Abstract

The isolation of microorganisms from ancient materials and the verification that they are as old as the materials from which they were isolated continue to be areas of controversy. Almost without exception, bacteria isolated from ancient material have proven to closely resemble modern bacteria at both morphological and molecular levels. This fact has historically been used by critics to argue that these isolates are not ancient but are modern contaminants introduced either naturally after formation of the surrounding material (for further details, see Hazen and Roeder 2001 and the reply by Powers, Vreeland, and Rosenzweig 2001) or because of flaws in the methodology of sample isolation (reviewed recently in Vreeland and Rosenzweig 2002). Such criticism has been addressed experimentally by the development of highly rigorous protocols for sample selection, surface sterilization, and contamination detection and control procedures. Using the most scrupulous and well-documented sampling procedures and contamination-protection techniques reported to date, Vreeland, Rosenzweig, and Powers (2000) reported the isolation of a sporeforming bacterium, Bacillus strain 2-9-3, from a brine inclusion within a halite crystal recovered from the 250-Myr-old Permian Salado Formation in Carlsbad, NM. As had been noted in earlier studies, a striking observation by Vreeland, Rosenzweig, and Powers (2000) was that the 16S rDNA of isolate 2-9-3 is 99% identical to that of Salibacillus marismortui, a bacterium isolated from the Dead Sea in 1936 (Arahal et al. 1999). In fact, Arahal et al. (1999) identified as S. marismortui three strains with 16S rDNA sequences differing by 0.01%, suggesting that isolate 2-9-3 might also be classified as S. marismortui. Two groups have since used phylogenetic analyses of 16S rDNA sequences to argue that isolate 2-9-3 is unlikely to be 250 Myr old. Graur and Pupko (2001) used a relative rate test to compare evolutionary rates of 16S rDNA on the branches leading to isolate 2-9-3 and S. marismortui and found no differences between the evolutionary rates. Considering the possibility that S. marismortui may also be ancient (Arahal et al. 1999; Vreeland, Rosenzweig, and Powers 2000), they also

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