Sample description, strains and environmental phylotypes
Environmental samples used for the quantification of mcrA and archaeal 16S rRNA genes are listed in Table 1. The methanogen strains and environmental phylotypes from anoxic marine sediments that were subjected to fluorescent PCR are described below. Methanothermococcus okinawensis IH1T and Methanotorris formicicus Mc-S-70T were stored at our laboratory, JAMSTEC. Methanobacterium formicicum DSM 1535T, Methanococcoides alaskaense DSM 17273T, Methanococcoides burtonii DSM 6242T, Methanoculleus chikugoensis JCM 10825T, Methanohalobium evestigatum DSM 3721T, Methanococcus vannielii JCM 13029T, Methanogenium organophilum DSM 3596T, Methanohalophilus mahii DSM 5219T, Methanohalophilus portucalensis DSM 7471T, Methanosalsum zhilinae DSM 4017T, Methanolobus vulcani DSM 3029T, Methanomethylovorans thermophila DSM 17232T, Methanosaeta concilii DSM 3671T, Methanosaeta harundinacea DSM 17206T, Methanosaeta thermophila DSM 6194T, Methanosarcina barkeri DSM 800T, Methanosarcina thermophila DSM 1825T, Methanothermus sociabilis JCM 10723T, Methanocaldococcus jannashii JCM 10045T, Methanothermococcus thermolithotrophicus JCM 10549T, Methanotorris igneus JCM 11834T and Methanopyrus kandleri JCM 9639T were obtained from DSMZ or JCM. Environmental mcrA clones from anaerobic marine sediments in the Nankai Trough, namely KM-m1.09 (AB233458) (ANME group cd), KM-m-4.10 (AB233466) (ANME group e) and KM-m-5.06 (AB233468) (ANME group a) (Nunoura et al., 2006), were also stored at our laboratory, JAMSTEC.
Table 1. Description of samples used in this study
|TDS-J||Thermophilic digested sludge||55||Municipal solid waste||Niigata, Japan||Yoneyama & Takeno (2002)|
|MDS||Mesophilic digested sludge||35||Municipal sewage waste||Toyama, Japan||This study|
|Garb||Mesophilic digested sludge||37||Municipal solid waste||Niigata, Japan||This study|
|Chem||Thermophilic digested sludge||52||Wastewater from food processing factory||Shizuoka, Japan||This study|
|#744C1-0 cm below sea floor||Methane seep sediments||<5||AOM community||The Nankai Trough||Nunoura et al. (2006)|
Nucleic acid extraction
The DNA assemblages from mesophilic and thermophilic digested sludges were extracted with ISOIL for Beads Beating (Nippon Gene Co. Ltd, Tokyo, Japan). The genomic DNA of isolates was extracted using an Ultra Clean Microbial DNA Kit (MO BIO Laboratories, Solana Beach, CA), and the DNA samples from cold seep sediments (744C0) was prepared as described previously (Nunoura et al., 2006).
Refinement of a primer and quantitative PCR system for mcrA
DNA sequences of mcrA from isolates and environmental phylotypes were aligned using arb version 20030822 (Ludwig et al., 2004). Conserved sequences were searched for using the ARB system, and a novel primer, ‘ME3MF’ (ATGTCNGGTGGHGTMGGSTTYAC), was designed.
In order to optimize fluorescent quantitative PCR conditions with the ME3MF and ME2r′ (TCATBGCRTAGTTDGGRTAGT) (Hales et al., 1996) primers, we used mcrA gene fragments cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA) from two strains of Methanobacteriales: Methanobacterium formicicum and Methanobacterium sociabillis; six strains of Methanococcales: Methanococcus vannielii, Methanococcus jannaschii, Methanothermococcus thermolithotrophicus, Methanothermococcus okinawensis, Methanotorris formicicus and Methanotorris igneus; two strains of Methanomicrobiales: Methanogenium organophilum and Methanoculleus chikugoensis; 11 strains of Methanosarcinales: Methanohalophilus mahii, Methanolobus vulcani, Methanosaeta harundinacea, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina thermophila, Methanohalobium evestigatum, Methanohalophilus portucalensis, Methanococcoides alaskaense, Methanococcoides burtonii, Methanosalsum zhilinae; one strain of Methanopyrales: Methanopyrus kandleri; and three environmental mcrA phylotypes probably from ANME archaea; KM-m1.09 (group c–d), KM-m-4.10 (group e) and KM-m-5.06 (group a–b) (Nunoura et al., 2006) as templates. The mcrA gene fragments from Methanococcus vannielii, Methanococcus jannaschii, Methanothermococcus thermolithotrophicus, Methanothermococcus okinawensis and Methanotorris formicicus were obtained using the ME1f′ (TCATBGCRTAGTTDGGRTAGT) (Hales et al., 1996) and ME2r′ primer set, those from Methanobacterium formicicum, Methanogenium organophilum, Methanosarcina barkeri, Methanosaeta harundinacea and Methanosaeta thermophila were amplified by using a primer set of ME3 (GGTGGHGTMGGWTTCACACA) (Hales et al., 1996) and ME2r′, and that from Methanopyrus kandleri was obtained based on genomic sequence (Slesarev et al., 2002).
Fluorescent PCR conditions using SYBR Premix Ex Taq (Takara Bio, Otsu, Japan) were optimized for a range of annealing temperatures (51–54 °C), annealing times (20–50 s) and additional MgCl2 concentrations (0 or 2.5 mM). The concentration of each primer and ROX reference dye II was 0.2 pmol μL−1 and 0.02 μL μL−1, respectively. For these runs, fluorescent PCR was performed using a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) with preheat, denaturing and extension conditions of 94 °C for 2 min, 94 °C for 40 s and 72 °C for 1 min, respectively. Fifty cycles of amplification and fluorescence detection were executed.
Once fluorescent PCR conditions had been optimized, we tested the detection limit of the assay system using cloned mcrA gene fragments from 11 representative methanogens: Methanobacterium formicicum, Methanococcoides alaskaense, Methanococcus vannielii, Methanogenium organophilum, Methanosaeta harundinacea, Methanosaeta thermophila, Methanosarcina barkeri, Methanobacterium sociabillis, Methanococcus jannaschii, Methanoculleus chikugoensis and Methanopyrus kandleri; and three environmental mcrA phylotypes of KM-m1.09, KM-m-4.10 and KM-m-5.06 as templates. When applying the optimized fluorescent quantitative PCR system to the environmental microbial community, a mixture of cloned mcrA gene fragment clones: Methanobacterium formicicum, Methanococcoides alaskaense, Methanococcus vannielii, Methanogenium organophilum, Methanosaeta harundinacea, Methanosaeta thermophila, Methanosarcina barkeri, Methanobacterium sociabillis, Methanococcus jannaschii and Methanoculleus chikugoensis with KM-m1.09 and KM-m-4.10 was used to obtain standard curves for each instance.
Amplification of mcrA and archaeal 16S rRNA genes from environmental DNA
For the environmental clone analysis of mcrA using the refined ME3MF and ME2r′ primer set, mcrA gene fragments were amplified using SYBR Premix Ex Taq (Takara Bio) under the optimized condition for the fluorescent PCR described below with 30–40 cycles of amplification. Archaeal 16S rRNA gene fragments were amplified from extracted environmental DNA by La Taq polymerase with GC buffer (Takara Bio). The oligonucleotide primers for the PCR amplification of the archaeal 16S rRNA gene were Arch21F and Arch958R (Lane, 1985; DeLong, 1992). Amplification conditions for the 16S rRNA gene have been described previously (Nunoura et al., 2006).
Gene fragments of the amplified mcrA and 16S rRNA gene were then cloned into the pCR2.1 vector. The inserts were directly sequenced by the dideoxynucleotide chain termination method using BigDye ver.3.1 (Applied Biosystems) in accordance with the manufacturer's recommendations.
For the environmental clone analysis, the similarity of 16S rRNA gene and mcrA sequences was analyzed using fastgroupII (http://biome.sdsu.edu/fastgroup/) (Yu et al., 2006) and the fasta algorithm of the dnasis software (Hitachi Software, Tokyo, Japan), and sequences with >97% and >96% identity, respectively, were assigned to the same clone type (phylotype).
Representative sequences for the mcrA clone analysis were subjected to a similarity search against the DDBJ/EMBL/GenBank database using the blast algorithm (http://blast.ddbj.nig.ac.jp/top-j.html). Representative 16S rRNA gene sequences were analyzed using arb version 20030822 (Ludwig et al., 2004) and categorized according to taxonomic hierarchy.
Quantification of the 16S rRNA gene
Quantification of the archaeal 16S rRNA gene from environmental microbial DNA was performed using a quantitative fluorescent PCR method with a 7500 Real Time PCR System, as described previously (Takai & Horikoshi, 2000) but with minor modifications. We used a prepared mixture of qPCR Quick GoldStar Mastermix Plus (Eurogentec, Seraing, Belgium) and concentrations for primers and probe were prepared following the manufacturer's instructions.
Nucleotide sequence accession numbers
The mcrA gene sequence obtained from the characterized methanogens, as well as mcrA and 16S rRNA gene sequences from the environmental DNA in this study were submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases under accession numbers AB353207–AB353262.