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Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal


Bacteria have been found associated with a variety of ancient samples1, however few studies are generally accepted due to questions about sample quality and contamination. When Cano and Borucki2 isolated a strain of Bacillus sphaericus from an extinct bee trapped in 25–30 million-year-old amber, careful sample selection and stringent sterilization techniques were the keys to acceptance. Here we report the isolation and growth of a previously unrecognized spore-forming bacterium (Bacillus species, designated 2-9-3) from a brine inclusion within a 250 million-year-old salt crystal from the Permian Salado Formation. Complete gene sequences of the 16S ribosomal DNA show that the organism is part of the lineage of Bacillus marismortui and Virgibacillus pantothenticus. Delicate crystal structures and sedimentary features indicate the salt has not recrystallized since formation. Samples were rejected if brine inclusions showed physical signs of possible contamination. Surfaces of salt crystal samples were sterilized with strong alkali and acid before extracting brines from inclusions. Sterilization procedures reduce the probability of contamination to less than 1 in 109.

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Figure 1: Halite crystals taken from the dissolution pipe at the 569 m (1850 ft) level of the Salado Formation (air intake shaft18 for the Waste Isolation Pilot Plant, Carlsbad, New Mexico) in October 1998.
Figure 2: Phylogenetic tree developed from the 16S rDNA sequence of unknown 2-9-3 compared with several other species of the family Bacillaceae.


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The authors acknowledge the following people who helped obtain the crystal samples for this research: D. Belski, N. Rempe, R. Carrasco, T. Garcia, D. Acevedo, S. Britain, E. Keyser, B. Kinsall, A. Morin and T. Padilla. This research was supported by the US National Science Foundation: Life in Extreme Environments Program (EAR Lexen).

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Correspondence to Russell H. Vreeland.

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Vreeland, R., Rosenzweig, W. & Powers, D. Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal. Nature 407, 897–900 (2000).

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