Scanning and transmission electron micrographs of biofilms, cells and hami.

<p>Left panels: MSI, right panel: SM. A: Scanning electron micrograph of MSI biofilm, showing SM1 euryarchaeal cells with defined distances and cell-cell connections. Bar: 1 µm. B: Scanning electron micrograph of SM biofilm, showing SM1 euryarchaeal cells with defined distances and fine-structured cell-cell connections. In-between: Bacterial filamentous and rod-shaped cells. Bar: 1 µm. C: Scanning electron micrograph of dividing SM1 euryarchaeal cell (MSI) with cell surface appendages. Bar: 200 nm. D: Scanning electron micrograph of dividing SM1 euryarchaeal cell (SM) with cell surface appendages. Bar: 200 nm. E: Transmission electron micrograph of cell surface appendages (hami) of SM1 euryarchaeal cells from the MSI biofilm. The hami carry the nano-grappling hooks, but besides that appear bare (square), without prickles (Moissl et al 2005). Bar: 100 nm. F: Transmission electron micrograph of cell surface appendages and matrix of SM1 euryarchaeal cells from the SM biofilm. The hami reveal the typical ultrastructure, with nano-grappling hooks and barbwire-like prickle region (square, Moissl et al 2005). Bar: 100 nm.</p

The conversion of biofilm to string-of-pearls community in the spring water originating from the subsurface.

<p>A: Biofilm. B: Intermediate transition state. C: String-of-pearls community. Row 1: Schematic drawings. Orange: SM1 euryarchaeal cocci, Green: Filamentous, sulfide-oxidizing bacteria. Row 2: Photographs and scanning electron micrograph (2B) of different stages. Row 3: FISH images of different stages (for MSI samples please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099801#pone.0099801-Probst1" target="_blank">[15]</a>; Archaea orange (CY3), Bacteria green (RG)). A: SM-BF, showing high dominance of Archaea. B: Attachment of archaea to filamentous bacteria. C: String-of-pearls communities with large archaeal colony and bacterial mantle. Arrows point to archaeal microcolonies, manteled by filamentous bacteria. It is proposed that attachment of SM1 Euryarchaeota to filamentous bacteria (B) mediates the transition from biofilm (A) to the string-of-pearls community (C). Scale bars: A3: 10 µm, B2: 1 µm B3: 10 mm, C3: 25 µm.</p

Scanning electron micrograph of filamentous bacterium and surrounded and cocooned by the SM1 euryarchaeal cells (SM-BF).

<p>Bar: 1 µm.</p

Detailed community profiling using PhyloChip G3 and SR-FTIR.

<p>A: Ordination analysis of PhyloChip G3 data based on weighted UniFrac measure of eOTU abundances followed by non-metric multidimensional scaling (NMDS). Stress for NMDS of archaeal eOTUs (#37): 0.0088. Stress for NMDS of bacterial eOTUs (#1300): 0.0223. B: Heatmap displaying significantly different families found between the two biofilm types, MSI-BF and SM-BF by PhyloChip G3 assay. Significance is based on aggregated HybScores of eOTUs on family level followed by a Welch-test. For false discovery detection please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099801#pone.0099801.s006" target="_blank">Fig. S6</a>. C: Ordination analysis of SR-FTIR data based on a linear discriminant analysis and principal component analysis (PCA-LDA) in the spectral region of 2800–3100 cm⁻¹ on the archaea spectra extracted from the maps from the three different locations. On the right there is the plot of PCA-LDA loadings. PCA-LDA1 explains for the 93.4% of the variance, PCA-LDA2 for 5.3% and PCA-LDA3 for 0.9%. Arrows point to the infrared signals used to explain the difference between the samples: 2975 cm⁻¹, 2965 cm⁻¹, 2924 cm⁻¹ and 2850 cm⁻¹. D: PCA-LDA in the spectral regions of 900–1280 cm⁻¹ and 2800–3100 cm⁻¹ on SR-FTIR spectra of the bacteria “pixels” from the chemical maps of the samples at the three different locations. On the right there is a plot of PCA-LDA loadings in the two spectral region of interest. PCA-LDA1 explains for the 54.5% of the variance, PCA-LDA2 for 28.6% and PCA-LDA3 for 7.3%. Arrows point to the main infrared signals used to explain the difference between the samples: 2958 cm⁻¹, 2925 cm⁻¹, 2870 cm⁻¹ and 2850 cm⁻¹, in the second panel 1250 cm⁻¹, 1110 cm⁻¹, 1080 cm⁻¹ and 1045 cm⁻¹.</p

Quantification of archaeal and bacterial signatures via qPCR, FISH and SR-FTIR (values in brackes give standard deviation).

<p>*data from Probst et al., 2013.</p><p>ND: Not Determined.</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

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

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