The Permian Bacterium that Isn't

Dan Graur and Tal Pupko

Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel

There is growing evidence for the presence of viable microorganisms in geological salt formations that are millions of years old. It is still not known, however, whether these bacteria are dormant organisms that are themselves millions of years old or whether the salt crystals merely provide a habitat in which contemporary microorganisms can grow, perhaps interspersed with relatively short periods of dormancy (McGenity et al. 2000Citation ). Vreeland, Rosenzweig and Powers (2000)Citation have recently reported the isolation and growth of a halotolerant spore-forming Bacillus species from a brine inclusion within a 250-Myr-old salt crystal from the Permian Salado Formation in New Mexico. This bacterium, Bacillus strain 2-9-3, was informally christened Bacillus permians, and a 16S ribosomal RNA gene was sequenced and deposited in GenBank under the name B. permians (accession number AF166093). It has been claimed that B. permians was trapped inside the salt crystal 250 MYA and survived within the crystal until the present, most probably as a spore. Serious doubts have been raised concerning the possibility of spore survival for 250 Myr (Tomas Lindahl, personal communication), mostly because spores contain no active DNA repair enzymes, so the DNA is expected to decay into small fragments due to such factors as the natural radioactive radiation in the soil, and the bacterium is expected to lose its viability within at most several hundred years (Lindahl 1993Citation ). In this note, we apply the-proof-of-the-pudding-is-in-the-eating principle to test whether the newly reported B. permians 16S ribosomal RNA gene sequence is ancient or not.

There are several reasons to doubt the antiquity of B. permians. The first concerns the extraordinary similarity of its 16S rRNA gene sequence to that of Bacillus marismortui. Bacillus marismortui was described by Arahal et al. (1999)Citation as a moderately halophilic species from the Dead Sea and was later renamed Salibacillus marismortui (Arahal et al. 2000Citation ). The B. permians sequence differs from that of S. marismortui by only one transition and one transversion out of the 1,555 aligned and unambiguously determined nucleotides. In comparison, the 16S rRNA gene from Staphylococcus succinus, which was claimed to be "25–35 million years old" (Lambert et al. 1998Citation ), differs from its homolog in its closest present-day relative (a urinary pathogen called Staphylococcus saprophyticus) by 19 substitutions out of 1,525 aligned nucleotides. Using Kimura's (1980)Citation two-parameter model, the difference between the B. permians and S. marismortui sequences translates into 1.3 x 10-3 substitutions per site along the two lineages. The rate of substitution for 16S ribosomal DNA in prokaryotes is remarkably uniform among diverse lineages and ranges between 1 x 10-8 and 5 x 10-8 substitutions per site per year (Ochman and Wilson 1987Citation ; Hillis and Dixon 1991Citation ; Munson et al. 1991Citation ; Clark et al. 1992Citation ; Moran et al. 1993Citation ; Aksoy, Pourhosseini, and Chow 1995Citation ; Bandi et al. 1995Citation ; Clark, Moran, and Baumann 1999Citation ; Ochman, Elwin, and Moran 1999Citation ). Applying these rates, we estimate the time of divergence between B. permians and S. marismortui to be about 13,000–65,000 years. We note, moreover, that the degree of divergence between B. permians and S. marismortui is smaller than the degree of divergence between two strains of S. marismortui (Arahal et al. 1999Citation ).

The second reason to doubt the antiquity of B. permians concerns the rate of evolution of its 16S rRNA gene. We used the relative-rate test (Sarich and Wilson 1973Citation ) with the phylogenetic weighting scheme of Robinson et al. (1998)Citation to test the number of substitutions on the B. permians branch against that on the S. marismortui branch, with Virgibacillus proomii as the outgroup (fig. 1 ). There was no significant difference in the numbers of nucleotide substitutions between the two lineages (P = 0.48). Were we to accept the antiquity of B. permians, we would have to conclude that the number of substitutions accumulated by S. marismortui during 250 Myr of evolution is equal to that accumulated by B. permians during the 3–7 days of active replication in the laboratory following its rescue from a 250-Myr evolutionary slumber.



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  Fig. 1.—Scaled neighbor-joining phylogenetic tree (Saitou and Nei 1987Citation ) from 16S rDNA sequences of Bacillus permians and closely related species. The alignment is available at http://kimura.tau.ac.il/~tal. Gray lines mark branches leading to nine groups of species (Bacillus marinus, Bacillus megaterium, Bacillus mycoides, Bacillus pseudofirmus, Bacillus halodurans, Bacillus sporothermodurans, Bacillus licheniformis, Bacillus subtilis, and Virgibacillus panthotenticus) for which two or more unambiguously alignable homologous sequences are available in the literature. Strains are shown in parentheses. Paralogous sequences are shown in brackets

 
Assuming, as Vreeland, Rosenzweig, and Power (2000)Citation did, that B. permians did not evolve for 250 Myr, all the differences between the two sequences would have to be attributed to at least 250 Myr of evolution in S. marismortui. Consequently, we must conclude that the rate of substitution in the 16S rRNA gene of S. marismortui is about 5 x 10-12 substitutions per site per year, i.e., a reduction of four orders of magnitude in comparison with the typical prokaryotic rate. Such a low rate of nucleotide substitution has never been encountered in nature, least of all in bacteria. Under the assumption that S. marismortui evolves at a rate that is typical of eubacteria, we must conclude that the time of divergence between B. permians and S. marismortui is quite short.

We note, however, that an alternative explanation to the modernity of B. permians may be raised. According to one such explanation, put forward by one of the co-authors of the Vreeland, Rosenzweig, and Powers (2000) report in a New York Times interview from October 19, 2000, S. marismortui may have also been trapped in salt for millions of years. In other words, the similarity between the two bacteria is explained by a lack of accumulation of nucleotide substitutions in both lineages due to the fact that both B. permians and S. marismortui are ancient. To test this possibility, we conducted a relative-rate test of B. permians and S. marismortui versus V. proomii, with two strains of Virgibacillus pantothenticus as outgroup (fig. 1 ). Again, we found no significant difference in the number of nucleotide substitutions between the two ingroup lineages (P = 0.74), and under the assumption of the molecular clock, we must conclude that B. permians, S. marismortui, and V. proomii are contemporaneous organisms. Of course, the claim may be raised that V. pantothenticus is also ancient, so we also performed the relative-rate test on B. permians, S. marismortui, and V. proomii against V. pantothenticus, with Bacillus halodenitrificans as the outgroup. Again, we found no significant difference in the mean numbers of nucleotide substitutions between the two ingroup lineages (P = 0.51). Interestingly, the results were robust to changes in the topology of the phylogenetic tree. We note, however, that this line of reasoning, i.e., assuming that the organisms with which we compare B. permians are also ancient, may be carried on ad infinitum.

Finally, we conducted an extensive phylogenetic study of B. permians and closely related species. From this study, we excluded genes whose sequenced lengths were shorter than that of the B. permians sequence (e.g., Bacillus circulans). We also excluded sequences that contained more than 1% ambiguously determined nucleotides (e.g., Salibacillus salexigens), as well as sequences that could not be easily aligned to the rest of the sequences (e.g., the 16S rRNA B paralog from B. subtilis strain 168). As an outgroup, we chose a bacterium from the Lactobacillaceae whose branch length was the shortest to the ingroup. Thus, we chose Trichococcus flocculiformis instead of Lactobacillus casei as in Vreeland, Rosenzweig, and Powers (2000)Citation . In the end, we were left with 57 closely related 16S rRNA gene sequences from 29 species. As is evident from figure 1 , B. permians does not occupy an ancestral position in the tree, and the degree of variation between B. permians and S. marismortui is much smaller than either the degree of variation among conspecific paralogous genes or the degree of variation among alleles from conspecific strains. In the analysis presented in figure 1 , many paralogous 16S rDNA sequences were excluded because they were only distantly related to the bulk of the sequences in the analysis. Thus, the relative degree of variation between B. permians and S. marismortui is even smaller than that shown in the figure.

Notwithstanding the heroic contamination checks by Vreeland, Rosenzweig, and Powers (2000)Citation , which included microscopical examinations, alkali and acid sterilizations, and UV radiations, the pudding just tastes too fresh to be Permian, and B. permians is most probably destined to join the growing list of such purportedly Methuselan specimens as the Miocene magnolia (Golenberg et al. 1990Citation ), the Cretaceous weevil (Cano et al. 1993Citation ), and the would-be dinosaur (Woodward, Weyland, and Bunnell 1994Citation ), which turned out to be contemporary artifacts (Austin, Smith, and Thomas 1997Citation ; Walden and Robertson 1997Citation ; Gutiérrez and Marín 1998Citation ).

Acknowledgements

We thank David Arahal, Tomas Lindahl, Nancy Moran, and Mitchell Sogin for their advice.

Footnotes

Ken Wolfe, Reviewing Editor

1 Address for correspondence and reprints: Dan Graur, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. graur{at}post.tau.ac.il Back

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Accepted for publication February 15, 2001.