RESUMEN
The Salar Grande in the Coastal Range of Northern Chile is a fossil evaporitic basin filled with almost pure halite (95% NaCl average). It is assumed that the basin has not received input of brines since the Pliocene (5.3 to 1.8 million years). Below 1 m the halite has remained undissolved since this time, whereas the upper layer has been dissolved and recrystallized by dripping fogs and occasional rainfall. We compared the archaeal community at different depths using both nested PCR and cultivation. The upper 10 cm of halite crust contained diverse haloarchaeal species, including several from new genera, but their provenance is unknown. For samples deeper in the core, a new and rigorous procedure for chemically sterilizing the surface of single halite crystals was developed. These halite crystals contained only species of the genus Halobacterium (Hbt.). Halobacterium salinarum-like sequences were detected by PCR, and evidence that they were from ancient DNA include: comparison with numerous negative controls; detection of 16S rRNA sequence differences in non-conserved regions, indicating genuine evolutionary mutations rather than PCR-cloning artefacts; independent isolation of Hbt. salinarum from ancient halite; and diverse mechanisms possessed by this species for minimizing radiation damage and thus enhancing its potential for long-term survival. Haloarchaea related to Hbt. noricense were obtained from enrichment cultures from ≈ 0.4 and 15.4 m depth. We investigated Hbt. noricense strain A1 and found that when trapped inside halite crystals its recovery was as rapid after 27 months of entombment as at day 0, faring much better than other extreme halophiles. A biogeographical investigation showed that Hbt. noricense-like organisms were: commonly found in surface-sterilized ancient halite, associated with salt mines, in halite crusts, and, despite a much more intense search, only rarely detected in surface environments. We conclude that some Halobacterium species are specialists at long-term survival in halite.
Asunto(s)
Archaea/clasificación , Archaea/fisiología , Biodiversidad , Microbiología Ambiental , Sales (Química) , Archaea/genética , Técnicas Bacteriológicas , Chile , Halobacterium/clasificación , Halobacterium/genética , Halococcus/genética , Halococcus/fisiología , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.