Comparison of 16S rRNA gene sequences of genus Methanobrevibacter

Abstract

Background: The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter.

Results: The phylogenetic analysis of the genus based on 786 bp aligned region from fifty-four representative sequences of the 120 available sequences for the genus revealed seven multi-member groups namely, Ruminantium, Smithii, Woesei, Curvatus, Arboriphilicus, Filiformis, and the Termite gut symbionts along with three separate lineages represented by Mbr. wolinii, Mbr. acididurans, and termite gut flagellate symbiont LHD12. The cophenetic correlation coefficient, a test for the ultrametric properties of the 16S rRNA gene sequences used for the tree was found to be 0.913 indicating the high degree of goodness of fit of the tree topology. A significant relationship was found between the 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) for the genus with the correlation coefficient (r) for logD and logS, and for [ln(-lnD) and ln(-lnS)] being 0.73 and 0.796 respectively. Our analysis revealed that for this genus, when S = 0.984, D would be <70% at least 99% of the times, and with 70% D as the species "cutoff", any 16S rRNA gene sequence showing <98% sequence similarity can be considered as a separate species. In addition, we deduced group specific signature positions that have remained conserved in evolution of the genus.

Conclusions: A very significant relationship between D and S was found to exist for the genus Methanobrevibacter, implying that it is possible to predict D from S with a known precision for the genus. We propose to include the termite gut flagellate symbiont LHD12, the methanogenic endosymbionts of the ciliate Nyctotherus ovalis, and rat feces isolate RT reported earlier, as separate species of the genus Methanobrevibacter.

Figure 1

Phylogenetic tree of the members of the genus Methanobrevibacter based on the 16S rRNA gene. The tree was generated using CLUSTAL W program [30] for sequence alignment (786 bp) and by the neighbor joining method using Kimura 2 parameter distances in MEGA 2.1 software. Only positions 286–1120 (E. coli numbering) were considered with Msp. stadtmanae as the outgroup. Numbers at nodes indicate percent bootstrap values above 50 (1000 replicates). Bar indicates Jukes-Cantor evolutionary distance. Bold letters indicate isolated strains whereas normal font indicates clones.

Figure 2

Relationship between the 16S rRNA gene sequence similarity (S) and the extent of DNA hybridization (D). (a) The correlation between logD and logs, (b) Complementary log log, i.e., [ln(-lnD) and ln(-lnS)] plot for the same, and (c) Probability of D for S from 0.90 to 1.00. The line was determined by linear regression of the values. The extent of DNA hybridization was measured by the membrane filter method and the data used for DNA hybridization was as reported earlier [8, 21, 22]. The Distribution of D was calculated from the equation ln(-lnD) = 0.5077 [ln(-lnS)] + 1.8999 and the SD of the residuals in D, which was 0.3498.

Figure 3

Alignment of sequences showing differences in the 16S rRNA gene sequence of endosymbiont of Nyctotherus ovalis strain NO Sequence for strain NO is shown in bold letters. The positions with differences in the 16S rRNA gene sequence of strain NO with other members are shown in bold-italics. See text for GenBank Accession numbers against the strain names used above. "*" indicates conserved positions amongst the sequences used.