RESULTADOS - La necesidad de la promulgación de una ley

Capítulo VII: La necesidad de la promulgación de una ley

III. RESULTADOS

The gene sequences obtained by different mcrA gene primer pairs were used to construct the phylogenetic trees from deduced amino acid sequences (Figure 3.11-3.14). The deeply branching Methanopyrus kandleri DSM 6324 (AF414042) was used as the out-group for these trees. All of the different groups identified by 16S rRNA gene primers were identified by mcrA gene primers and they clustered with the respective reference sequences. Since all the mcrA reference sequences were not available in the GenBank at the time of this study, some of the sequences were determined using pure cultures of methanogens such as Methanobrevibacter sp. SM9, Methanobrevibacter sp. NT7, Methanobrevibacter sp. 31A, Methanobrevibacter thaueri DSM11955 and

Methanimicrococcus blatticola DSM 13328. Details of the mcrA gene sequences of the pure cultures are shown in Appendix 1.

In the phylogenetic tree constructed using the sequences obtained from primer pair M-P1, all five groups which were identified in the 16S rRNA gene libraries clustered together with their reference sequences. The group of sequences that clustered separately from known methanogens were assumed to be the RCC group. This group has several sub-groups with high bootstrap values suggesting that it consists of different genera or species. This similar feature was observed in the 16S rRNA gene sequences of the RCC group. There were some sequences that clustered with mrtA gene sequences of

Methanosphaera stadtmanae, and they are assumed to be from Methanosphaera

species. None of the mrtA sequences from our sample clustered with cultured

Methanosphaera species suggesting that rumen Methanosphaera species are different from the cultured human strain. Subgroups of Methanosphaera spp. have high bootstrap values, which suggests that more than one species of Methanosphaera exists in the rumen. This observation is consistent with the observation of 16S rRNA gene sequences of Methanosphaera species. Similarly, the mcrA gene sequences of rumen

Methanimicrocoocus species clustered separately from cultured Methanimicrococcus

species suggesting that rumen Methanimicrococcus species are also different from cultured species.

The phylogenetic analysis of sequences from primer pair M-P2 found three groups of methanogens which clustered with their reference sequences accordingly (Figure 3.12). No mrtA sequences were amplified by this primer pair. In addition, there were no sequences representing uncultured groups when using this primer pair.

Interestingly, there was one sequence from Methanomicrobium spp. among the sequences of the primer pair M-P3 and this sequence clustered with the mcrA sequence of Methanomicrobium mobile (Figure 3.13). There were some unidentifiable sequences (not the RCC-like sequences) amplified by M-P3 (Figure 3.13). These sequences dominated the clone library constructed by the primer pair M-P4 (Figure 3.14). These sequences are distantly clustered with mrtA sequences of known methanogens. Unfortunately, there are not many mrtA sequences available in the GenBank yet. As such, not much information was obtained about these sequences. Their phylogenetic affiliation remains uncertain.

91 Mbb. gottschalkii Methanosphaera Mbb. ruminantium Methanimicrococcus RCC

92 Figure 3.5. Inferred phylogenetic relationships between partial 16S rRNA genes (C2SP1001- C2SP1048) obtained by the primer pair 109f and 915r (P1) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference 16S rRNA gene sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. The tree was constructed from an alignment of 760 nucleotide positions. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 0.05 nucleotide substitutions per nucleotide position. The 16S rRNA genes of

Methanocaldococcus jannaschii (GenBank accession M59126), Methanococcus vannielii (M36507) and Methanothermococcus thermolithotrophicus (M59128) were used as out-group sequences, and are not shown in the figure.

93 Mbb. gottschalkii Mbb. ruminantium Methanosphaera Methanimicrococcus RCC

94 Figure 3.6. Inferred phylogenetic relationships between partial 16S rRNA genes (C2SP2001- C2SP2059) obtained by the primer pair 630f and 803r (P2) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference 16S rRNA gene sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. The tree was constructed from an alignment of 185 nucleotide positions. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 0.02 nucleotide substitutions per nucleotide position. The 16S rRNA genes of

Methanimicrococcus

Methanosphaera

96 Figure 3.7. Inferred phylogenetic relationships between partial 16S rRNA genes (C2SP3001- C2SP3061) obtained by the primer pair 1af and 1100r (P3) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference 16S rRNA gene sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. The tree was constructed from an alignment of 1022 nucleotide positions. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 0.02 nucleotide substitutions per nucleotide position. The 16S rRNA genes of

97 Mbb. ruminantium Methanosphaera Mbb. gottschalkii RCC Methanimicrococcus

98 Figure 3.8. Inferred phylogenetic relationships between partial 16S rRNA genes (C2SP4001- C2SP4047) obtained by the primer pair 109f and 1386r (P4) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference 16S rRNA gene sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. The tree was constructed from an alignment of 1180 nucleotide positions. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 0.05 nucleotide substitutions per nucleotide position. The 16S rRNA genes of

99 Mbb. gottschalkii

Mbb. ruminantium

Methanosphaera

100 Figure 3.9. Inferred phylogenetic relationships between partial 16S rRNA genes (C2SP5001- C2SP5046) obtained by the primer pair 915af and 1386r (P5) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference 16S rRNA gene sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. The tree was constructed from an alignment of 445 nucleotide positions. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 0.02 nucleotide substitutions per nucleotide position. The 16S rRNA genes of

101 Mbb. ruminantium Mbb. gottschalkii Methanosphaera RCC Methanimicrococcus

102 Figure 3.10 Inferred phylogenetic relationships between partial 16S rRNA genes (C2SP6001- C2SP6049) obtained by the primer pair 86f and 1340r (P6) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference 16S rRNA gene sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. The tree was constructed from an alignment of 1185 nucleotide positions. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 0.02 nucleotide substitutions per nucleotide position. The 16S rRNA genes of

103 Mbb. gottschalkii Mbb. ruminantium Methanosphaera Methanimicrococcus mcrA group 1

104 Figure 3.11. Inferred phylogenetic relationships between deduced amino acid sequences of mcrA genes (C2SMP1001- C2SMP1051) obtained by the primer pair ML1 and ML2 (MP1) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference mcrA gene amino acid sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 10% sequence divergence. The mcrA genes of

Methanopyrus kandleri (GenBank accession AF 414042) was used as out-group sequence, and are not shown in the figure.

105

Mbb. ruminantium

Mbb. gottschalkii

106 Figure 3.12. Inferred phylogenetic relationships between deduced amino acid sequences of mcrA genes (C2SMP2001- C2SMP2049) obtained by the primer pair ME1 and ME2 (MP2) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference mcrA gene amino acid sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 10% sequence divergence. The mcrA genes of

Methanopyrus kandleri (GenBank accession AF 414042) was used as out-group sequence, and are not shown in the figure.

107 Methanomicrococcus Methanomicrobium Unidentified mrtA Mbb. gottschalkii Mbb. ruminantium

108 Figure 3.13. Inferred phylogenetic relationships between deduced amino acid sequences of mcrA genes (C2SMP3001- C2SMP3064) obtained by the primer pair MCRf and MCRr (MP3) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference mcrA gene amino acid sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 5% sequence divergence. The mcrA

genes of Methanopyrus kandleri (GenBank accession AF 414042) was used as out- group sequence, and are not shown in the figure.

109

Unidentified mrtA

110 Figure 3.14. Inferred phylogenetic relationships between deduced amino acid sequences of mcrA genes (C2SMP4001- C2SMP4014) obtained by the primer pair MCRmf and MCRr (MP4) from the rumen sample of cow (C2) fed with lucerne silage and barley meal and reference mcrA gene amino acid sequences. In the parentheses are the GenBank accessions numbers for reference sequences. The superscript T designates a type strain. Bootstrap values (>70%) are shown at selected nodes, and are percentages based on 1000 resamplings. The scale bar indicates 10% sequence divergence. The

mcrA genes of Methanopyrus kandleri (GenBank accession AF 414042) was used as out-group sequence, and are not shown in the figure.

111

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