“Methanoplasmatales,” Thermoplasmatales-Related Archaea in Termite Guts and Other Environments, Are the Seventh Order of Methanogens

Cubitermes ugandensis and Ophiotermes sp. were collected in Kakamega Forest Reserve (Kenya), and Macrotermes michaelseni was collected near Kajiado (Kenya). Trinervitermes sp. and Alyscotermes trestus originated from the campus of the Jomo Kenyatta University of Agriculture and Technology, Gachororo, Kenya. Only worker caste termites were used for this work. The wood-feeding cockroaches Salganea esakii and Panesthia angustipennis were collected in the vicinity of the Keta Shrine in Ishikawa Prefecture, Japan, by Kiyoto Maekawa, Toyama University. The millipede Anadenobolus sp. was obtained from a commercial breeder (b.t.b.e. Insektenzucht, Schnürpflingen, Germany). All animals were kept in plastic containers at room temperature in the dark.

The hindguts of 10 to 20 termites were dissected with sterile fine-tipped forceps, pooled in 2-ml tubes containing 750 μl sodium phosphate buffer (120 mM; pH 8.0), and homogenized. Homogenates of individual cockroach hindguts were prepared in a similar manner. DNA was prepared using a bead-beating protocol combined with phenol-chloroform extraction. The homogenate was transferred to a 2-ml bead-beating vial, and 250 μl sodium dodecyl sulfate (SDS) solution (10% SDS, 0.5 M Tris-HCl, pH 8.0, 0.1 M NaCl), and 0.7 g heat-sterilized zirconia-silica beads (0.1-mm diameter; Carl Roth, Karlsruhe, Germany) was added. Cells were lysed by shaking with a cell disruptor (FastPrep-24; MP Biomedicals, Ilkirch, Germany) for 45 s at a velocity of 6.5 m/s. Cell debris was sedimented by centrifugation at 20,000 × g for 4 min. The supernatant was extracted with 1 volume of phenol-chloroform-isoamyl alcohol (24:24:1 by volume; pH 8.0). After a second centrifugation step, the supernatant was extracted with 1 volume of chloroform-isoamyl alcohol (24:1 [vol/vol]) and centrifuged again in a 2-ml phase lock gel heavy tube (Eppendorf, Hamburg, Germany). The DNA was precipitated by mixing the aqueous phase with 2 volumes of polyethylene glycol (PEG) solution (30% PEG 6000 in 1.6 M NaCl). After centrifugation for 30 min, the pellet was washed with 500 μl ice-cold ethanol (70%) and dried under vacuum. DNA was dissolved in 50 μl elution buffer (MinElute PCR Purification Kit; Qiagen, Hilden, Germany), checked photometrically for purity (Nanodrop; PeqLab, Erlangen, Germany), quantified fluorimetrically (Qubit; Invitrogen, Eugene, OR), and stored at −20°C.

16S rRNA genes were amplified using either the archaeon-specific primer pair Ar109f (5′-AMDGCTCAGTAACACGT-3′) (25) and Ar912r (5′-CTCCCCCGCCAATTCCTTTA-3′) (35) or the archaeon-specific primer Ar109f and the prokaryote-specific primer 1490R with the modification of Hatamoto et al. (23) (5′-GGHTACCTTGTTACGACTT-3′), a combination that yields only archaeal 16S rRNA genes (43). Each PCR mixture (50 μl) contained reaction buffer, 2.5 mM MgCl₂, 1 U Taq DNA polymerase (all Invitrogen, Carlsbad, CA), 50 μM deoxynucleoside triphosphate mixture, 0.3 μM each primer, 0.8 mg/ml bovine serum albumin, and 1 μl DNA extract. The PCR program consisted of an initial denaturation step (94°C for 3 min) followed by 32 cycles of denaturation (94°C for 20 s), annealing (52°C for 20 s), and extension (72°C for 50 s) and a final extension step (72°C for 7 min). For the amplification of the mcrA gene, the primer pair mcrA-f (5′-GGTGGTGTMGGATTCACACARTAYGCWACAGC-3′) and mcrA-r (5′-TTCATTGCRTAGTTWGGRTAGTT-3′; 37) was used; the reaction mixture and the PCR protocol were the same as described above, except for the annealing temperature (53.5°C) and the cycle number (35) and a decreased ramp temperature rate of 1°C/s. The PCR products were purified and cloned as described by Schauer et al. (52).

The 16S rRNA gene sequences obtained in this study were imported into the current Silva database (version 106) (48; http://www.arb-silva.de) using the ARB software package (34). Sequences from other studies that were not included in Silva were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/). The sequences were automatically aligned, and the alignments were refined manually. A 30% consensus filter was used to exclude highly variable positions. Phylogenetic trees of almost full-length sequences (1,250 bp) were calculated using RAxML, a maximum-likelihood method (56). Tree topology and node support (100 bootstraps) were tested using the maximum-parsimony method (DNAPARS) implemented in ARB. The mcrA gene sequences were imported into a seed alignment complemented with sequences of unknown origin that were retrieved from the NCBI database. Trees were calculated at the amino acid level (140 amino acids) using PhyML, a maximum-likelihood method (21) implemented in ARB. Tree topology and node support (100 bootstraps) were tested using the maximum-parsimony method (PROTPARS) implemented in ARB.

Enrichment cultures were set up in anoxic, bicarbonate-buffered AM5 medium under an atmosphere of N₂-CO₂ (80:20 [vol/vol]) (4), but dithiothreitol (DTT) was omitted. The basal medium was supplemented with Casamino Acids (2 g/liter), coenzyme M (10 mg/liter), cysteine (2 mM), and palladium on activated charcoal (10 ml/liter) and (optionally) with yeast extract (2 g/liter) or rumen fluid (10%). The medium (4.5 ml) was dispensed into 15-ml rubber-stoppered glass vials, and hydrogen gas (5 ml) was added to the headspace. Substrates were added from sterile stock solutions (final concentrations): formate (50 mM), methanol (50 mM), acetate (30 mM), or xylan (9 g/liter). Tubes were inoculated (0.5 ml) with gut homogenates of C. ugandensis or Anadenobolus sp. prepared in basal medium (1 gut per ml), and the tubes were incubated at 30°C in the dark. The methane content in the headspace was measured every week. The culture headspace (0.2 ml) was sampled with a gas-tight syringe, and the methane content was analyzed using a gas chromatograph with a flame ionization detector (38).

DNA was extracted from the enrichment culture (2 ml) (see above), and the copy numbers of archaeal 16S rRNA genes were determined by quantitative real-time PCR (qPCR) as described by Kemnitz et al. (28) using the primers A364aF (5′-CGGGGYGCASCAGGCGCGAA-3′) (6) and A934b (5′-GTGCTCCCCCGCCAATTCCT-3′) (20). Bacterial 16S rRNA genes were quantified as described by Stubner (57) using the primer pair 519fc (5′-CAGCMGCCGCGGTAANWC-3′) and 907r (5′-CCGTCAATTCMTTTRAGTT-3′) (31). In addition, the bacterial community structure of the sample was determined by 454 pyrotag sequencing as described elsewhere (30).

The sequences obtained in this study were submitted to GenBank. The accession numbers are JX266062 to JX266091 for 16S rRNA genes and JX266092 to JX266145 for mcrA genes from hindgut homogenates. The accession numbers for the corresponding genes of strains MpT1 and MpM2 are JX266068, JX266097, JX648297, and JX648298.

Analysis of the archaeal 16S rRNA gene sequences from the hindguts of several higher termites revealed a diverse community of methanoarchaea, consisting of Methanobacteriales, Methanosarcinales, and Methanomicrobiales, although not all lineages were represented in each species (Table 1). In addition, each termite species yielded a substantial proportion of clones that clustered with a deep-branching lineage distantly related to Thermoplasmatales previously obtained from termite guts and other intestinal environments. A detailed analysis of the entire archaeal diversity in the different termite species will be published in a different context (J. O. Nonoh, K. Paul, D. K. Ngugi, and A. Brune, unpublished data).

Clone libraries of the mcrA genes amplified from the same samples not only yielded the mcrA genes expected of the methanogens identified in the rRNA-based analysis, but each contained an additional cluster of mcrA genes of unknown origin (Table 1). In each termite, the distribution patterns of the different clone groups were in agreement with the assumption that the novel mcrA genes stemmed from the uncultured lineage of Thermoplasmatales.

Since the 16S rRNA gene sequences obtained in the first data set were relatively short (800 bp), the phylogenetic resolution was not sufficient for the requirements of our study. Therefore, we also constructed smaller clone libraries with the primer pair Ar109f and Ar1490r for the termites C. ugandensis and Ophiotermes sp. and for the cockroaches S. esakii and P. angustipennis to obtain longer sequences (1,380 bp), together with those already present from previous studies, for all lineages of Thermoplasmatales-related archaea affiliated with termites and cockroaches. We included 16S rRNA gene sequences from all established lineages of Euryarchaeota from the Silva database and GenBank, including all sequences of uncultured Thermoplasmatales obtained in previous studies. The resulting phylogenetic trees showed the same major lineages of methanoarchaea previously documented by others, with the Thermoplasmatales and their uncultured relatives clearly falling within the radiation of methanogens, confirming the paraphyletic character of methanoarchaea as a taxonomic group (Fig. 1).

The sequences of Thermoplasmatales-related archaea obtained from termites and cockroaches fell into a distinct clade of clones obtained exclusively from intestinal environments that was clearly separated from previously published clades containing sequences from diverse marine and freshwater habitats. Next, relatives of this clade were clones previously obtained from an anaerobic digestor (Vadin Group) (18). Within the intestinal cluster, the sequences from insect guts formed two distinct lineages, each comprising both termite-specific and cockroach-specific lineages, with well-supported subclusters reflecting the phylogeny of their respective hosts. Other lineages in the intestinal cluster consisted of clones from vertebrate guts, which were previously obtained from the intestinal tracts of cattle, wallabies, chickens, and humans (see the introduction), and clones obtained from a manure pit.

To test the phylogenetic positions of the novel mcrA genes obtained in this study, we added the sequences to a comprehensive set of mcrA sequences from public databases, comprising all major lineages of methanogens and including all mcrA genes of uncertain origin from environmental studies. Phylogenetic analysis confirmed the presence of mcrA genes in insect guts belonging to representatives of the orders Methanosarcinales, Methanobacteriales, and Methanomicrobiales, which was in agreement with the results of the 16S rRNA analysis (Fig. 2). The clones of unknown origin obtained from termite guts (Table 1) and from the guts of the cockroaches S. esakii and P. angustipennis (this study) formed two distinct insect-specific lineages in a larger cluster of mcrA genes from intestinal habitats, including cows, wallabies, pigs, chickens, and humans. Also, the mcrA genes from the intestinal tracts of termites and cockroaches formed well-supported subclusters reflecting the phylogeny of their respective hosts.

As in case of the 16S rRNA gene sequences of Thermoplasmatales-related archaea, the novel mcrA genes from intestinal environments were most closely related to clones from an anaerobic digestor and clearly separated from other, previously published clades containing sequences from diverse marine and freshwater habitats, including additional sequences of intestinal origin.

A hindgut homogenate of C. ugandensis was inoculated into basal medium with or without yeast extract with optional additions of methanol, formate, or xylan and incubated under a headspace containing H₂ and CO₂. After a lag phase of several weeks, the culture containing methanol and yeast extract started to form CH₄. No methane formation was observed under any other conditions, even after 6 months of incubation, and also not if rumen fluid was added to the cultures. Subsequent transfers of the culture on the same medium led to robust CH₄ formation (up to 17-kPa headspace partial pressure); rumen fluid was not required. Transfers of the enrichment culture to medium lacking methanol showed no methanogenesis; transfers to medium containing methanol in the absence of H₂ produced much less methane than with H₂. No methanogenesis occurred with acetate as the sole substrate (Fig. 3).

After the initial transfers, the culture already consisted mostly of small, roundish cells (ca. 0.6 to 1 μm in diameter) (Fig. 4A). DNA was extracted from several subcultures, and the archaeal 16S rRNA genes were amplified using specific primers (Ar109f and 1490R). Each PCR product could be sequenced without cloning, and the sequencer traces indicated that in each case only a single phylotype of archaea was present. The sequences obtained from the different subcultures were identical. Phylogenetic analysis revealed that the archaeal 16S rRNA sequence (phylotype MpT1) fell into the apical cluster of putative methanogens consisting exclusively of clones from higher termites (Fig. 1, TC-1a). Also, the mcrA genes amplified from the same samples yielded identical sequences, which fell into the corresponding cluster of novel mcrA genes in the phylogenetic tree (Fig. 2, TC-1a).

When the abundance of archaeal 16S rRNA genes in the enrichment culture had increased to 64% (based on the total copy numbers of archaeal and bacterial 16S rRNA, determined by qPCR), the bacterial contaminants remaining in the enrichment culture were determined by 454 pyrotag sequencing. Classification of the bacterial sequences revealed that the bacteria remaining in the enrichment culture represented several lineages of so-far uncultivated Clostridiales (Fig. 5). All attempts to isolate strain MpT1 in pure culture have so far been unsuccessful.

Meanwhile, we also obtained a second methanogenic enrichment culture from the hindgut homogenate of a millipede (Anadenobolus sp.), using the same medium and enrichment strategy as for strain MpT1. The culture accumulated even larger amounts of methane (45 kPa in the headspace) than strain MpT1. Again, the PCR products obtained with specific primers for archaeal 16S rRNA genes and mcrA genes could be sequenced without cloning, which indicated that this enrichment culture was also dominated by a single strain of methanogens. Strain MpM2 had the same coccoid morphology as strain MpT1 but was slightly larger (Fig. 4B); both strains did not show the typical cofactor F₄₂₀ autofluorescence of many methanogens. Phylogenetic analysis showed that the 16S rRNA sequence of strain MpM2 also fell into the intestinal cluster of the novel methanogens within the radiation of sequences from termites and cockroaches (Fig. 1, TC-1 and CC-1). The mcrA gene of strain MpM2 clustered with the corresponding mcrA genes of the TC-1 subcluster (Fig. 2).

Previous studies of archaeal diversity in the hindguts of C. orthognathus (subfamily Termitinae) and Nasutitermes takasagoensis (subfamily Nasutitermitinae) had revealed the presence of four major lineages of Euryarchaeota in higher termites: Methanosarcinales, Methanomicrobiales, Methanobacteriales (for references, see reference 5), and a deep-branching clade distantly related to Thermoplasmatales (16, 42). Clones from the same lineages were also recovered from C. ugandensis and Ophiotermes sp., Trinervitermes sp., M. michaelseni, and A. trestus (this study), which indicated that representatives of the clade are consistently present in all subfamilies of higher termites. In addition, clones of this lineage were also obtained from the wood-feeding cockroaches S. esakii and P. angustipennis (22; this study), which are distantly related to termites.

Interestingly, the novel archaea from insect guts form two distinct lineages, each comprising clones from higher termites and wood-feeding cockroaches that seem to be specific for their respective hosts. The general absence of the group from lower termites is in agreement with previous studies reporting that the insects are exclusively colonized by members of the genus Methanobrevibacter (45); the single clone of Thermoplasmatales-related archaea obtained from Reticulitermes speratus (54) is affiliated with cluster TC-1b (Fig. 1).

The tree topologies of the 16S rRNA genes of novel archaea (Fig. 1) and the mcrA genes of unknown origin (Fig. 2) strongly resemble each other. A simplified tanglegram of the two trees illustrates that the phylogenetic positions of the major clusters of 16S rRNA and mcrA genes match perfectly (Fig. 6). This is true for all studies that reported both 16S rRNA and mcrA clones from the same environments: termite and cockroach guts (this study), wallaby gut (15), human gut (39, 40), and salt marsh sediment (7). In addition, other opposing clusters in the tree contain clones that originated from the same (e.g., the rumen) or related (i.e., from the guts and the manure of farm animals) habitats. Also the internal topologies of the respective groups are highly coincident (Fig. 1 and 2), which provides strong support for the idea that the sequence pairs from different animals originated from the same archaeal lineages. This is corroborated further by the similar clone frequencies of 16S rRNA and mcrA genes in the corresponding libraries of different termite gut species (Table 1), although the results are probably affected by differences in copy numbers of the 16S rRNA gene in Methanosarcinales and Methanobacteriales (1).

Further proof of the methanogenic nature of the new lineage came from the successful enrichment of strains MpT1 and MpM2, the only archaea present in the highly methanogenic enrichment cultures from termite and millipede guts. The 16S rRNA and mcrA gene sequences of both strains cluster with corresponding clones obtained from the guts of termites and cockroaches (Fig. 1 and 2, TC-1 and CC-1). They are part of the “intestinal cluster” of putative methanogens that also comprises clones from the rumen (RCC) (58) and the human gut (40). More distant relatives are found in anaerobic digestors (VADIN), rice field soil (RC-III), sediments, and other terrestrial environments (TMEG-1 and -2). Since matching mcrA genes were obtained from most of these habitats, it is safe to assume that all these lineages are methanogenic.

The final piece of evidence for the methanogenic nature of the new lineage was provided by the study of Dridi et al. (12), which was published during the revision stage of the present study. They isolated and described a new genus and species of methanogens, M. luminyensis, from human feces and reported that its 16S rRNA gene sequence was most closely related to several clones of uncultured Thermoplasmatales previously obtained from the digestive tracts of various mammals. They claimed that these clones and their isolate represent a new order of methanogens, but their phylogenetic analysis was superficial and comprised only a limited set of taxa. Our detailed phylogenetic analysis of both 16S rRNA and mcrA genes revealed that M. luminyensis is not a member of the vertebrate clones in the intestinal cluster (Fig. 1 and 2), which comprises most of the clones previously obtained from the digestive tracts of mammals. Instead, the isolate falls within the radiation of RC-III, where it clusters with clones from rice field soil (20, 8) and a single clone previously obtained from the human gut (39).

The methanogenic character of euryarchaeota in RC-III had been suggested earlier by Kemnitz et al. (28), who observed that the abundance of RC-III clones in a methanogenic enrichment culture from rice field soil was reduced by the addition of bromoethanesulfonate (BES), a specific inhibitor of methanogenesis. Considering the methanogenic character of M. luminyensis and the fact that mcrA sequences corresponding to RC-III have been obtained from rice paddies and other soils (Fig. 6), it is likely that all members of RC-III are methanogens.

The tanglegram (Fig. 6) shows that the most basal cluster in the new lineage of mcrA genes belongs to marine benthic group D, based on the matching positions of two sets of 16S rRNA and mcrA genes (ARC-7 and MCR-2) obtained from the same salt marsh samples (7). Although there are no mcrA genes matching the deeper-branching lineages, this may be due to the general lack of mcrA-based studies of methanogenic diversity, particularly in marine habitats. Therefore, it is not possible to predict whether the deeper-branching lineages are also methanogenic.

Interestingly, we observed a consistent and moderately supported sister group position of the novel mcrA genes and those of the ANME-1 group, an uncultivated lineage of methane-oxidizing archaea that may involve a methyl-CoM reductase in anaerobic methane oxidation (29). However, in view of the methanogenic properties of M. luminyensis (12) and our enrichment cultures and the cumulative evidence for the absence of methane oxidation in termite guts (47), a methanotrophic character of this novel lineage of archaea can be excluded.

In the highly enriched cultures of strains MpT1 and MpM2, methanogenesis was strongly stimulated by the simultaneous supply of both H₂ and methanol. The small amount of methane formation in the enrichment culture containing only methanol is most likely due to hydrogen formation by the clostridial members of the enrichment culture during fermentation of substrates stemming from yeast extract. Although a final statement on the substrate requirements will have to wait until these strains have been brought into pure culture, it seems that the metabolism of strains MpT1 and MpM2 (intestinal cluster) resembles that of M. luminyensis (RC-III) and obligately H₂-requiring methylotrophic methanogens from other lineages, like Methanosphaera stadtmanae (Methanobacteriales) and Methanomicrococcus blatticola (Methanosarcinales). Interestingly, such organisms have so far been isolated exclusively from the intestinal tracts of humans (12, 41) and cockroaches (55). It is likely that this mode of methanogenesis is an adaptation to the intestinal habitat.

In a study of the archaeal diversity in the hindgut of the termite N. takasagoensis, the relative abundance of uncultured Thermoplasmatales (Fig. 1, clusters TC-1a and TC-2) increased when the animals were fed with xylan (42), a substrate that contains substantial amounts of O-methylated glucuronic acid residues (49). It is possible that the apparent enrichment of these methanogens was an indirect effect caused by methanol formation during the fermentative breakdown of xylan.

Although it has been repeatedly proposed that environmental clones distantly affiliated with the Thermoplasmatales represent a separate order of methanogens (15, 39), none of these studies provided enough evidence to substantiate this claim. Analysis of the entire set of sequences available to date clearly documents the diversity of the new lineage, including numerous habitat-specific clades and its sister group relationship to the Thermoplasmatales, and provides robust evidence for the presence of mcrA genes in all members. The methanogenic nature of the lineage is further corroborated by the isolation of M. luminyensis (12) and the enrichment cultures of strains MpT1 and MpM2 (this study). Based on this evidence, we propose the provisional name “Methanoplasmatales” for the entire deep-branching lineage of euryarchaeota outlined in Fig. 6. Although a first representative of the lineage has been isolated and described (12), we suggest postponing a formal description of any higher taxa until further representatives have been obtained in culture, their cell envelopes have been characterized, and the presence of mcrA genes in the basal lineages (particularly the marine groups) has been assessed.

Considering the apparently obligate hydrogen dependence of methanol reduction both in M. luminyensis and in the enrichment cultures, it may be promising to also use such combinations of methanogenic substrates for enrichments from other habitats. There are several other deep-branching lineages of euryarchaeota that may also be methanogenic, and even more diversity may be present because of a bias of commonly used PCR primers against hitherto undetected lineages (60). This is underlined by two studies of archaeal diversity in termite guts that had failed to detect clones affiliated with “Methanoplasmatales.” In one case (46), this was most likely due to a mismatch in the reverse primer to the consensus sequence of “Methanoplasmatales,” whereas in the other case (11), the sequence of the forward primer differed slightly from that of the forward primer successfully used by Hara et al. (22).

It is striking that the majority of the mcrA genes of the “Methanoplasmatales” have so far been retrieved only from intestinal samples. The fact that there are only a few clones from other environments may simply be due to the lack of such studies, particularly in marine environments. More cultivation efforts are required to expand our knowledge about this novel group of methanogens, not least to investigate their metabolic relationship to Thermoplasmatales, a clade of Euryarchaeota that may have experienced a secondary loss of the capacity for methanogenesis (3).