Microbes Environ. Vol. 19, No. 3, 221–226, 2004 http://wwwsoc.nii.ac.jp/jsme2/

Isolation and Detection of from the Gut of Higher

PINSURANG DEEVONG1,2, SATOSHI HATTORI1, AKINORI YAMADA1,3, SAVITR TRAKULNALEAMSAI2, MORIYA OHKUMA1,4*, NAPAVARN NOPARATNARAPORN2 and TOSHIAKI KUDO1,5

1 Environmental Molecular Biology Laboratory, RIKEN, Wako, Saitama 351–0198, Japan 2 Department of Microbiology, Kasetsart University, Bangkok 10900, Thailand 3 Center for Ecological Research, Kyoto University, Kamitakanami-Hiranocho, Otsu 520–2113, Japan 4 PRESTO, Japan Science and Technology Agency, Wako, Saitama 351–0198, Japan 5 Graduate School of Integrated Science, Yokohama City University, Tsurumi, Yokohama 230–0045, Japan

(Received June 24, 2004—Accepted July 1, 2004)

Four strains of hydrogenotrophic methanogens showing narrow ranges of utilizable substrates were isolated in pure cultures from the guts of various feeding groups of (phylogenetically) higher termites. An analysis of the 16S rRNA gene sequence revealed that three strains were closely related to Methanobacterium bryantii (99% nucleotide identity), the other to the . The latter was related to a clone identified previ- ously from the gut of a higher without cultivation (clone MPn19) and Methanobrevibacter arboriphilicus (99.0 and 97.9% nucleotide identity, respectively) but distinct from the identified in lower termites. The specific detection of related methanogens in the gut population by nested-PCR indicated that every termite harbored the species of Methanobrevibacter. However, the related to the Methanobacterium strain was not detected in two termite species from which the Methanobacterium strains were isolated, suggesting that they are less prevalent in the gut community.

Key words: Methanobrevibacter, Methanobacterium, symbiosis, termite

Termites harbor methanogenic (methanogens) in studied fully. Three species of Methanobrevibacter have their guts and are one of the few terrestrial arthropods that been isolated from a lower termite, Reticulitermes emit methane6). Methane emission from termites has often flavipes12,13). To our knowledge, however, there is no other been debated to be a significant source of global atmospher- publication on the isolation of methanogens from the ter- ic methane, since termites are abundant in terrestrial ecosys- mite gut. Methanogen diversity in the termite gut has been tems particularly in tropical regions3,20). Termites are gener- investigated using culture-independent molecular analyses ally divided into two, so-called lower and higher termites. based on the 16S rRNA gene. Earlier molecular analyses The higher termites (Termitidae) exist in a great biomass used the lower termite Reticulitermes speratus15,18). Howev- density in tropical regions and show considerable variation er, our current knowledge of gut methanogens has expanded in their feeding behavior, which is not limited to wood-feed- to cover phylogenetically diverse species of termite15,18) and ing. Some feed exclusively on soil while others cultivate in relation to their localization in the gut5,22). Methanogens and consume fungi. Nevertheless, all known termites have a belonging to Methanobrevibacter are found in both lower dense and diverse gut microbial community that aids in and higher termites, whereas higher termites often harbor digestion14). more diverse methanogens. It is noted that the methanogen Methanogens in the termite gut, though often cited as im- sequences reported so far from the termite gut form unique portant components of the gut community, have yet to be phylogenetic clusters. Methanogen 16S rRNA gene sequences from the guts of xylophagous cockroaches 7) * Corresponding author; E-mail: [email protected], Tel: 81–48– have also been assigned to these clusters . 467–9648; Fax: 81–48–462–4672 Here, we isolated and cultivated methanogen strains be- 222 DEEVONG et al. longing to the genera Methanobrevibacter and Methanobac- 3'), Ar1000R (5'-TCTCGCTCGTTGCCTGACT-3') and terium from various feeding groups of higher termites Ar1000F (5'-AGTCAGGCAACGAGCGAGA-3'). DNA se- in Thailand. The presence of related methanogens in the quencing was performed with the ABI PRISM BigDye Ter- termite gut was examined by the diagnostic nested-PCR minator Cycle Sequencing Ready Reaction kit (Applied approach. Biosystems Japan, Tokyo) and with an ABI model 3700 au- tomatic sequence analyzer. The sequence data were com- Materials and Methods pared with 16S rRNA gene sequences in the databases. Se- quences were aligned using the CLUSTAL X package Termites version 1.8323), then checked manually. Nucleotide posi- Termites used here were the wood-feeding termite tions of ambiguous alignments were omitted from the sub- Microcerotermes crassus (Termitinae), a soil-feeding sequent phylogenetic analysis. Construction of a neighbor- termite Pericapritermes sp. (Termitinae), the soil/wood joining tree and a bootstrap analysis (with 1,000 resam- interface feeding termite Termes comis (Termitinae), and plings) for evaluation of tree topology were carried by the fungus-growing termite Macrotermes gilvus (Macro- MEGA version 2.111). termitinae). The former species was collected in Nakhon Ratchasima, Thailand, whereas the latter three were collected Nested-PCR in Pathum Thani, Thailand. Only the worker castes of each The mixed-population DNA of the termite gut communi- termite species were used. ty was extracted as described previously10). The methanogen 16S rRNA gene was first amplified from the extracted DNA Media and isolation using PCR primers M23FB and M1382R. The PCR product The anaerobic medium described by Hattori et al.8) was was used as a template for the subsequent nested-PCR. The used with slight modifications. Dithiothreitol (2 mM) was PCR primers used for the nested-PCR were either used as a reducing agent instead of cystein and sodium dis- MBB157F and MBB722R or MB183F and MB722R. The ulfide. The medium was supplemented with acetate (2 mM) target nucleotide sequences of the primers are shown in Fig. and co-enzyme M (10 M). Methanogens were grown 2. The nested-PCR with the MBB primers was performed on H2/CO2 (80:20 v/v; 100 kPa) at 30LC. The guts were after 1 min denaturation at 94LC, for 30 cycles at 94LC for dissected and squeezed under a stream of nitrogen gas and 30 sec, 68LC for 45 sec, and 72LC for 2 min, plus a final inoculated into the medium. The enrichment was repeated elongation step of 10 min at 72LC. For the nested-PCR with by inoculating a serial dilution of the first culture. MB primers, 64LC instead of 68LC was used. The Then, methanogens were purified by successive anaerobic nested-PCR products were purified and cloned into pGEM-T colony formations with roll tubes. The cells of methanogens vector (Promega), and nine to sixteen clones in each of the were detected by epi-fluorescence microscopy of F420. The nested-PCR products were sequenced with a T7 primer concentration of each of substrate used to test the growth (Promega). of the methaonogen isolates was 10 mM except 40 mM for formate. Nucleotide sequence data The nucleotide sequence data determined here will ap- 16S rRNA gene amplification and sequencing pear in the DDBJ database under accession numbers The cells of methanogens in the pure cultures were har- AB181816–AB181819 for the isolated strains and vested by centrifugation and DNA was extracted with an AB181820–AB181832 for the representative clones of the ISOPLANT II kit (Nippon Gene, Tokyo, Japan). The nested-PCR products. methanogen 16S rRNA gene was amplified using PCR primers M23FB and M1382R15,22). The PCR was performed, Results after 1 min denaturation at 94LC, with 30 cycles at 94LC for 30 sec, 52LC for 45 sec, and 72LC for 2 min, plus a final The enrichment of methanogens was achieved in each of elongation step of 10 min at 72LC. The PCR product was the four termite cases when using H2/CO2 as a substrate. purified with a Wizard PCR Prep DNA Purification kit Four strains Mc30, Ps21, Tc3, and Mg38 were isolated in (Promega, Madison, USA) and used for direct sequencing. pure cultures from M. crassus, Pericapritermes sp., T. co- Sequencing primers used were M520R (5'-TACCGCG- mis, and M. gilvus, respectively. The cells of strain Mc30 GCGGCTGGC-3'), 800F (5'-ATTAGATACCCGGGTAG- were coccobacilli and those of the other three strains were Methanogens from Higher Termites 223

Fig. 1. Phylogenetic positions of strains isolated from the guts of higher termites based on the 16S rRNA gene sequence. The database accession numbers in DDBJ and Ribosomal RNA Database Project of the reference sequences are shown in parentheses. The databases’ sequences with less than 1,000 bp were excluded from the inference. Methanothermobacter thermautotrophicum and Methanothermus fervidus were used as outgroups. Bootstrap values above 50% are shown for each node. The scale bar represents 0.02 substitutions per nucleotide position. Taxa that shared the target sequences of the primers MBB157F and MB183F are labeled with a closed circle and a closed square, respectively. Note that the sequence information for the regions corresponding to these primers is not available in the clones from the termite gut. Taxa that share the target sequences of primers MBB722R and MB722R are labeled with an open circle and an open square, respectively.

rods. The purity of isolates was checked by microscopy and MPn1916) (99.0% nucleotide identity), a clone TS1 of an by the absence of growth in a medium without growth sup- endosymbiont from a ciliate Trimyema compressum portive substrates for methanogens (i.e. using N2 instead of (published only in the database; 98.3% nucleotide identity), H2) or in a nutrient broth (L-broth). We confirmed the purity and two strains of Methanobrevibacter arboriphilicus by analyzing 16S rRNA gene sequences of thirty colonies (each with 97.9% nucleotide identity). A clone from a from a single pure culture, obtaining the identical sequence soil-feeding termite Cubitermes orthognathus, P3-Ar-125) in each case. (AF293559, ca. 790 bp overlap), was also related to strain The phylogenetic analysis of the isolated strains (Fig. 1) Mc30 (98.6% nucleotide identity), although this sequence clearly indicated that strain Mc30 belonged to the genus was not included in the phylogenetic analysis shown in Fig. Methanobrevibacter. The closest relatives of strain Mc30 1 due to its short length. Strain Mc30 was distantly related were a clone from the termite Pericapritermes nitobei, to three species of lower termite, M. cuticularis, M. curva- 224 DEEVONG et al.

Fig. 2. Alignment of 16S rRNA gene sequences of the regions corresponding to the primers for nested-PCR. Bases identical to the sequence of strain Mc30 are shown by dots. The target sequences of the primers are also shown. The numbers of the primers correspond to the position of the Methanobacterium bryantii sequence. MBB, Methanobrevibacter and MB, Methanobacterium.

tus, and M. filiformis as well as the clones from lower ter- The forward primer for strain Mc30 (MBB157F) shared the mites (less than 97.2% nucleotide identity). target sequence with M. cuticularis, but not with the other Three strains Ps21, Tc3, and Mg38 were closely related members of . The identity of the target to each other showing more than 99.7% nucleotide identity. sequence in the clones from termites including clone These strains were clearly assigned to the genus MPn19 was unclear because the sequence corresponding to Methanobacterium, and closely related to M. bryantii and this region was not available. The forward primer for strains M. ivanovii (more than 97.7% nucleotide identity). Ps21, Tc3, and Mg38 (MB183F) shared the target sequence Physiological properties were characterized in the repre- with M. bryantii RiH2 and M. uliginosum Kf1-F1 but not sentative strains Mc30 and Ps21. Strain Mc30 utilized H2/ with the other Methaobacteriaceae members. The nested- CO2 or formate for growth but not 2-propanol and 2-bu- PCR specifically detected the related population as shown tanol. Strain Ps21 utilized H2/CO2, 2-propanol, or 2-butanol in the control amplifications of the isolated strains (Fig. 3, for growth but not formate. Both strains were unable to uti- lanes 1–6). The Methanobrevibacter species related to lize methanol, ethanol, trimethylamine, pyruvate, and glu- strain Mc30 was detected more or less in every four termite cose for growth. The temperature range for growth of strain species. The Methanobacterium species related to strains Mc30 was 20–40LC with optimum growth at 37LC and a Ps21, Tc3, and Mg38 was detected in M. crassus but not in specific growth rate of 4.3P103 h1. That of strain Ps21 was Pericapritermes sp. and M. gilvus. In the case of T. comis, 15–42LC with the optimum at 30LC and a specific growth the amplification was weak but significant. rate of 6.5P103 h1. The pH range for the growth of strain In order to confirm the amplification of related sequenc- Mc30 was 5.5 to 8.4 (optimum at 7.0), while that of strain es, the nested-PCR product was cloned and several clones Ps21 was 5.5 to 8.5 (optimum at 7.4). Both strains grew were analyzed as to their DNA sequences in each of the am- with 2% NaCl and with 3% KCl in the medium. plifications. In the case of the nested-PCR for the Methano- The presence of species related to the strains isolated in brevibacter-related species, clones showing 97.5–99.2% the gut community was investigated by diagnostic nested- nucleotide identity to the sequence of strain Mc30 were PCR approaches. Specific primers were designed for the identified from each of the nested-PCR. Each of the nested- nested-PCR (Fig. 2). The forward primer recognized the tar- PCR products for Methanobrevibacter also contained a get more specifically than the reverse primer in each case. clone group that showed more than 97.2% nucleotide identi- Methanogens from Higher Termites 225

the narrow spectrum of utilizable substrates support their assignment to these two genera9). The hydrogenotrophic character of the isolates suggests an advantage in the gut en- vironment, since the gut shows high levels of hydrogen pro- duction. The acetate produced by fermentation is known to accumulate in the gut, and methyl-groups contained in lig- nin are also considered to be abundant in the gut. However, the isolates were unable to utilize these substrates for their growth. Moreover, we tried to cultivate methanogens using acetate, methanol, and methylamine as growth substrates but could not obtain an enriched culture of methanogens. Extreme alkalinity of the gut is frequently observed in higher termites1). In fact, some alkaliphilic bacteria related to Bacillus and Paenibacillus have been identified in the gut of higher termites17,21). The alkaline gut contents are also known to be rich in K rather than Na and many alkali- philes isolated from the termite gut show a preference for K for their growth17,21). The methanogen isolates, however, showed neither an alkaliphilic feature nor a strong prefer- ence for K. Although the localization of methanogens in the gut compartments needs to be determined, the posterior Fig. 3. Diagnostic nested-PCR for the detection of Methanobrevi- bacter-related (A) and Methanobacterium-related (B) methano- portions of the gut do not have a high pH and Methanobac- gen species. Lanes 1 and 2 are plasmid DNA of clones, lanes 3–6 teriaceae are shown to be abundant in these areas at least in are genomic DNA of strains isolated in this study, and lanes 7–10 the case of C. orthognathus5). are DNA extracted from the gut community of termites. Lane 1, Quantitative hybridization with family-level probes tar- clone MNt116) belonging to Methanomicrobiales; lane 2, clone geting the 16S rRNA of methanogens revealed that mem- MPn416) belonging to Methanosarcinaceae; lane 3, strain Mc30; lane 4, strain Mg38; lane 5, strain Ps21; lane 6, strain Tc3, lane 7, bers of Methanobacteriaceae are most abundant in the gut Microcerotermes crassus lane 8, Macrotermes gilvus; lane 9, community in many termites2). Culture-independent clone Pericapritermes sp.; lane 10, Termes comis. Lane M is molecular analyses of PCR-amplified methanogen 16S rRNA genes   size standards ( /HindIII digest plus X174/HaeIII digest [Toyo- from the gut community also indicated the presence of bo]). The sizes of the standards from top to bottom are 23130, 9416, 6557, 4361, 2322, 2027, 1353, 1078, 872, 603 and 310 bps. Methanobrevibacter in the soil-feeding termites P. nitobei and C. orthognathus5,16). One of the clone groups from these termites was closely related to strain Mc30. The common ty to each other and 96.8–94.7% nucleotide identity to the presence of species related to strain Mc30 was confirmed sequence of strain Mc30. They were closely related to the here in four higher termite species by the nested-PCR ap- clone from the termite C. orthognathus, P3-Ar-15) proach. Methanobrevibacter species related to strain Mc30 (AF293556; 97.5–98.9% nucleotide identity). All the clones are probably common in the gut of higher termites. The of the nested-PCR for the Methanobacterium-related spe- presence of Methanobrevibacter members is also common cies in M. crassus and T. comis showed more than 99.2% in lower termites. However, the constituents of Methano- nucleotide identity to the sequence of strain Ps21. brevibacter differ between lower and higher termites at least at the species level. In contrast to Methanobrevibacter, the presence of mem- Discussion bers of the genus Methanobacterium in the termite guts has We successfully isolated methanogens in pure cultures not been reported so far. Here, three strains of Methanobac- from the termite gut. To our knowledge, this is the first pa- terium were isolated from three termite species. Nested- per describing the isolation of methanogens from higher ter- PCR further showed their presence in M. crassus. Thus, the mites. Based on a 16S rRNA gene analysis, the methano- Methanobacterium strains are common to higher termites. gens were found to belong to either Methanobrevibacter or Methanobacterium strains were successfully isolated from Methanobacterium. Their H2/CO2-utilizing character and M. gilvus and Pericapritermes sp., although related species 226 DEEVONG et al. could not be detected by the nested-PCR in these two ter- 4: 20. 5) Friedrich, M.W., D. Schmitt-Wagner, T. Lueders and A. Brune. mites. The result suggests that these methanogens exist as 2001. Axial differences in community structure of Crenarchaeota relatively minor populations, since the nested-PCR is gener- and in the highly compartmentalized gut of the ally considered to be highly sensitive. Probably, the cultiva- soil-feeding termite Cubitermes orthognathus. Appl. Environ. Microbiol. 67: 4880–4890. tion was very sensitive but caused significant bias. 6) Hackstein, J.H.P. and C.K. Stumm. 1994. Methane production in Leadbetter and Breznak12) and Brauman et al.2) mention terrestrial arthropods. Proc. Natl. Acad. Sci. USA 91: 5441–5445. 7) Hara, K., N. Shinzato, M. Seo, T. Oshima and A. Yamagishi. that Yang et al. reported the isolation of M. arboriphilicus 2002. Phylogenetic analysis of symbiotic archaea living in the gut and M. bryantii in the guts of two Nasutitermes termite spe- of xylophagous cockroaches. Microbes Environ. 17: 185–190. cies (Termitidae, Nasutitermitinae), referring to the abstract 8) Hattori, S., Y. Kamagata, S. Hanada and H. Shoun. 2000. Ther- macetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, of an annual meeting. Lacking their detailed information, thermophilic, syntrophic acetate-oxidizing bacterium. Int. J. Syst. we could not compare them with the strains isolated in Evol. Microbiol. 50: 1601–1609. 9) Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley and S.T. Will- this study. However, this may also support the presence of iams. 1994. Bergey’s manual of determinative bacteriology, 9th related methanogens in the guts of higher termites. ed., pp. 719–736. The Williams & Wilkins Co., Baltimore. The 16S rRNA gene sequence showing less than 98% nu- 10) Hongoh Y., M. Ohkuma and T. Kudo. 2003. Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes cleotide identity can be considered a separate species in the speratus (Isoptera; Rhinotermitidae). FEMS Microbiol. Ecol. 44: genus Methanobrevibacter4), suggesting that strain Mc30 231–242. 11) Kumar, S., K. Tamura, I.B. Jakobson and M. Nei. 2001. MEGA2: represents a novel species of this genus. In addition to Molecular Evolutionary Genetics Analysis software. Bioinfor- Methanobrevibacter, previous culture-independent analyses matics 17: 1244–1245. revealed that methanogens of Methanosarcinaceae and 12) Leadbetter, J.R. and J.A. Breznak. 1996. Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter Methanomicrobiales are present in the gut of higher curvatus sp. nov., isolated from the hindgut of the termite Reticu- termites5,16), but the cultivation of them has not been accom- litermes flavipes. Appl. Environ. Microbiol. 62: 3620–3631. 13) Leadbetter, J.R., L.D. Crosby and J.A. Breznak. 1998. Methano- plished yet. Although the characterization of these as-yet- brevibacter filiformis sp. nov., a filamentous methanogen from uncharacterized methanogens and quantitative analyses of termite hindguts. Arch. Microbiol. 169: 287–292. their composition are necessary, this study contributes to 14) Ohkuma, M. 2003. Termite symbiotic systems: efficient bio-recy- cling of lignocellulose. App. Microbiol. Biotechnol. 61: 1–9. the understanding of methanogen diversity in the gut of 15) Ohkuma, M., S. Noda, K. Horikoshi and T. Kudo. 1995. Phyloge- termites. ny of symbiotic methanogens in the gut of the termite Reticuliter- mes speratus. FEMS Microbiol. Lett. 134: 45–50. 16) Ohkuma, M., S. Noda and T. Kudo. 1999. Phylogenetic relation- Acknowledgments ships of symbiotic methanogens in diverse termites. FEMS Mi- crobiol. Lett. 171: 147–153. This work was partially supported by grants for the Bio- 17) Ohkuma, M., H. Simizu, T. Thongaram, S. Kosono, K. Moriya, S. Trakulnaleamsai, N. Noparatnaraporn and T. Kudo. 2003. An architect Research Program and the Eco Molecular Science alkaliphilic and xylanolytic Paenibacillus isolated from the gut of Research Program from RIKEN. P. D., S. H. and A. Y. are a soil-feeding termite. Microbes Environ. 18: 145–151. 18) Shinzato, N., T. Matsumoto, I. Yamaoka, T. Oshima and recipients of the Asian Program Associate, the Special A. Yamagishi. 1999. Phylogenetic diversity of symbiotic Postdoctoral Fellowship and the Junior Research Associate methanogens living in the hindgut of the lower termite Program of RIKEN, respectively. We thank T. Inoue, Y. Reticulitermes speratus analyzed by PCR and in situ hybridization. Appl. Environ. Microbiol. 65: 837–840. Hongoh and T. Johjima for termite collection and valuable 19) Shinzato, N., T. Matsumoto, I. Yamaoka, T. Oshima and A. comments. Yamagishi. 2001. Methanogenic symbionts and the locality of their host lower termites. Microbes Environ. 16: 43–47. 20) Sugimoto, A., T. Inoue, I. Tayasu, L. Miller, S. Takeichi and T. Abe. 1998. Methane and hydrogen production in a termite-sym- References biont system. Ecol. Res. 13: 241–257. 21) Thongaram, T., S. Kosono, M. Ohkuma, Y. Hongoh, M. Kitada, 1) Bignell, D.E. and P. Eggleton. 1995. On the elevated intestinal 42 T. Yoshinaka, S. Trakulnaleamsai, N. Noparatnaraporn and T. pH of higher termites (Isoptera: Termitidae). Insectes Soc. : Kudo. 2003. Isolation and characterization of alkaliphiles from 57–69. the gut of higher termites in Thailand: a niche in the termite gut 2) Brauman, A., D.J. Eggleton, D.E. Bignell, J.A. Breznak and M.D. and its unexpected salt preference. Microbes Environ. 18: 152– Kane. 2001. Molecular phylogenetic profiling of prokaryotic 159. communities in guts of termites with different feeding habits. 22) Tokura, M., M. Ohkuma and T. Kudo. 2000. Molecular phyloge- FEMS Microbiol. Ecol. 35: 27–36. ny of methanogens associated with flagellated protists in the gut 3) Brauman, A., M.D. Kane, M. Labat and J.A. Breznak. 1992. and with the gut epithelium of termites. FEMS Microbiol. Ecol. Genesis of acetate and methane by gut bacteria of nutritionally 33: 233–240. diverse termites. Science 257: 1384–1387. 23) Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin and 4) Dighe, A.S., K. Jangid, J.M. Gonzalez, V.J. Pidiyar, M.S. Patole, D.G. Higgins. 1997. The CLUSTAL_X windows interface: flexi- D.R. Ranade and Y.S. Shouche. 2004. Comparison of 16S rRNA ble strategies for multiple sequence alignment aided by quality gene sequences of genus Methanobrevibacter. BMC Microbiol. analysis tools. Nucleic Acids Res. 25: 4876–4882.