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.     

Microorganisms with novel dissimilatory (bi)sulfite reductase genes are widespread and part of the core microbiota in low-sulfate peatlands

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions.Peatlands contain 15% to 30% of the global soil carbon (13, 79) and represent a net carbon sink that has contributed to global cooling in the past 8,000 to 11,000 years (21). While peatlands are generally resilient to external perturbation, it is predicted that long-term global changes such as warming, decreased precipitation, and increased atmospheric deposition of reactive nitrogen and sulfur compounds will transform peatlands into new ecosystem types, accompanied by unforeseeable changes in the carbon balance (17). The carbon loss from peatlands is mediated largely by the anaerobic microbial decomposition of organic matter to the greenhouse gases carbon dioxide and methane (36), and it is estimated that 10 to 20% of the globally emitted methane is derived from peatlands (30, 87). Primary and secondary fermentation and subsequent methanogenesis are considered to be the main carbon degradation processes because of the absence or limited availability of alternative electron acceptors. However, other microbial processes, such as denitrification and dissimilatory iron and sulfate reduction, can occur together with methanogenesis in the same peat soil fraction and contribute considerably to anaerobic carbon mineralization (4, 5, 43, 44). Fluctuations in environmental conditions on short- and long-term scales govern trophic interdependencies among microorganisms. Transitions between synergistic (e.g., the syntrophic interspecies transfer of hydrogen/formate) and antagonistic (e.g., competition for the same substrates) microbial interactions determine the extent of carbon flow diversion away from methanogenesis. A prime example is the suppression of microorganisms catalyzing methanogenic carbon degradation by sulfate-reducing microorganisms (SRM) that are energetically favored in the competition for substrates such as acetate, alcohols, and hydrogen (22, 81, 82). While sulfate concentrations are generally low in peatlands (10 to 300 μM), ongoing sulfate reduction proceeds at rates (2.5 to 340 nmol cm⁻³ day⁻¹) that are comparable to rates in sulfate-rich environments such as marine sediments (5, 40, 41). It was previously proposed that such high sulfate reduction rates are fueled by an anoxic recycling of reduced sulfur compounds via the so-called “thiosulfate shunt” (5). The alternative replenishment of the sulfate pool by the reoxidation of reduced sulfur species in the presence of oxygen is dependent on the vegetation type and alternating periods of precipitation and drought (14, 18, 64, 68, 86). In addition, increasing global atmospheric sulfur pollution and acid precipitation contribute to terrestrial sulfate pools and are predicted to repress methane emissions from peatlands by up to 15% within the first third of this century (22).Given the significance of dissimilatory sulfate reduction in peatlands, it is surprising that most information about the identity of microorganisms catalyzing this process in peatlands is derived from studies of a single model fen system (Schlöppnerbrunnen) located in the forested Lehstenbach catchment (Bavaria, Germany). Different redox processes such as fermentation (25), methanogenesis (29), denitrification (63), Fe(III) reduction (69), and sulfate reduction (2, 51) are present and have been studied at this site (4). The atmospheric deposition of sulfur originating from the combustion of soft coal in Eastern Europe until the 1990s led to the accumulation of sulfur species in the soils of this catchment. Although air pollution affecting this site has decreased in recent years (39), historically deposited sulfate stored in upland soils can desorb and is then transported via groundwater flow into the fen, where it drives dissimilatory sulfate reduction (1). DNA stable isotope probing using in situ concentrations of typical ¹³C-labeled degradation intermediates (mixture of lactate, acetate, formate, and propionate) has shown that a low-abundance Desulfosporosinus species, representing on average only 0.006% of the total bacterial and archaeal 16S rRNA genes, has the potential to be responsible for a substantial part of the sulfate reduction in the studied fen. However, a large fraction of the sulfate reduction observed in situ still remains unexplained (67). Other microorganisms that are potentially involved in sulfate reduction were previously detected in this fen by using 16S rRNA gene- and dsrAB-based diversity analyses. Few of these dsrAB sequences were affiliated with the previously described SRM genera Desulfomonile and Syntrophobacter, but most of the retrieved dsrAB sequences may derive from new taxa, as they represent novel lineages without cultivated representatives (51, 67, 73). Microorganisms that respire sulfite or sulfate anaerobically depend on the dsrAB-encoded key enzyme dissimilatory (bi)sulfite reductase for energy conservation, and thus, these genes have been widely used as markers for PCR-based molecular diversity studies of this guild (16, 38, 46, 84). However, some organisms that are phylogenetically related to SRM but that have seemingly lost the ability for sulfite/sulfate reduction can also harbor dsrAB. The dsrAB sequences of these organosulfonate reducers (45) or syntrophs (32) can be amplified by the commonly used DSR1F-DSR4R PCR primer mix (50). DNA stable isotope probing experiments targeting dsrAB in incubations with a mixture of ¹³C-labeled lactate, acetate, formate, and propionate could therefore not unambiguously link members of the novel dsrAB lineages to sulfate reduction in the Schlöppnerbrunnen peatland (67). Besides their unknown identity and ecophysiological function, additional important questions regarding the ecology of these enigmatic dsrAB-containing microorganisms remain unanswered: what is their actual abundance in peatlands, are they a stable part of the microbial peatland community or do they occur only sporadically, and are they endemic to the Schlöppnerbrunnen fen site or more widely distributed in different types of wetlands? Using a set of molecular ecology tools, we address these questions in this study and demonstrate that some dsrAB-containing microorganisms are widespread in peatlands and can thrive in these systems in considerable numbers.     

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 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 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.     

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.