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. 2003 Apr;69(4):2153-60.
doi: 10.1128/AEM.69.4.2153-2160.2003.

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

Peter P Sheridan  1 , Vanya I Miteva, Jean E Brenchley
Affiliations

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

Peter P Sheridan et al. Appl Environ Microbiol. 2003 Apr.

Abstract

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 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees 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.

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Figures

FIG. 1.
FIG. 1.
Phylogenetic relationships of the 24 rRNA gene sequences obtained from the community cultures and 28 closely related sequences, based on a distance analysis (neighbor-joining algorithm with Jukes-Cantor model; 1,000 bootstrap replicates performed). Accession numbers are provided in Materials and Methods. Asterisks, clones supposed to represent new species or genera. str., strain.
FIG. 2.
FIG. 2.
Distribution of 16S rRNA gene clones according to ARDRA patterns and phylogenetic groups. Sequenced and nonsequenced fragments with identical ARDRA patterns were placed in the same group.
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