Beyond Chloride Brines: Variable Metabolomic Responses in the Anaerobic Organism Yersinia intermedia MASE-LG-1 to NaCl and MgSO4 at Identical Water Activity

Abstract

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.

Keywords: compatible solutes; magnesium sulfate; metabolome; sodium chloride; stress response.

Figure 1

Growth curve of Y. intermedia MASE-LG-1 cultivated under the three investigated conditions applied for the metabolome experiments; optimal anoxic conditions (30°C, pH 7, 0 M NaCl, 0 M MgSO₄), NaCl salt stressed (30°C, pH 7, 0.4 M NaCl) and MgSO₄ salt stressed (30°C, pH 7, 0.7 M MgSO₄). Growth was determined by direct cell counting. The images display the morphological change when grown in control conditions and in MgSO₄.

Figure 2

Volcano plots displaying significance (y-axis) vs. fold-change (x-axis). Metabolite data for Y. intermedia MASE-LG-1 grown under non-stressed conditions (control, C) and salt-stressed condition with identical water availability in NaCl and MgSO₄. Pairwise comparison of (A) control and NaCl relative to control, (B) control and MgSO₄ relative to control, and (C) NaCl and MgSO₄ relative to NaCl. The plot is generated by plotting the negative log (base 10) of the adjusted p-value (<0.05) on the y-axis. Significant and insignificant peaks are represented by dark blue and light blue data points, respectively.

Figure 3

Pie-chart representing the percentage and biological categories of the identified or putatively annotated metabolites based upon their putative functions. Assignment was performed using KEGG database. (A) Data are comprised of 385 peaks, the three smallest sections represent energy metabolism, xenobiotics drugs etc., xenobiotics biodegradation and metabolism with <1%. (B) Detailed information about the various lipid compounds. * depicts peaks of metabolites that are involved in the lipid metabolism but are grouped within a different chemical class.

Figure 4

Visualization of metabolites that are significantly changed (adjusted p-value < 0.05) in the salt-stressed sample NaCl on the KEGG metabolic pathway map using Pathway Projector (Kono et al., 2009). Color code for pathway categories: aqua represents glycan biosynthesis and metabolism, blue represents carbohydrate metabolism, green represents lipid metabolism, red represents nucleotide metabolism, purple represents energy metabolism, yellow represents amino acid metabolism, pink represents metabolism of cofactors and vitamins, dark red represents biosynthesis of secondary metabolites, orange represents metabolism of other amino acids, and magenta represents biodegradation and metabolism of xenobiotics. Blue circles indicate a decrease and red circles an increase in the salt-stressed sample compared to the control. Orange circles depict metabolites only found in the salt stress sample, whereas cyan circles depict metabolites only found in the control sample. Size indicates the relative increase but is cut off at 3 due to overlay issues. Pairwise comparison of (A) control and NaCl relative to control, (B) control and MgSO₄ relative to control, and (C) NaCl and MgSO₄ relative to NaCl.

Figure 5

Visualization of metabolites that are significantly changed (adjusted p-value < 0.05) in salt stressed sample NaCl on the KEGG global map: Biosynthesis of amino acids 01230. Green lines indicate pathways identified for yin631. Orange lines indicate pathways where metabolites have been identified. Yellow circle: putatively annotated metabolite but not significantly changed. Black circle: identified metabolite, i.e., the peak matches a standard, but is not significantly changed. Arrows indicate increase (red) or decrease (blue) in salt stress samples. Black outline of the arrows depicts an identified metabolite, while no outline indicates a putatively annotated metabolite.

Figure 6

Visualization of metabolites that are significantly changed (adjusted p-value < 0.05) in salt stressed sample MgSO₄ on the KEGG global map: Biosynthesis of amino acids 01230. Green lines indicate pathways identified for yin631. Orange lines indicate pathways where metabolites have been identified. Yellow circle: putatively annotated metabolite but not significantly changed. Black circle: identified metabolite, i.e. the peak matches a standard, but is not significantly changed. Arrows indicate increase (red) or decrease (blue) in salt stress samples. Black outline of the arrows depicts an identified metabolite, while no outline indicates a putatively annotated metabolite.

Figure 7

Visualization of metabolites that are significantly changed (adjusted p-value < 0.05) under both salts relative to NaCl on the KEGG global map: Biosynthesis of amino acids 01230. Green lines indicate pathways identified for yin631. Orange lines indicate pathways where metabolites have been identified. Yellow circle: putatively annotated metabolite but not significantly changed. Black circle: identified metabolite, i.e., the peak matches a standard, but is not significantly changed. Arrows indicate increase (red) or decrease (blue) in MgSO₄ stressed samples. Black outline of the arrows depicts an identified metabolite, while no outline indicates a putatively annotated metabolite.

Figure 8

Heatmap of detected putative osmolytes. The individual intensity values (n = 3, the numbers at the top of each column indicate the replica) of each condition, e.g., control, NaCl, and MgSO₄ are shown. Metabolite levels are coloured according to relative intensity (blue = low, red = high). Asterisks mark significant different results.