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Characterization of Microbial Diversity in Hypersaline Environments by Melting Profiles and Reassociation Kinetics in Combination with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

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Abstract

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community “genome” had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.

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Acknowledgements

The electron microscopy work was done at the Laboratory for Electron Microscopy, University of Bergen. We thank Mikal Heldal for contributing the electron microscopy work. The T-RFLP analyses were performed at the laboratory for Oral Microbiology, University of Bergen. We thank Professor Vidar Bakken for using the ABI DNA sequencer and Kristi Øvreås for technical assistance on the T-RFLP analysis. This work has been financed by the European Union through contract MAS3-CT97-0154 “MIDAS” and the Norwegian Research Council (NFR). Finally, we thank Miguel Cuervo-Arango, owner of the Bras-del-Port salterns, for his kind help.

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Authors and Affiliations

  1. Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway

    L. Øvreås, F. L. Daae & V. Torsvik

  2. División de Microbiología, Universidad Miguel Hernández, Campus San Juan, San Juan de Alicante, 03550 Alicante, Spain

    F. Rodríguez-Valera

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  1. L. Øvreås
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Correspondence to L. Øvreås.

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Øvreås, L., Daae, F., Torsvik, V. et al. Characterization of Microbial Diversity in Hypersaline Environments by Melting Profiles and Reassociation Kinetics in Combination with Terminal Restriction Fragment Length Polymorphism (T-RFLP) . Microb Ecol 46, 291–301 (2003). https://doi.org/10.1007/s00248-003-3006-3

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