metatranscriptomics should be a powerful tool to dissect this complex, multi-layered symbiotic system.

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus‐growing termite Macrotermes gilvus

The fungus‐growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T‐RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T‐RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood‐feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co‐evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.

Phylogenetic diversity, localization, and cell morphologies of members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, recently discovered bacterial groups dominant in termite guts.

ABSTRACT Recently we discovered two novel, deeply branching lineages in the domain Bacteria from termite guts by PCR-based analyses of 16S rRNA (Y. Hongoh, P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo, Appl. Environ. Microbiol. 71:6590-6599, 2005). Here, we report on the specific detection of these bacteria, the candidate phylum TG3 (Termite Group 3) and a subphylum in the phylum Fibrobacteres, by fluorescence in situ hybridization in the guts of the wood-feeding termites Microcerotermes sp. and Nasutitermes takasagoensis. Both bacterial groups were detected almost exclusively from the luminal fluid of the dilated portion in the hindgut. Each accounted for approximately 10% of the total prokaryotic cells, constituting the second-most dominant groups in the whole-gut microbiota. The detected cells of both groups were in undulate or vibroid forms and apparently resembled small spirochetes. The cell sizes were 0.2 to 0.4 by 1.3 to 6.0 μm and 0.2 to 0.3 by 1.3 to 4.9 μm in the TG3 and Fibrobacteres, respectively. Using PCR screenings with specific primers, we found that both groups are distributed among various termites. The obtained clones formed monophyletic clusters that were delineated by the host genus rather than by the geographic distance, implying a robust association between these bacteria and host termites. TG3 clones were also obtained from a cockroach gut, lake sediment, rice paddy soil, and deep-sea sediments. Our results suggest that the TG3 and Fibrobacteres bacteria are autochthonous gut symbionts of various termites and that the TG3 members are also widely distributed among various other environments.