Table1.docx

<p>Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium Yersinia intermedia MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO₄) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO₄ and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that Yersinia intermedia MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.</p

Image1.PDF

<p>Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium Yersinia intermedia MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO₄) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO₄ and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that Yersinia intermedia MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.</p

Survival after combined stresses (desiccation and radiation).

<p>Survival of <i>Y</i>. <i>intermedia</i> MASE-LG-1 after desiccation and irradiation in combination (A) and desiccation and irradiation in the presence of oxygen (B). N₀: Viable cells without desiccation / irradiation. N: Viable cells after desiccation / irradiation (n = 3). (A) White squares: Cells were exposed to ionizing radiation under anoxic conditions in liquid culture medium. Black circles: Cells were desiccated (24 h) under anoxic conditions and subsequently exposed to ionizing radiation under anoxic conditions. Grey circles: Cells were exposed to ionizing radiation under anoxic conditions and subsequently desiccated (24 h) under anoxic conditions. (B) Black circles: Cells were desiccated (24 h) under anoxic conditions and subsequently exposed to ionizing radiation under anoxic conditions. White triangles: Cells were desiccated (24 h) under anoxic conditions and subsequently exposed to ionizing radiation under oxic conditions. White square: Survival of <i>Y</i>. <i>intermedia</i> MASE-LG-1 without desiccation and irradiation treatment.</p

Survival of <i>Y</i>. <i>intermedia</i> MASE-LG-1 after exposure to desiccation, vacuum and Martian atmosphere.

<p>N₀: viable cells without desiccation / exposure to vacuum, N: viable cells after desiccation / exposure to vacuum (n = 3). Black: Cells were desiccated on glass slides under anoxic conditions. Light grey: Cells were desiccated on quartz discs under anoxic conditions and exposed to vacuum (10⁻⁵ Pa) within the Trex-Box. Dark grey: Cells were desiccated on quartz discs under anoxic conditions and exposed to Martian atmosphere (Mars gas at a pressure of 10⁻³ Pa) within the Trex-Box.</p

Influence of perchlorates.

<p>Influence of perchlorates on tolerance to desiccation (A) and ionizing radiation (B). N₀: Viable cells without desiccation / irradiation, N: Viable cells after desiccation / irradiation. Recovery was performed under standard cultivation conditions without perchlorate (n = 3). Asterisks denote significant difference (<i>p</i> < 0.05) to the control (survival after desiccation without perchlorates). (A) Black columns: Cells were exposed (15 min) to 0.5% perchlorate (0.5% NaClO₄ = 35.6 mM; 0.5% Ca(Cl₄)₂ = 20.9 mM; 0.5% Mg(ClO₄)₂ = 22.4 mM) before desiccation treatment (24 h, anoxic conditions). Grey columns: Cells were exposed (15 min) to 1.0% perchlorate (1.0% NaClO₄ = 71.2 mM; 1.0% Ca(Cl₄)₂ = 41.9 mM; 1.0% Mg(ClO₄)₂ = 44.8 mM) before desiccation treatment (24 h, anoxic conditions). (B) Cells were exposed (15 min) to the indicated perchlorates before treatment with ionizing radiation up to 800 Gy. Black circles: 0.5% Mg(ClO₄)₂; White circle: 1% Mg(ClO₄)₂; Black triangle: 0.5% Na(ClO₄); White triangle: 1% Na(ClO₄); Black square 0.5% Ca(ClO₄)₂; White square: 1% Ca(ClO₄)₂.</p

Overview of components and concentrations at which growth / survival was detectable after exposure of 15 minutes.

<p>Overview of components and concentrations at which growth / survival was detectable after exposure of 15 minutes.</p

Overview of performed single and combined stress tests with strain MASE-LG-1.

<p>Overview of performed single and combined stress tests with strain MASE-LG-1.</p

Influence of nutrient limitation.

<p>Influence of nutrient limitation on tolerance to desiccation (A) and ionizing radiation (B). N₀: Viable cells without desiccation / irradiation, N: Viable cells after desiccation / irradiation. Recovery was performed under standard cultivation conditions (n = 3). (A) Black columns: growth in diluted medium after standard cultivation time (24 h). MASE I medium including all supplements was diluted 1:10 / 1:50 before inoculation. Grey columns: survival of <i>Y</i>. <i>intermedia</i> MASE-LG-1 in diluted medium (1:10 / 1:50) after desiccation (24 h) under anoxic conditions. Asterisks denote significant difference (<i>p</i> < 0.05) to the control (desiccation in full medium). (B) Cells grown under a limited set of nutrients were exposed to ionizing radiation up to 800 Gy under anoxic conditions. White squares: MASE I cultivation medium including all supplements without dilution. Black circles: MASE I cultivation medium, including all supplements was diluted 1:10 before inoculation. Grey circles: MASE I cultivation medium, including all supplements was diluted 1:50 before inoculation.</p

The Trex-Box.

<p>The desiccated cells on quartz glass discs can be positioned (arrow 1) inside the open Trex-Box. The box can be connected to various pumping systems (arrow 2) (A). Closed Trex-Box is ready for exposure to combined stresses e.g. radiation and vacuum (B).</p

Survival after desiccation and radiation.

<p>Survival of <i>Y</i>. <i>intermedia</i> MASE-LG-1 after anoxic desiccation (A) and after exposure to ionizing radiation (B). N_0: Viable cells without desiccation / irradiation, N: Viable cells after desiccation / irradiation (n = 3). (A) Cells were applied on glass slides and dried under anoxic conditions up to 190 days. (B) Cells were exposed to ionizing radiation up to 1000 Gy in liquid culture medium under anoxic conditions.</p