Genome analyses of uncultured TG2/ZB3 bacteria in 'Margulisbacteria' specifically attached to ectosymbiotic spirochetes of protists in the termite gut.

We investigated the phylogenetic diversity, localisation and metabolism of an uncultured bacterial clade, Termite Group 2 (TG2), or ZB3, in the termite gut, which belongs to the candidate phylum 'Margulisbacteria'. We performed 16S rRNA amplicon sequencing analysis and detected TG2/ZB3 sequences in 40 out of 72 termite and cockroach species, which exclusively constituted a monophyletic cluster in the TG2/ZB3 clade. Fluorescence in situ hybridisation analysis in lower termites revealed that these bacteria are specifically attached to ectosymbiotic spirochetes of oxymonad gut protists. Draft genomes of four TG2/ZB3 phylotypes from a small number of bacterial cells were reconstructed, and functional genome analysis suggested that these bacteria hydrolyse and ferment cellulose/cellobiose to H2, CO2, acetate and ethanol. We also assembled a draft genome for a partner Treponema spirochete and found that it encoded genes for reductive acetogenesis from H2 and CO2. We hypothesise that the TG2/ZB3 bacteria we report here are commensal or mutualistic symbionts of the spirochetes, exploiting the spirochetes as H2 sinks. For these bacteria, we propose a novel genus, 'Candidatus Termititenax', which represents a hitherto uncharacterised class-level clade in 'Margulisbacteria'. Our findings add another layer, i.e., cellular association between bacteria, to the multi-layered symbiotic system in the termite gut.

Phylogenetic Diversity and Single-Cell Genome Analysis of "Melainabacteria", a Non-Photosynthetic Cyanobacterial Group, in the Termite Gut.

Termite guts harbor diverse yet-uncultured bacteria, including a non-photosynthetic cyanobacterial group, the class "Melainabacteria". We herein reported the phylogenetic diversity of "Melainabacteria" in the guts of diverse termites and conducted a single-cell genome analysis of a melainabacterium obtained from the gut of the termite Termes propinquus. We performed amplicon sequencing of 16S rRNA genes from the guts of 60 termite and eight cockroach species, and detected melainabacterial sequences in 48 out of the 68 insect species, albeit with low abundances (0.02-1.90%). Most of the melainabacterial sequences obtained were assigned to the order "Gastranaerophilales" and appeared to form clusters unique to termites and cockroaches. A single-cell genome of a melainabacterium, designated phylotype Tpq-Mel-01, was obtained using a fluorescence-activated cell sorter and whole genome amplification. The genome shared basic features with other melainabacterial genomes previously reconstructed from the metagenomes of human and koala feces. The bacterium had a small genome (~1.6 Mb) and possessed fermentative pathways possibly using sugars and chitobiose as carbon and energy sources, while the pathways for photosynthesis and carbon fixation were not found. The genome contained genes for flagellar components and chemotaxis; therefore, the bacterium is likely motile. A fluorescence in situ hybridization analysis showed that the cells of Tpq-Mel-01 and/or its close relatives are short rods with the dimensions of 1.1±0.2 µm by 0.5±0.1 µm; for these bacteria, we propose the novel species, "Candidatus Gastranaerophilus termiticola". Our results provide fundamental information on "Melainabacteria" in the termite gut and expand our knowledge on this underrepresented, non-photosynthetic cyanobacterial group.

Draft Genome Sequences of Geomicrobium sp. Strains JCM 19037, JCM 19038, JCM 19039, and JCM 19055, Isolated from Aquatic Samples.

Haloalkaliphilic strains JCM 19037, JCM 19038, JCM 19039, and JCM 19055, closely related to Geomicrobium sediminis, were isolated from aquatic samples, and their draft genome sequences were determined. The genome information of these four strains will be useful for studies of their physiology and ecology.

Draft Genome Sequences of Vibrio sp. Strains Isolated from Tetrodotoxin-Bearing Scavenging Gastropod.

Vibrio sp. strains JCM 18905 and JCM 19053 were isolated from a tetrodotoxin (TTX)-bearing scavenging gastropod, and Vibrio sp. strain JCM 18904 was isolated from a sea cucumber. All these are closely related to Vibrio alginolyticus. Their comparative genome information is useful for studies of TTX production in bacteria.

Draft Genome Sequences of Psychrobacter Strains JCM 18900, JCM 18901, JCM 18902, and JCM 18903, Isolated Preferentially from Frozen Aquatic Organisms.

Four Psychrobacter strains, JCM 18900, JCM 18901, JCM 18902, and JCM 18903, related to either Psychrobacter nivimaris or Psychrobacter cibarius, were isolated from frozen marine animals. The genome information of these four strains will be useful for studies of their physiology and adaptation properties to frozen conditions.

Draft Genome Sequences of Cyclodextrin-Producing Alkaliphilic Bacillus Strains JCM 19045, JCM 19046, and JCM 19047.

Bacillus strains JCM 19045, JCM 19046, and JCM 19047 are alkaliphiles that produce ß-cyclodextrin from starch. They are related to Bacillus xiaoxiensis and Bacillus lehensis. The genome information for these three strains will be useful for studies of the physiological role of cyclodextrin and cyclodextrin production.

Physiological characterization of aerobic culturable bacteria in the intestine of the sea cucumber Apostichopus japonicus.

Various aerobic culturable bacteria (1,133 isolates) were isolated from the gut of Apostichopus japonicus (black adult, green adult, black small, green small, black juvenile, and green juvenile sea cucumbers) and from the sea sediment and the seawater using different culture conditions and without enrichment culture. By molecular analysis of partial 16S rRNA gene sequences of 231 isolates, they were tentatively affiliated with 53 described species in the phyla Firmicutes (42 species), Proteobacteria (9 species) and Actinobacteria (2 species). Eighteen species were often found among the intestines and the sea sediment. High diversity was observed in the genus Bacillus (20 species), Oceanobacillus and Virgibaillus but there were no isolates affiliated to members of the genus Vibrio, well-known sea pathogens. There were no clear differences in the bacterial communities among the hosts varied in size and color. Most isolates showed various polysaccharide degradation activities, suggesting their possible contributions in the digestion of organic matters in the gut.

Diversity and function of aerobic culturable bacteria in the intestine of the sea cucumber Holothuria leucospilota.

Sea cucumbers play an important role in nutrient cycling of marine ecosystems by consuming sediments and moving sand, thus occupying a similar niche to earthworms in terrestrial ecosystems. However, our understanding of microbial diversity and functions associated with sea cucumbers is meager. Here, we isolated 141 bacterial strains under aerobic conditions using various media from the intestine of Holothuria leucospilota, a common sea cucumber in Japanese warm waters. By partial 16S rRNA gene sequences of the isolates, the isolates were tentatively affiliated with 55 described species. Among them, 23 species were common between 2 individuals of H. leucospilota. High diversity was observed in the genera Bacillus and Vibrio, which are often found in marine sediments, marine animals and other various environments. Most isolates showed various polysaccharide degradation activities and were able to grow under or were tolerant of anaerobic condition. We suggest that these aerobically isolated bacteria can play a role in digestion of detritus in aerobic and/or anaerobic regions of the intestine.

Phylogenetic analysis of cellulolytic enzyme genes from representative lineages of termites and a related cockroach.

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis.

Heterologous expression and characterization of an endoglucanase from a symbiotic protist of the lower termite, Reticulitermes speratus.

RsSymEG, an endoglucanase of glycosyl hydrolase family (GHF) 7 encoded by a transcript isolated from the symbiotic protist of the termite Reticulitermes speratus, is expressed in Aspergillus oryzae. Interestingly, purified RsSymEG1 has a relatively higher specific activity (603 micromol min(-1) mg(-1) protein) and V(max) value (769.6 unit/mg protein) than previously reported data for GHF7 endoglucanase of Trichoderma ressei. It also has the same K(m) value (1.97 mg/ml) with Clostridium cellulolyticum enzymes that contain cellulose binding module, a property indicative of high affinity to substrate, though no cellulose binding module is found within it. Thin-layer chromatography analysis revealed that RsSymEG1 preferentially hydrolyzes the beta-1,4-cellulosic linkage of cellodextrins into cellobiose and glucose.

Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus.

Cryptocercus cockroaches and lower termites harbour obligate, diverse and unique symbiotic cellulolytic flagellates in their hindgut that are considered critical in the development of social behaviour in their hosts. However, there has been controversy concerning the origin of these symbiotic flagellates. Here, molecular sequences encoding small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase were identified in the symbiotic flagellates of the order Trichonymphida (phylum Parabasalia) in the gut of Cryptocercus punctulatus and compared phylogenetically to the corresponding species in termites. In each of the monophyletic lineages that represent family-level groups in Trichonymphida, the symbionts of Cryptocercus were robustly sister to those of termites. Together with the recent evidence for the sister-group relationship of the host insects, this first comprehensive study comparing symbiont molecular phylogeny strongly suggests that a set of symbiotic flagellates representative of extant diversity was already established in an ancestor common to Cryptocercus and termites, was vertically transmitted to their offspring, and subsequently became diversified to distinct levels, depending on both the host and the symbiont lineages.

Transcriptome analysis of the digestive organs of Hodotermopsis sjostedti, a lower termite that hosts mutualistic microorganisms in its hindgut.

Microorganisms dwell symbiotically in the termite hindgut. In this study, we identified genes that contribute to the role of the host in maintaining this symbiotic relationship with microorganisms. Body tissue and digestive organs (salivary gland, foregut, midgut, and hindgut) dissected from the lower termite Hodotermopsis sjostedti were used for the analyses. The transcriptomes in these organs were investigated using expressed sequence tag (EST) analysis. The cDNA libraries from the salivary gland and foregut included not only cellulase genes, but also several genes involved in glucose production, heme-cellulose degradation, chitin degradation, the innate immune system, and anti-microbial activity. We compared the expression level of these genes in the organs and body by real-time quantitative RT-PCR. Real time RT-PCR analyses confirmed that the genes associated with cellulose degradation, innate immunity, and anti-microbial proteins are much more strongly expressed in the salivary gland than in other tissues. Our results identify functional genes used by the host in the termite symbiotic system.

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.

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 microm and 0.2 to 0.3 by 1.3 to 4.9 microm 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.

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach.

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host.

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 cloning and characterization of a cellulase gene from a symbiotic protist of the lower termite, Coptotermes formosanus.

The endo-beta-1,4-glucanase gene was cloned from a cDNA library constructed from the mixed population of symbiotic protists in the hindgut of the lower termite, Coptotermes formosanus, using the lambda ZAP II vector. The recombinant phage library was screened for cellulolytic activity by the Congo red staining procedure. The nucleotide sequence comprised 941 nucleotides including a polyA tail sequence and showed high sequence similarity with endoglucanase genes belonging to glycosyl hydrolase family 5. Determination of the 5' end of the cellulase gene using the 5'RACE method showed that the full-length cDNA comprised a 921-bp ORF, encoding a putative 33,620 Da protein. The organismal source of this cellulase gene was identified using PCR with gene-specific primers and whole-cell in situ hybridization as the smallest symbiotic hypermastigote protist, Spirotrichonympha leidyi. The optimal pH and temperature of the cellulase heterologously expressed in Escherichia coli were 5.8-6.0 and 70 degrees C, respectively. The Km and Vmax values on carboxymethyl cellulose (CMC) substrate were 1.90 mg/ml and 148.2 units/mg protein, respectively.

Molecular phylogeny of Asian termites (Isoptera) of the families Termitidae and Rhinotermitidae based on mitochondrial COII sequences.

The families Termitidae and Rhinotermitidae are the most evolved and diverse groups of the social insects, termites (Order Isoptera), showing elaborated morphology and complex behavior. Molecular phylogeny of termites with the emphasis on these families was examined by Bayesian and maximum-likelihood analyses based on DNA sequence of mitochondrial cytochrome oxidase II (COII) gene of 31 genera sampled in Asia (mainly Thailand and Japan) along with those reported previously. Termitidae was monophyletic and originated from within polyphyletic Rhinotermitidae. Among the four subfamilies of Termitidae, Macrotermitinae was monophyletic suggesting a single common origin of fungus-growing habit characteristic for this subfamily, and was placed in the basal position in the family. A group consisting of other subfamilies Termitinae and Nasutitermitinae, though some important groups were still untouched, was the most apical but neither Termitinae nor Nasutitermitinae formed a monophyletic lineage. It was implied that, as defense systems of the soldier castes, the appearance of snapping mandibles has occurred at a single event, but the development of nasus for chemical secretion has probably not. Our tree provides some evidence concerning contradictions in the previously proposed phylogeny of termites.

Molecular phylogeny of symbiotic basidiomycetes of fungus-growing termites in Thailand and their relationship with the host.

Termitomyces-related symbiotic basidiomycetes in the nests of fungus-growing termites (Macrotermitinae) of several genera in Thailand were cultivated and analyzed phylogenetically based on the DNA sequence of nuclear ribosomal RNA genes. The relationships of the symbiotic fungi with host termites and their locality were apparently complex, supporting intricate mechanisms for the termites to acquire the symbionts.