Methanocella paludicola gen. nov., sp. nov., a methane-producing archaeon, the first isolate of the lineage ‘Rice Cluster I’, and proposal of the new archaeal order Methanocellales ord. nov.

Rice paddy fields are one of the major sources of the greenhouse gas methane, contributing about 10–25 % of global methane emission (Dentener et al., 2001; Neue, 1993). Methane emission from rice paddy fields is attributed to methanogenic Archaea (methanogens), the key organisms responsible for the final step of the anoxic degradation of organic substances. The Rice Cluster I (RC-I) lineage, which hitherto has been comprised solely of 16S rRNA gene clone sequences, plays a key role in methane production in rice paddy fields (Lu & Conrad, 2005; Conrad et al., 2006). Despite their numerical significance and cosmopolitan dispersal, RC-I methanogens have so far escaped isolation.

Recently, we have successfully isolated a novel methanogenic archaeon, designated strain SANAE^T, belonging to the RC-I lineage from a Japanese rice paddy field. Enrichment and isolation of strain SANAE^T was achieved by application of the co-culture approach with Syntrophobacter fumaroxidans as the hydrogen-producing syntrophic partner (Sakai et al., 2007). In a previous publication we described the isolation of strain SANAE^T. Here we give a detailed description of strain SANAE^T, including morphological, physiological and genetic properties. We propose that strain SANAE^T is the type strain of a novel species within a novel genus with the name Methanocella paludicola gen. nov., sp. nov. In addition, we propose the order Methanocellales ord. nov., with the newly isolated methanogen as a representative of the new order.

The basal medium used in this study was prepared as described previously (Sekiguchi et al., 2000). All cultivations were performed at 37 °C in 50 ml serum vials containing 20 ml medium (pH at 25 °C, 7.0) under an atmosphere of N₂/CO₂ (80/20, v/v) without shaking, unless otherwise mentioned. Growth and substrate utilization was determined by monitoring the optical density of the cultures (at 600 nm) and the concentration of methane. Incubations were stopped after 3 months. The pH, temperature and salinity range of strain SANAE^T were determined in standard media containing 0.01 % yeast extract and 1 mM acetate. The gas phase contained H₂ at approximately 150 kPa. Temperature tests were carried out between 18 and 60 °C, pH tests between pH 5.5 and 8.0, and salinity tests between 1 and 30 g NaCl l⁻¹ (17–513 mM). During the pH test, the medium was routinely monitored to observe if the initial pH conditions were changed or not. Salinity tests were performed using the same medium described above (which already contains 33 mM Na⁺ and 14 mM Cl⁻). Antibiotics were evaluated with cultures supplemented at final concentrations of 100 μg antibiotic ml⁻¹. Antibiotic susceptibility tests were carried out at 37 °C for a period of 2 months. All measurements were performed in triplicate.

Cell morphology and motility were examined by a phase-contrast microscope (Olympus model BX50F). Gram staining was carried out according to the method of Hucker (Doetsch, 1981). The susceptibility of the isolate to detergents was tested with SDS at concentrations ranging from 0.01 to 2.5 %, and cell lysis was determined by microscopic observation of cell integrity. Transmission electron microscopy was performed as described previously (Sekiguchi et al., 2003). Short-chain fatty acids, alcohols, methane, H₂ and carbon dioxide were measured as described previously (Imachi et al., 2000, 2002, 2006). The G+C content of the genomic DNA was determined by the method of Kamagata & Mikami (1991).

All procedures for DNA extraction, PCR amplification and sequencing have been reported previously (Imachi et al., 2006). PCR was carried out using primer pairs Arch21F (DeLong, 1992) and 1490R (Weisburg et al., 1991), and ME1 and ME2 (Hales et al., 1996), for amplification of the 16S rRNA and mcrA genes, respectively. Phylogenetic analyses were performed using the arb program (Ludwig et al., 2004). A 16S rRNA gene-based tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) for sequences having >1000 nt. Subsequently, shorter sequences were inserted into the tree without changing the tree topology by using the parsimony insertion tool of the arb program. 16S rRNA gene sequence similarity values were calculated using the Calculate Matrix function of the arb program with Jukes and Cantor correction (Jukes & Cantor, 1969; Ludwig et al., 2004). A deduced McrA amino acid sequence-based phylogenetic tree was constructed by the neighbour-joining method implemented in the arb program with 265 amino acid positions and the percentage acceptance mutations (PAM) distance correction. For both phylogenetic trees, the tree topologies were evaluated by using bootstrap resampling analysis (Felsenstein, 1985) for 1000 replicates with the neighbour-joining, maximum-parsimony and maximum-likelihood methods as described previously (Sekiguchi et al., 2006). Fluorescence in situ hybridization was performed as described by Sekiguchi et al. (1998). To detect strain SANAE^T cells, a 16S rRNA-targeted oligonucleotide probe specific for strain SANAE^T, SANAE1136 (Sakai et al., 2007), was used.

Cells of strain SANAE^T were non-motile, rod-shaped, 1.8–2.4 μm long and 0.3–0.6 μm wide, and occurred singly (Fig. 1⇓). Coccoid cells were observed in a late-exponential phase culture with H₂ or formate (Fig. 2⇓). Fluorescence in situ hybridization probe targeting of SANAE^T cells demonstrated that both cell types belong to strain SANAE^T (Fig. 2⇓). Cells of strain SANAE^T autofluoresced in epifluorescence microscopy, indicating the presence of the methanogen-specific coenzyme F₄₂₀. The cells stained Gram-negative and resisted disruption by less than 2 % (w/v) SDS. Colonies of strain SANAE^T in a deep agar medium were white to cream, reaching a diameter of 1–1.5 mm after 6 months incubation with H₂ as a substrate.

Strain SANAE^T required acetate as carbon source. Yeast extract supported growth; however, it was not required. H₂ and formate (40 mM) supported growth and methane production. The following substrates did not support growth and/or methane production: pyruvate (20 mM), lactate (20 mM), acetate (20 mM), 1-propanol (5 mM), 2-propanol (5 mM), ethanol (5 mM), 1-butanol (5 mM), 2-butanol (5 mM), cyclopentanol (5 mM), methanol (20 mM), methylamine (10 mM), trimethylamine (10 mM), dimethylamine (10 mM) and propionate (20 mM).

Strain SANAE^T grew between 25 and 40 °C with an optimum at 35–37 °C. The pH range for growth was between 6.5–7.8, with an optimum at around pH 7.0. The strain could grow at NaCl concentrations ranging from 0–1 g NaCl l⁻¹ (0–17 mM). In medium containing 2–4 g NaCl l⁻¹ (34–68 mM), a small amount of methane was detected; however, growth was inhibited because methane production was not increased. Methane production and growth were completely inhibited by 5 g NaCl l⁻¹ (86 mM). Under optimal conditions (pH 7.0, 37 °C), the doubling time, calculated from methane production rates, was 4.2 days. The strain tolerated ampicillin, penicillin G, vancomycin, kanamycin, bacitracin and streptomycin. Growth was inhibited by rifampicin, tetracycline and chloramphenicol.

The DNA G+C content of strain SANAE^T was 56.6 mol%. Analysis of its 16S rRNA gene sequence affiliates strain SANAE^T with sequences in the RC-I lineage (previously published in Sakai et al., 2007). Hitherto, this lineage, which falls between the orders Methanosarcinales and Methanomicrobiales (Fig. 3⇓), was exclusively composed of sequences retrieved from clone libraries. The 16S rRNA gene sequence similarities between strain SANAE^T and members of Methanosarcinales were in the range 80.0–82.8 %, and those between the strain and members of Methanomicrobiales ranged from 77.5 to 82.4 %. The closest cultured relative of strain SANAE^T was Methanomethylovorans hollandica (82.8 % similarity). In addition to the 16S rRNA gene-based analysis, we also determined the partial sequence of the mcrA gene in strain SANAE^T (681 bp) and constructed a phylogenetic tree based on the deduced amino acid sequence of mcrA genes (Fig. 4⇓). In this tree also, strain SANAE^T affiliates with a member of the RC-I lineage. The closest relative based on McrA amino acid sequence was Methanosalsum zhilinae (71.8 % similarity).

Phenotypic and genetic analyses in this study showed that strain SANAE^T is an H₂-utilizing methanogen and that it should be considered to be a member of the RC-I lineage. Previous culture-dependent and -independent investigations have provided evidence that members of the RC-I lineage utilize H₂ (Lueders et al., 2001; Sizova et al., 2003; Erkel et al., 2005, 2006; Lehmann-Richter et al., 1999). Erkel et al. (2006) constructed a complete genome sequence of an RC-I methanogen from an enrichment culture growing on H₂ and revealed that the RC-I methanogen had a full set of genes involved in methanogenesis from H₂/CO₂. Abundant populations of RC-I Archaea were detected in an acidophilic methanogenic consortium supplemented with H₂ as sole energy source (Sizova et al., 2003). The 16S rRNA gene sequences from those RC-I enrichments were determined and are available from the Genbank/EMBL/DDBJ databases as AM114193 for the metagenome (the genome contained three rrn operons) and AF524853 for the acidophilic consortium. The sequence similarities between these RC-I enrichments and strain SANAE^T were in the range 92.2–94.2 %, indicating that strain SANAE^T and these RC-I organisms are phylogenetically distinct from each other, at least at the species or genus level.

Since the discovery of RC-I, it has been recognized as a different genotypic group from the orders Methanosarcinales and Methanomicrobiales because of the low sequence similarity values with those of the two aforementioned orders (Großkopf et al., 1998). In fact, as described above, the 16S rRNA gene sequence similarities between strain SANAE^T and members of the Methanosarcinales and Methanomicrobiales were also low. Additionally, the bootstrap values of both 16S rRNA gene-based and McrA amino acid sequence-based phylogenetic trees solidly supported the three lineages, Methanosarcinales, Methanomicrobiales and RC-I (Figs 3⇑ and 4⇑). These results suggest that RC-I, containing strain SANAE^T, is certainly a monophyletic taxon at order level within the class ‘Methanomicrobia’ (Garrity et al., 2003). Additionally, the physiological properties of strain SANAE^T distinguish it clearly from members of the order Methanosarcinales, which utilize acetate and simple methylated compounds in contrast to strain SANAE^T. However, the phenotypic distinction between strain SANAE^T and members of the Methanomicrobiales is not so straightforward as both consume hydrogen and formate exclusively and have similar responses to pH, temperature and salinity. They differ, however, in cell shape (Table 1⇓).

Based on these phenotypic and phylogenetic data, we propose that strain SANAE^T is the type strain of a novel species within a novel genus, for which we propose the name Methanocella paludicola gen. nov., sp. nov. We also propose the status of order for the RC-I lineage and propose the name Methanocellales ord. nov.