Charly Favreau : Physiological response of the haloarchaea Halobacterium salinarum trapped within halite crystals: Methodological developments and ex situ molecular study

At the beginning of the 20th century, microorganisms were discovered within salt crystals called halites (NaCl). They were trapped in the fluid inclusions during the formation of the crystals by evaporation from the liquid environment. These halophilic microorganisms are supposed to be preserved and even remain viable after several million years, even if the exact duration of viability is still controversial. The molecular mechanisms allowing the acclimation of halophiles within halites have remained an open question for decades. The lack of "-omics" studies is explained by the analytical challenge of salt crystals due to the high amount of NaCl and the presumed hermetic nature of the crystal system. The main questions associated with the molecular response concern the viability, activity, metabolism and structural modifications of the cells after their entrapment. Answering these questions requires first of all the resolution of analytical problems that focus on two major aspects: the removal of all contaminating cells and organic biomolecules (including proteins) from the crystal surfaces and the realization of selective extractions of biomolecules directly from the cells contained in the halite fluid inclusions with sufficient rapidity to avoid the change of physiology during the extraction.

This PhD project first focused on the laboratory production and characterization of a halite model containing the model haloarchaeon Halobacterium salinarum NRC-1. These crystals were then used to develop a new molecular toolbox, applicable to halite crystals and compatible with their high salt content. These methods allow the cleaning of the crystal surface of any residual cells and proteins as well as the extraction of proteins directly contained in the fluid inclusions without impacting the cellular physiology. The use of these methods on the laboratory halite model then allowed the first molecular examination of the early acclimation of H. salinarum contained in the fluid inclusions. Analysis of its proteome after two months of entrapment revealed a high degree of similarity to that of stationary phase liquid cultures. While central metabolism proteins are part of the shared proteome between liquid culture extracts and halite fluid inclusions, ribosomal proteins show a strong down-regulation. Regarding cell mobility, archaellum and gas vesicle proteins are either absent or less abundant in halite samples. In fluid inclusions, some transporters differ from those in liquid cultures, suggesting altered interactions between cells and the fluidic microenvironment within the crystals.

The molecular study of the physiological response to early trapping has generated a number of hypotheses related to the early acclimatization of H. salinarum within halite crystals, although these hypotheses have yet to be verified on natural models. The methodologies developed in this PhD project represent a real advance for the scientific community with the production of the necessary tools to study the physiology of microorganisms embedded in halite crystals, whether in laboratory models or in natural terrestrial samples. These methods are also of interest in the search for potential biosignatures preserved in halite fluid inclusions on other planets like Mars.