Makoto Mitsumori, National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan (e-mail: email@example.com).
Aims: The object of the present study is isolation of methyl coenzyme-M reductase (MCR) genes (mcrA) from the bovine rumen fluid and determination of phylogenetical placements of the genes to investigate mechanisms of methanogenesis in the rumen from a point of view of mcrA genes.
Methods: Genes for methanogen-specific MCR were isolated from the bovine rumen by PCR amplification. The deduced amino acid sequences were fitted to the alignments of mcrA gene products from the referred sequences.
Significance and Impact of the Study: Although the deduced amino acid sequences of mcrA genes, isolated from the bovine rumen in the present study, were close to that of Methanobrevibacter ruminantium, these amino acid sequences did not fall into known clusters of MCR. The findings suggest that methanogenesis in the rumen would be partially carried out by unknown methanogens.
Methane is biologically produced by the metabolism of the large and diverse group of methanogenic micro-organisms, methanogens, which are phylogenetically placed exclusively as members of the archaea domain. They inhabit typical anaerobic environments, such as wetlands, sediments, geothermal springs and the digestive tracts of mammals (Garcia et al. 2000). Methane produced by methanogens is then released into atmosphere. It has been estimated that methane production by ruminants is about 15% of total atmospheric methane emissions (Moss 1993). Moreover, methanogenesis in the rumen represents a 2–12% energy loss of intake (Czerkawski 1969).
Because the final step of methane production by methanogens is catalysed by methyl coenzyme-M reductase (MCR), and is assumed to be a common enzyme in methanogens (Ermler et al. 1997). Moreover, as amino acid sequences of MCR show high similarity to each other, MCRs have been available as the targets for the detection of methanogenes like 16S rDNA genes (Luton et al. 2002). To date, mcrA genes were used to analyse the diversity of the methanogenic community from rice field soil (Lueders et al. 2001) or landfill (Luton et al. 2002). Although methanogens in the rumen were detected by amplification of 16S rDNA fragments for analysing their phylogenetical placements (Whitford et al. 2001), there is no report concerning to mcrA genes from the rumen.
In the present study, we attempt to isolate mcrA genes from the rumen by PCR, and to determine phylogenetical placements of the genes.
Materials and methods
Animals and sample collection
Two Holstein cows were used in the present study. One Holstein cow (body weight, 150 kg) in Tokyo University of Agriculture and Technology (TUAT) was given a mixture (2 : 1) of hay and concentrate twice a day. Rumen fluid was drained from the Holstein cow by a stomach-tube before a morning feed (Bayaru et al. 2001). Another ruminally fistulated, nonlactating Holstein cow (body weight, 640 kg) at the National Institute of Livestock and Grassland Science (NILGS) was given 2·65 kg of timothy hay, 0·84 kg of steam-flaked corn, and 0·46 kg soya bean meal twice a day. Rumen fluid was taken 2 h after feeding through the fistula by a suction pump. A 1-ml portion of the rumen fluid was centrifuged at 14 000 g for 5 min at 4°C to collect bacterial cells. Pellets of the bacterial cells were stored at −20°C until use in the following experiments.
DNA extraction and PCR amplification
DNA was extracted from the pelleted bacterial cells by using a QIAamp DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's instruction. The extracted DNA was used as a template for PCR using primers (Hales et al. 1996), ME1 (5′-GCMATGCARATHGGWATGTC-3′) and ME2 (5′-TCATKGCRTAGTTDGGRTAGT-3′). They were designed to amplify a DNA fragments encoding the MCR. The PCR amplification was performed with an ExTaq PCR kit (Takara, Kyoto, Japan) using 1 μl of a template (10 ng μl−1) in 25 μl of reaction solution. The PCR amplification was carried out as follows: 1 cycle at 95°C for 3 min, 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min. The PCR products were separated by electrophoresis on 1·5% agarose gels and stained with ethidium bromide. Target DNA fragments, approx. 760 bp, were purified using a QIAEX®II Gel Extraction Kit (Qiagen GmbH).
Cloning, sequencing and phylogenic analysis of amplified DNA fragments
The purified PCR products were cloned with a TA Cloning KitTM (Invitrogen, Carlsbad, CA, USA) according to the method recommended by the manufacture, and transformants were randomly picked up. The inserts of plasmids within transformants were identified using Insert CheckTM (Toyobo, Tokyo, Japan) according to the manufacture's instructions. DNA sequences of inserts within the plasmids were determined by Shimazu Co., Ltd (Kyoto, Japan). Sequence data of the inferred amino acid was aligned by use of CLUSTAL W ver.1.7 (Thompson et al. 1994) with reference to the sequences obtained from the GenBank using neighbour-joining method (Saitou and Nei 1987). The tree was evaluated by using the bootstrap test based on 1000 resamplings (Felsenstein 1985).
Methanogen-specific DNA fragments were amplified from DNA extracted from the bovine rumen fluid by PCR with primers targeting mcrA genes. The amplified fragments from two bovine rumen fluids, which were approx. 760 bp, are shown in Fig. 1. The fragments were cloned into TA cloning vector for sequencing.
Thirty-five clones, of which TUAT1 to TUAT17 and NILGS1 to NILGS18 were originated from the bovine in TUAT and NILGS, respectively, were sequenced. From a comparison of the deduced amino acid sequences, 22 clones were split into the following four groups on the basis of sequence identities: group 1, TUAT17 and NILGS16; group 2, NILGS9 and NILGS10; group 3, TUAT6, TUAT7 and TUAT13; group 4, TUAT1, TUAT2, TUAT9, TUAT10, TUAT14, TUAT15, TUAT16, NILGS2, NILGS3, NILGS4, NILGS5, NILGS7, NILGS11, NILGS12 and NILGS17. The other 13 clones had different sequences from above groups. Whereas DNA lengths of TUAT10 and TUAT14 were 723 bp, the fragment size of the other clones was 722 bp. Nucleotide sequences have been deposited in GenBank under the accession numbers AB125111 to AB125135.
The phylogenetic placements of the deduced amino acid sequences are shown in Fig. 2. The phylogenetic placements of the DNA sequences were almost the same with Fig. 2 (data not shown).
The sequences obtained in the present study were placed in the same cluster. Although the sequences did not share a high degree of sequence similarity with reference sequences, Methanobrevibacter ruminantium was found to be the closest to the present clones with sequence identities of 83·9 to 88·3%.
Although 17 sequences of the deduced amino acids have been obtained from the 35 clones in the present study, these sequences were placed in the same cluster, which was relatively close to M. ruminantium, in phylogenetical placements (Fig. 2). As the targeting DNA band, approx. 720 bp, of NILGS was thicker than that of TUAT, it has been assumed that the ratio of methanogen population possessing mcrA genes to total bacterial population in NILGS is higher than that in TUAT (Fig. 1). However, phylogenetical placements of clones obtained from the two bovine rumen did not show significant difference, except that the clones from NILGS showed slightly broader diversity (Fig. 2).
16S rDNA genes have been used for analysis of methanogens in the bovine rumen. Tajima et al. (2001) showed the presence of a novel cluster of methanogens, distantly related to thermoacidophilic archaea, by analysis of 16S rDNA sequences amplified from the bovine rumen fluid. Whitford et al. (2001) also indicated the presence of four clusters of methanogens in the rumen on the basis of 16S rDNA sequences. Two of them, MbrI and MbrII, were 98·5–98·8 and 97·2–97·7% similar to the 16S rDNA sequence of M. ruminantium respectively. The DNA sequences of mcrA genes and their deduced amino acid sequences, isolated in this study, showed 85·2–88·1 and 83·9–88·3% similarities to those of M. ruminantium. It has been known that mcrA genes could be used as phylogenetic tool for the specific detection and the identification of methanogenes, because the phylogeny of the mcrA genes and 16S rDNA from the recognized orders of methanogens clearly had a strong similarity (Luton et al. 2002). The mcrA genes isolated in this study, therefore, would be placed in MbrI and/or MbrII clusters reported by Whitford et al. (2001) by means of phylogenetic placements (Fig. 2).
However, the mcrA genes presented in this study have been the first mcrA genes isolated from the rumen, and have created a novel cluster, which does not fall into known clusters of mcrA genes. Thus, it has been presumed that a kind of methanogens possessing mcrA genes, clustered around the mcrA genes isolated in this study, inhabit the bovine rumen and would play roles in methane production. Further work is needed to determine species of methanogens possessing the novel mcrA genes, precisely.
This work was partially supported by the Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries (STAFF) and Grant-in-Aid (No. 11695071) for Scientific Research from Ministry of Education, Science, Sports and Culture in Japan.