Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.     

Phylogeny of not-yet-cultured spirochetes from termite guts.

Comparisons of 16S rDNA sequences were used to determine the phylogeny of not-yet-cultured spirochetes from hindguts of the African higher termite, Nasutitermes lujae (Wasmann). The 16S rRNA genes were amplified directly from spirochete-rich hindguts by using universal primers, and the amplified products were cloned into Escherichia coli. Clones were screened with a spirochete-specific DNA probe. Analysis of 1,410 base positions of the 16S rDNA insert from one spirochete clone, designated NL1, supported its assignment to the genus Treponema, with average interspecies similarities of ca. 85%. The sequence of NL1 was most closely related (ca. 87 to 88% similarity) to sequences of Spirochaeta stenostrepta and Spirochaeta caldaria and to a previously published sequence (ca. 87% similarity) of spirochetal clone MDS1 from the Australian lower termite, Mastotermes darwiniensis (Froggatt). On the basis of 16S rRNA sequence comparisons and individual base signatures, clones NL1 and MDS1 clearly represent two novel species of Treponema, although specific epithets have not yet been proposed. The gross morphology of NL1 was determined from in situ hybridization experiments with an NL1-specific, fluorescently labeled oligonucleotide probe. Cells were approximately 0.3 to 0.4 by 30 microns in size, with a wavelength and amplitude of about 10 microns and 0.8 to 1.6 micron, respectively. Moreover, electron microscopy of various undulate cells present in gut contents confirmed that they possessed ultrastructural features typical of spirochetes, i.e., a wavy protoplasmic cylinder, periplasmic flagella, and an outer sheath. The sequence data suggest that termite gut spirochetes may represent a separate line of descent from other treponemes and that they constitute a significant reservoir of previously unrecognized spirochetal biodiversity.     

Phylogenetic foundation of spirochetes

The spirochetes are free-living or host-associated, helical bacteria, some of which are pathogenic to man and animal. Comparisons of 16S rRNA sequences demonstrate that the spirochetes represent a monophyletic phylum within the bacteria. The spirochetes are presently classified in the Class Spirochaetes in the order Spirochetales and are divided into three major phylogenetic groupings, or families. The first family Spirochaetaceae contains species of the genera Borrelia, Brevinema, Cristispira, Spirochaeta, Spironema, and Treponema. The second family Brachyspiraceae contains the genus Brachyspira (Serpulina). The third family Leptospiraceae contains species of the genera Leptonema and Leptospira. Novel spirochetal species, or phylotypes, that can not be presently cultivated in vitro, have been identified from the human oral cavity, the termite gut, and other host-associated or free-living sources. There are now over 200 spirochetal species or phylotypes, of which more than half is presently not cultivable. It is likely that there is still a significant unrecognized spirochetal diversity that should be evaluated.     

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Coevolution of symbiotic spirochete diversity in lower termites.

The phylogenetic relationships of symbiotic spirochetes from five dry-wood feeding lower termites (Cryptotermes cavifrons, Heterotermes tenuis, Kalotermes flavicollis, Neotermes mona, and Reticulitermes grassei) was compared to those described in previous reports. The 16S rDNA bacterial genes were PCR-amplified from DNA isolated from intestinal samples using a spirochete-selective primer, and the 16S amplicons were cloned into Escherichia coli. Sequences of the cloned inserts were then used to determine closest relatives by comparison with published sequences. Clones sharing more than 97% sequence identity were grouped into the same phylotype. Forty-three new phylotypes were identified. These termite whole-gut-spirochetes fell into two previous defined clusters, designated as Treponema Clusters I and II, and one new Cluster III. Thirty-seven phylotypes were grouped in Cluster I. Cluster II comprised three phylotypes, two from Reticulitermes grassei (LJ029 and LJ012) and one from Heterotermes tenuis (LQ016). Three phylotypes, LK057, LK050 and LK028, were affiliated to Cluster III. Members of Cluster I showed the following characteristics: (i) spirochete phylotypes from a particular species of termite were more closely related to each other than to phylotypes of other termite species; (ii) spirochetes obtained from different genera of the same family, such as Cryptotermes sp., Kalotermes sp., and Neotermes sp., all from the family Kalotermitidae, were also related to each other. It was therefore concluded that spirochetes are specific symbionts that have coevolved with their respective species of termites, are stably harbored, and are closely related to members of the same termite family.     

Origin of symbiotic gut spirochetes as key players in the nutrition of termites

Termites harbour symbiotic spirochetes in their hindguts, which have long been considered treponemes, although they represent separate lines of descent from known species of Treponema. ‘Termite gut treponemes’ have a mutualistic relationship with the host termites with their physiological properties including CO₂-reductive acetogenesis, from which the resulting acetate fulfils most of the respiratory requirement of the host. Song and co-workers showed that a spirochetal isolate (strain RmG30) from a Madeira cockroach represents the earliest branching lineage of extremely diverse termite (Treponema) cluster I and was a simple homolactic fermenter, suggesting that CO₂-reductive acetogenesis exhibited by some members of termite cluster I originated via horizontal gene transfer. Phylogenomic and 16S rRNA sequence-based phylogenetic analyses indicated a deeply-branched sister clade containing termite cluster I was distinguishable as a family-level lineage. In this context, a new family, ‘Termitinemataceae’ has been proposed for this clade. Strain RmG30 has been designated as the type strain of Breznakiella homolactica gen. nov. sp. nov. named after John A. Breznak, an American microbiologist distinguished in termite gut microbiology. The study has posed important questions for the future, including the actual roles of the termite spirochetes in each termite lineage and the evolutionary process of their physiological properties.     

Phylogenetic relationships of symbiotic methanogens in diverse termites

Termites harbor symbiotic microorganisms in their gut which emit methane. The phylogeny of the termite methanogens was inferred without cultivation based on nucleotide sequences of PCR-amplified 16S ribosomal RNA genes. Seven methanogen sequences from four termite species were newly isolated, and together with those previously published, these sequences were phylogenetically compared. The termite methanogen sequences were divided into three clusters. Two clusters of sequences, derived from the gut DNA of so-called higher termites, were related to methanogens in the orders Methanosarcinales or Methanomicrobiales. All of the sequences in the case of lower termites were closely related to the genus Methanobrevibacter. However, most of the termite symbionts were found to be distinct from known methanogens. They are not dispersed among diverse methanogen species, but rather formed unique lineages in the phylogenetic trees.     

Candidatus Symbiothrix dinenymphae: bristle-like Bacteroidales ectosymbionts of termite gut protists

Many reports have stated that flagellated protists in termite guts harbour ectosymbiotic spirochetes on their cell surface. In this study, we describe another bristle-like ectosymbiont affiliated with the order Bacteroidales. The 16S rRNA phylotype Rs-N74 predominates among Bacteroidales clones obtained from the gut of the termite Reticulitermes speratus. An Rs-N74 phylotype-specific probe was designed in this study and used for detection of the corresponding bacteria in the gut by fluorescence in situ hybridization (FISH) analysis. Surprisingly, the signals were detected specifically from the bristle-like 'appendages' of various flagellate species belonging to the genus Dinenympha; these 'appendages' had been believed to be spirochetal ectosymbionts or structures of the protists. The Rs-N74 bacteria attached to the cell surface of the protists by a tip and coexisted with the spirochetal ectosymbionts. An electron micrograph revealed their morphology to be similar to a typical Bacteroidales bacterium. This bacterium is proposed to represent a novel genus and species, 'Candidatus Symbiothrix dinenymphae', phylogenetically affiliated with a cluster consisting exclusively of uncultured strains from termite guts. A Bacteroidales-specific probe for FISH further revealed that this type of symbiosis exists also in various other protists, including parabasalids and oxymonads, and is widespread in termite guts.     

Formyltetrahydrofolate synthetase gene diversity in the guts of higher termites with different diets and lifestyles

In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The "higher" termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the "lower" termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches.     

GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.     

Comparison of bacterial communities in the alkaline gut segment among various species of higher termites

The first proctodeal (P1) segment in the hindgut of certain higher termites shows high alkalinity. We examined the bacterial diversity of the alkaline P1 gut segments of four species of higher termites by T-RFLP and phylogenetic analyses based on PCR-amplified 16S rRNA genes. The bacterial community of the P1 segment was apparently different from that of the whole gut in each termite. Sequence analysis revealed that Firmicutes (Clostridia and Bacilli) were dominant in the P1 segments of all four termites; however, the phylogenetic compositions varied among the termites. Although some of the P1 segment-derived sequences were related to the sequences previously reported from the alkaline digestive tracts of other insects, most of them formed phylogenetic clusters unique to termites. Such termite P1 clusters were distantly related to known bacterial species as well as to sequences reported from alkaline environments in nature. We successfully obtained enrichment cultures of Clostridia- and Bacilli-related bacteria, including putative novel species under anaerobic alkaline conditions from the termite guts. Our results suggest that the alkaline gut region of termites harbors unique bacterial lineages and are expected to be a rich reservoir of novel alkaliphiles yet to be cultivated.     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites.

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.     

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.     

Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Lignocellulose degradation by microorganisms from termite hills and termite guts: A survey on the present state of art

Abstract: In several aspects termites are a fascinating group of insects having attracted the interest of many researchers. They exhibit a complex social behavior and caste differentiation occurring elsewhere only among the hymenoptera. In an enlarged part of the hindgut, the paunch, termites have established a unique symbiotic association with prokaryotic and eukaryotic microorganisms. A similar flora is also found in wood-eating roaches of the genus Cryptocercus . The study of symbiosis between termites and their intestinal microbes is of general interest, because due to this symbiotic interaction termites can feed on complex biopolymers such as wood. Flagellates and bacteria occur in the gut of lower termites, while higher termites possess only bacteria. In particular spirochetes are abundant in the termite gut. Apart from spirochetes and other more common bacteria, actinomycetes, yeasts and fungi have also been isolated from different species of termites. This review summarizes the distinct role of the intestinal flora in degradation of wood components such as cellulose, hemicellulose and lignin.     

Diet is the primary determinant of bacterial community structure in the guts of higher termites

The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically ‘lower termites’ are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood‐feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus‐level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host‐specific distribution of the bacterial lineages in termite guts are best explained by diet‐related differences in the availability of microhabitats and functional niches.     

Folate cross-feeding supports symbiotic homoacetogenic spirochetes

Treponema primitia, an H2-consuming CO2-reducing homoacetogenic spirochete in termite hindguts, requires an exogenous source of folate for growth. Tetrahydrofolate (THF) acts as a C1 carrier in CO2-reductive acetogenesis, a microbially mediated process important to the carbon and energy requirements of termites. To examine the hypothesis that other termite gut microbes probably supply some form of folate to T. primitia in situ, we used a bioassay to screen for and isolate folate-secreting bacteria from hindguts of Zootermopsis angusticollis, which is the host of T. primitia. Based on morphology, physiology, and 16S rRNA gene sequences, the major folate secretors were identified as strains of Lactococcus lactis and Serratia grimesii. During growth, these isolates secreted 5-formyl-THF at levels up to 146 ng/ml, and their cell-free culture fluids satisfied the folate requirement of T. primitia strains in vitro. Analysis of Z. angusticollis hindgut fluid revealed that 5-formyl-THF was the only detectable folate compound and occurred at an in situ concentration (1.3 mug/ml) which was more than sufficient to support the growth of T. primitia. These results imply that cross-feeding of 5-formyl-THF by other community members is important for growth of symbiotic hindgut spirochetes and thus termite nutrition and survival.     

Genomic Analysis Reveals Multiple [FeFe] Hydrogenases and Hydrogen Sensors Encoded by Treponemes from the H<Subscript>2</Subscript>-Rich Termite Gut

We have completed a bioinformatic analysis of the hydrogenases encoded in the genomes of three termite gut treponeme isolates: hydrogenotrophic, homoacetogenic Treponema primitia strains ZAS-1 and ZAS-2, and the hydrogen-producing, sugar-fermenting Treponema azotonutricium ZAS-9. H₂ is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The spirochetes encoded 4, 8, and 5 [FeFe] hydrogenase-like proteins, identified by their H domains, respectively, but no other recognizable hydrogenases. The [FeFe] hydrogenases represented many sequence families previously proposed in an analysis of termite gut metagenomic data. Each strain encoded both putative [FeFe] hydrogenase enzymes and evolutionarily related hydrogen sensor/transducer proteins likely involved in phosphorelay or methylation pathways, and possibly even chemotaxis. A new family of [FeFe] hydrogenases (FDH-Linked) is proposed that may form a multimeric complex with formate dehydrogenase to provide reducing equivalents for reductive acetogenesis in T. primitia. The many and diverse [FeFe] hydrogenase-like proteins encoded within the sequenced genomes of the termite gut treponemes has enabled the discovery of a putative new class of [FeFe] hydrogenase proteins potentially involved in acetogenesis and furthered present understanding of many families, including sensory, of H domain proteins beyond what was possible through the use of fragmentary termite gut metagenome sequence data alone, from which they were initially defined.     

Identifying the core microbial community in the gut of fungus‐growing termites

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass‐degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus‐growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus‐growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454‐pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus‐level taxa was present in all termite species and accounted for 56–68% of the species‐specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus‐level ecological niches. Finally, we show that gut communities of fungus‐growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus‐growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus‐growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.