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Applied and Environmental Microbiology, April 2003, p. 2153-2160, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2153-2160.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

Peter P. Sheridan,{dagger} Vanya I. Miteva,* and Jean E. Brenchley

NASA Astrobiology Institute and Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Received 17 July 2002/ Accepted 20 January 2003

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Pennsylvania State University, 211 South Frear, University Park, PA 16802. Phone: (814) 865-3330. Fax: (814) 865-3330. E-mail: vim1{at}psu.edu.

{dagger} Present address: Department of Biological Sciences, Idaho State University, Pocatello, ID 83209.

Applied and Environmental Microbiology, April 2003, p. 2153-2160, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2153-2160.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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