Comparison of halobacteria from geographically separated Permo-Triassic salt deposits and longevity studies

Research project P 13995 Halobacteria from Permian salt Helga STAN-LOTTER 11.10.1999 This proposal addresses the question of the preservation of viable microorganisms in terrestrial subsurface salt sediments over great geological times. Two main approaches will be pursued, which might also shed light on the general phenomen of - microbial longevity: First, a thorough census of the microbial populations from several salt sediments in different geographic areas, but of similar age, will be taken, using classical and molecular techniques. This should indicate if only certain species or genera are culturable, if there is evidence for unculturable species, and how the isolates relate to known halophilic microorganisms. It is expected that a correlation between distribution of species with geographical and sedimentary features, such as ion composition and presence of potential energy sources, might emerge. Samples of salt sediments of Permian and Triassic age will be compared with respect to their microbial content. Rock salt and/or bacterial isolates from the Alpine basin (Germany, Austria) and the Zechstein shelf sea (England) are already available; samples from the Salado formation (New Mexico and Texas) will be obtained during a field trip. Several physico-chemical parameters of the sediments will be determined, following dissolution or grinding of the rock salt. Culturable strains will be examined by chemotaxonomic and molecular studies; any novel strains will be deposited in culture collections. The presence of non-culturable halophilic phylotypes will be assessed by analysis of amplifiable small-subunit ribosomal RNA genes. The results are expected to provide information on the microbial biodiversity in Permian and Triassic rock salt from areas, which are thought to have experienced similar climatic conditions during the time of their formation, and to strengthen the evidence for the antiquity of the rock salt flora. The second part of the investigation attempts to elucidate possible strategies for long term survival of halophilic microorganisms, which could lead to the identification of molecules associated with the state of dormancy. In model systems, selected halobacteria will be examined for starvation responses. Such experiments have not yet been performed with any halobacteria. Survival times following storage in the absence of energy sources will be determined; the influence of the ionic environment, including similar composition as fluid inclusions of rock salt, on starvation survival will be assessed. Starvation might influence the morphology of cells, the whole cell protein composition and various cellular functions. A portion of this study, consisting of an international field trip and subsequent workshops, will be performed under the National Science Foundation (NSF) program on Life in Extreme Environments (LExEn) in the USA.

Extremely halophilic (salt loving) viable microorganisms (haloarchaea) were repeatedly isolated from Alpine subterranean salt deposits, which are believed to have been deposited about 250 millions years ago. Although it is not known if the haloarchaea are of the same age as the rock salt, they can be considered as long-time survivors under conditions of high salinity and desiccation. Some strains, which belong to the genus Halococcus, were very similar in their properties, suggesting an extensive degree of relatedness. These results were consistent with the notion that the halophilic strains from different geographical sites could be the remnants of populations which once inhabited the ancient hypersaline seas. Rock salt samples from the Salado and Castile formations in New Mexico and Texas, which are of similar age as the Alpine salt deposits, were also investigated for their microbial content. Salt bore cores from deep drilling operations, which stem from 650 m depth, were found to contain haloarchaeal strains, some of which represented novel species. Besides classical methods for the cultivation of haloarchaea, molecular approaches were also used. Extracted DNA from the rock salt was investigated for the presence of the 16S rRNA genes, which are diagnostic for bacterial or haloarchaeal species. About 120 sequences of these genes were obtained, which were classified in 12 groups. The similarity to known haloarchaeal species can be expressed in percent; several groups showed similarity values below 90-95%, which suggested the presence of novel strains. Two groups were 98-99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to the known strain Halobacterium salinarum, respectively. None of the groups was completely identical to any known haloarchaeal 16S rRNA sequences. The results suggested that an unexpected amount of novel sequences of 16S rRNA genes were present in Alpine Permo-Triassic salt sediments, some of which are similar, but not identical, to those of known haloarchaea. The apparent longevity of microorganisms in ancient rock salt could have impacts on the search for extraterrestrial life. Several findings of halite in outer space, e.g. on Martian meteorites, were reported, and thus a specific search for the possibility of halophilic microorganisms should be considered.