Long-term survival of halophilic microorganisms entombed in fluid inclusions of halite

Hypersaline environments are subjected to dynamic environmental conditions which can result in the precipitation of salts, including halite. Microbial communities can get trapped inside fluid inclusions of halite, primarily haloarchaea. The entombment can be an advantage, including the protection afforded by halite to shield against intense radiation, which complements the inherent radiation resistance of many haloarchaea. We tested the capacity of the haloarchaeal species, Halobacterium salinarum NRC-1 and Halobacterium noricense A1, and the halophilic bacterial species, Salinibacter ruber M13, to tolerate UV and ionising radiation when in halite crystals. All species survived UV irradiation when in halite crystals and, with different lag phases, they even survived an ionising radiation dose of 5 kGy. Halobacerium salinarum NRC1 was entombed in halite crystals, and its proteome examined at 42 days of entombment compared with growth prior to entombment. This strain down-regulated proteins involved in transcription and translation, suggesting that its energy is primarily devoted to maintenance. In contrast to all other ribosomal proteins detected, ribosomal protein L13 had an elevated abundance at day 42, suggesting that it has a role connected with long-term survival. We also found the up-regulation of several proteins, highlighting the existence of on-going anaerobic metabolic activities. We explored the biodiversity of the Dead Sea entrapped in halite deposited from 1980 to present, and to this end, in November 2021, we collected samples of water and halite and the DNA was extracted. Archaeal and bacterial 16S rRNA genes were amplified from these halite crystals and water samples. Overall, we have gained insights into the radiation resistance of entombed halophiles, started to understand the mechanisms by which Haloarchaea survive inside halite, and indication about which Haloarchaea preferentially survive in halite in the environment. The findings of this study have implications about the survival of life on Earth, and opens the possibility of life elsewhere.