Correspondence: Hiroyuki Imachi, Subsurface Geobiology Advanced Research (SUGAR) Team, Extremobiosphere Research Program, Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan. Tel.: +81 46 867 9709; fax: +81 46 867 9715; e-mail: firstname.lastname@example.org
Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.
The anaerobic oxidation of methane (AOM) process is one of the major sinks for methane on earth and is driven by uncultured archaeal anaerobic methanotrophs (ANMEs) (see references in a review by Krüger & Treude, 2005). The reaction has been widely identified in anaerobic marine (sulphate-rich) sediments and thus most of the previous studies on AOM have focused on marine ecosystems. Other than the marine ecosystems, recently, Eller et al. (2005) and Alain et al. (2006) have demonstrated that the previously uncultured archaeal components phylogenetically related to the marine ANMEs are also distributed in terrestrial environments (i.e. freshwater lake and terrestrial mud volcanoes, respectively) and are potentially associated with the in situ AOM activity. These findings suggest that the ANMEs might be widely distributed not only in marine sediments but also in terrestrial anoxic environments and play a more important role in the global carbon cycle than previously expected. Thus, the detection and quantification protocols of the phylogenetically diverse ANMEs in various environments are to be established. Here, we design specific primers targeting 16S rRNA genes of different phylogenetic groups of ANMEs. Using the newly designed primers, a quantitative PCR method is also established. Using these primers and a quantitative PCR technique, the detection and quantification of ANMEs are tested in methane-rich but sulphate-limited environmental samples such as methanogenic sludge, rice field soil and a natural gas field.
Materials and methods
PCR primer design
For the construction of each ANME group-specific primer, we collected all sequences affiliated with the ANMEs from the public database, and designed specific primers based on the aligned sequences using the probe design tool of the arb program (Ludwig et al., 2004). The specificity of new primers was confirmed using blast (Altschul et al., 1997) and the ARB-SILVA database (http://www.arb-silva.de). The primers used in this study are shown in Table 1.
Table 1. 16S rRNA gene-targeted PCR primers used in this study
Sequence (5′ to 3′)
E. coli position
These primers consist of a mixture of each taxonomic group targeted primers at an equal amount (mol).
The primer does not cover the GBHS clone group within the ANME-1 (see also Fig. 1).
A total of 12 environmental samples were collected and analysed in this study (Table 2). All the samples examined were from methanogenic environments. The gas field sediment and formation water samples were obtained from sand separators in commercial gas-producing wells. The samples MOB4 and MOB7 were obtained from the same gas–water production well as described in Mochimaru et al. (2007) and at the same time. The chemical properties of the gas field samples MOB4 and MOB7 were described in a previous report (Mochimaru et al., 2007). The rice field soils and lotus field sediment were collected from a depth of 10–20 cm. Methanogenic sludges were taken from full-scale reactors. The temperature, pH, oxidation-reduction potential (ORP), dissolved oxygen (DO) and conductivity of the samples were measured using a multisensor system (W-23XD, Horiba) in the fields.
Table 2. Environmental samples analyzed in this study
DNA extraction, PCR, cloning and phylogenetic analysis
Extraction of DNA from environmental samples was performed using an ISOL for Beads Beating kit (Nippon Gene) according to the manufacturer's instructions. PCR amplification was performed using the TaKaRa Ex Taq (TaKaRa Bio Inc.), and the reaction mixtures for PCR were prepared according to the manufacturer's instruction including c. 5 ng of template DNA in a 25-μL PCR mixture. The concentration of template DNA was measured using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and a spectrofluorophotometer (LS55, Perkin Elmer). The PCR conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s and extension at 72 °C for 1 min. The annealing temperature using the ANME-specific primers was empirically optimized using the clones of each taxonomic ANME group described below. When using the ANME-specific primers in combination with Ar109f and 1492R primers (Table 1), the annealing temperature was set at 52 °C. The amplification of archaeal 16S rRNA genes with the archaeal universal primers Ar109f/Ar912r (Table 1) was performed concurrently as the positive control. As additional positive controls, the representative 16S rRNA gene clone of each ANME group, except for the Guaymas Basin hydrothermal sediment (GBHS) clone group within the ANME-1, was used. The clones for the ANME-1, -2a, -2b, -2c and -3 were obtained from methane seep sediments of the Nankai Trough stored in our laboratory. The PCR products were checked on a 1.5% agarose gel using an ethidium bromide stain, then purified using a MinElute PCR purification kit (Qiagen) and subsequently cloned into Escherichia coli using a TOPO TA cloning kit (Invitrogen). Ten clones were randomly picked from each clone library for sequencing. The 16S rRNA gene sequences were determined using a BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems) and an automated sequence analyzer (3130xl Genetic Analyzer, Applied Biosystems). The phylogenetic analysis of the 16S rRNA gene sequence obtained was performed as described previously (Imachi et al., 2006).
Real-time PCR quantification
Quantitative PCR was performed with a 7500 Real-Time PCR System (Applied Biosystems) using the SYBR Premix Ex Taq II Perfect Real Time (TaKaRa Bio Inc.). A reaction mixture for PCR was prepared according to the manufacturer's instructions, including c. 0.1 or 1 ng of template DNA. The 16S rRNA gene-targeted primer sets used in this study are shown in Table 3. For the construction of template standards for each primer set, we used a dilution series of 16S rRNA gene PCR products of ANME-1 and -2c clones, and E. coli, which were obtained with the archaeal primer set of Arch21F/1492R or the bacterial primer set of 8F/1492R. These PCR products were used in every real-time PCR analysis to calculate the number of 16S rRNA genes. Template DNA was quantified spectrofluorometrically using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). The optimal PCR conditions including the annealing temperatures were empirically determined for each primer set (Table 3). The quantification limit was determined as the lowest copy number of the dilution series of 16S rRNA gene PCR products, which could yield a linear correlation coefficient (R2) of >0.99 in the standard curve. Because the ANME2c-1411R primers had a single mismatch with the sequences of Methanogenium organophilum (DSM 3596) and Methanomicrobium mobile (DSM 1539), we also examined the specificity using the genomic DNA extracted from the reference microorganisms. We confirmed that the designed primer sets provided no positive amplification of the 16S rRNA genes from all the genomic DNA of reference microorganisms as compared with the negative control (i.e. no additional template). The PCR conditions were as follows: initial denaturation at 95 °C for 10 s, followed by 40 cycles of denaturation at 95 °C for 5 s, 30 s of annealing (temperatures are shown in Table 3) and extension at 72 °C (the times are shown in Table 3). Two types of experiments were performed to check the specificity of the real-time PCR assays. First, the melting curve analysis (60–90 °C) was performed after each amplification step. Second, the PCR products were confirmed by gel electrophoresis and subsequent clone library analysis.
Table 3. 16S rRNA gene-targeted primers used for quantitative real-time PCR
Numbers in parentheses represents GenBank accession numbers or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) culture collection number.
The 16S rRNA gene sequences reported in this study have been deposited in the GenBank/EMBL/DDBJ database under accession no. AB461386–AB461393. The clones used as the positive control of the PCR amplifications are AB461389–AB461393.
Results and discussion
Previous studies have indicated that the ANMEs were classified into three 16S rRNA gene-based phylogenetic groups called the ANME-1, ANME-2 and ANME-3 (see references in a review by Krüger & Treude, 2005). Several primers and probes targeting 16S rRNA gene sequences of the ANMEs have been developed (Boetius et al., 2000; Girguis et al., 2003; Knittel et al., 2005; Niemann et al., 2006). Therefore, we evaluated the specificity and coverage of these published primers using the match probes tool of the arb program. Among them, the ANME3-1249 primer for the ANME-3 (Niemann et al., 2006) could cover almost all sequences of ANME-3 and had specificity for the target group. The AR468f primer for the ANME-2c (Girguis et al., 2003) could cover almost all sequences of the target group, but the primer was not strictly specific to the ANME-2c. The primer sequence also matches with three sequences belonging to the ANME-2a (Supporting Information, Fig. S1). We attempted to design alternative primers of AR468f for the ANME-2c, but it was still difficult to design a primer perfectly specific to the ANME-2c sequences. Consequently, the primers AR468f and ANME3-1249 were used for the specific detection of ANME-2c and ANME-3 in this study, respectively (Table 1). In addition, the other primers and probes (i.e. ANME-1-350, EelMS932, ANME-2a-647, ANME-2c-622, ANME-2c-760 and AR736r) could not fully cover or were not sharply specific to the sequences of many ANMEs deposited in the public database (Fig. S1). Therefore, new primers were designed based on ANMEs 16S rRNA gene sequences (Table 1, Fig. S2). For the ANME-1 and -2 groups, we designed for each subgroup of ANME-1 (GBHS group and the others) and ANME-2 (subgroup a, b and c), due to the difficulty in finding primers fully covering all the sequences of the whole ANME-1 and ANME-2 groups, respectively.
The newly designed primers were applied to the PCR-based molecular survey of 12 environmental samples (Table 2). Previous studies reported that potential AOM activity was observed in methanogenic sludge and rice field soil (Zehnder & Brock, 1980; Murase & Kimura, 1994). Thus, we collected four methanogenic sludges and three rice field soil samples. In this survey, we also used Ar109f (an archaeal universal primer) and 1492R (a bacterial and archaeal universal primer) in combination with the specific primers of the ANMEs. The PCR amplification by these universal primers would provide the longer sequence information of the potential ANMEs detected by the specific primers. We conducted the PCR screening with 18 primer sets for 12 environmental samples (i.e. a total of 216 PCR reaction conditions) in duplicate PCR tubes. As a result, three phylotypes potentially affiliated with the ANME groups were obtained from the only natural gas field sample MOB4, i.e. one ANME-1 and two ANME-2c phylotypes were retrieved (Fig. 1). These ANME-1 and -2c clones were obtained from the primer sets ANME1-395F/ANME1-1417R and ANME2c-AR468f/ANME2c-1411R, respectively. No chimeric signature was identified among these sequences. The phylotype MOB4-1-1 belonged to ANME-1 and was most closely related to the sequence from hydrothermal sediments in the Guaymas Basin clones Gba1r013 and Gba1r010 (Teske et al., 2002) (both with similarity values of 97%). The phylotypes MOB4-2c-1 and MOB4-2c-2 of the ANME-2c were phylogenetically very closely related to each other (sequence similarity 99%). The most closely related clonal sequences of the phylotypes were fos0644c1 and HydBeg05, both of which were retrieved from methane seeps, offshore of Oregon (Knittel et al., 2005; Meyerdierks et al., 2005) (both with similarity values of 99%). In addition, two primer sets of Ar109f/ANME1-1417R and ANME2a-426F/1492R gave PCR products from the thermophilic methanogenic sludge samples JE-MDS and SO-MDS, lotus field sediment sample NS-LF and gas field sample NA5, but these sequences were not related to the ANMEs. The sequences amplified by Ar109f/ANME1-1417R were similar to an uncultured archaeal linage ARC I (Chouari et al., 2005), and the sequences taken by the ANME2a-426F primer belonged to the genera Methanosaeta and Methanosarcina (data not shown).
Because the ANMEs phylotypes were successfully amplified from the natural gas field samples, the abundance of the ANME phylotypes in the microbial communities was estimated using the quantitative PCR technique. The relative abundance of the targeted ANME group was estimated against the total 16S rRNA gene number (defined as the sum of the numbers obtained by bacterial and archaeal primer sets) in the DNA extract. The relative abundance of bacterial and archaeal 16S rRNA genes was 98.0% and 2.0%, respectively. The 16S rRNA genes of the ANME-1 and ANME-2c were detected in the quantitative real-time PCR assays, but the numbers were below the quantification limit (<102 copies μL−1), i.e. showing <0.02% of the total prokaryotic 16S rRNA gene communities. In the previous study of the same natural gas field of MOB4, the 16S rRNA gene-based clone analysis detected the many methanogens sequences related to Methanobacterium spp., but none of the ANMEs' sequences (Mochimaru et al., 2007). Thus, our newly designed primers detected the possible existence of the ANMEs in the natural gas field sediments. However, it was also suggested that the phylogenetic diversity and abundance of the ANMEs might be very small in the microbial communities of the habitats.
The newly designed primers are more specified to each of the phylogenetic groups of the ANMEs and would thus be very useful to survey the distribution of ANMEs, because most of the previously reported primers/probes could not fully cover or could not sharply amplify the targeted group (Figs S1 and S2). Among the environment samples examined in this study, the 16S rRNA gene sequences of the ANMEs were detected only in the natural gas field sediments. This result does not imply that the potential AOM activity in the terrestrial environment samples could be limited to the natural gas field sediments. As suggested recently (Raghoebarsing et al., 2006; Ettwig et al., 2008), the AOM function in terrestrial habitats could be driven by previously unidentified bacterial and/or archaeal phylotypes other than the ANMEs often obtained from marine environments. Nevertheless, the environmental distribution pattern of the ANME phylotypes may represent a common environmental feature of the habitats for the ANMEs. The natural gas fields examined in this study are located in the old marine forearc basin and are based on the subsurface gas reservoirs in the turbidite sandstones (e.g. Maekawa et al., 2006). In addition, the fields are highly influenced by ancient seawater (Mochimaru et al., 2007). Thus, the natural gas field is geologically and geochemically relevant to the seawater and/or the marine sediments in the past. A similar presumption was also provided in a previous study demonstrating the existence of the ANME 16S rRNA gene sequences in the terrestrial mud volcanoes, with marine palaeoenvironmental backgrounds (Alain et al., 2006). However, the detailed distribution patterns of the ANMEs need to be surveyed further in nonmarine anaerobic environments.
We gratefully acknowledge Hiroshi Iwamoto, Yoshiyuki Tazaki, Yasuyoshi Tomoe, Yoshito Tanabe, Takako Watanabe, Hidenobu Takahashi, Hideki Inaba and Yasunori Tanji for their contributions to the sampling. We also thank Yuki Kasai, Hanako Oida and Hisako Hirayama for their help. This study was partly supported by grants from the Japan Society for the Promotion of Science, and by the Ministry of Education, Culture, Sports, Science and Technology, Japan.