Hyperosmotic salt stress elicits complex and predominantly species-specific global metabolic responses.

<p>(A) Principal component analysis (PCA) was performed based on log₂ metabolite ion fold-changes upon low (IC₁₀, L), medium (IC₂₅, M) or high (IC₅₀, H) salt stress relative to unstressed controls. For each species the three stress intensity points are connected by triangular patches for visualization purposes. Patches and labels are colored according to taxonomic classification as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.g001" target="_blank">Fig 1A</a>. (B) Loading plot of metabolites underlying the PCA shown in panel A. Selected metabolites with large coefficients are highlighted. Note that metabolite annotations are based on accurate mass and can be ambiguous; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.s001" target="_blank">S1 Data</a> for complete annotations. (C) Numbers of strongly and significantly responding metabolites in each analyzed species, grouped either by the lowest stress intensity under which a change was observed (gray bars) or by change direction (magenta and blue bars). (D) Histogram of the number of species in which metabolite ions were affected by the individual salt stress intensities (black, dark gray and light gray curves) or by at least one stress intensity (magenta curve).</p

Correlation analysis of metabolite responses with salt tolerance.

<p>(A) Correlation of metabolite fold-changes in response to strong salt stress with IC₅₀ values of the different organisms was assessed by Pearson’s correlation coefficient R. For each metabolite, the upper quartile x_0.75 of absolute log₂ fold-changes across all species was plotted against R. Only metabolites detected in more than 10 species were considered. Metabolites with |R| > 0.5 and x_0.75 > 1 are highlighted in blue (anticorrelating metabolites) and pink (correlating metabolites), and the names of representative compounds are listed. The full correlation data is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.s002" target="_blank">S2 Data</a>. (B) Visualization of metabolites correlating with salt tolerance on the KEGG metabolic pathway map using PathwayProjector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.ref047" target="_blank">47</a>]. Color intensity of metabolites indicates strength of positive (pink) or negative (blue) correlation, and size indicates x_0.75. Key pathways are highlighted and labeled. (C) Correlation of fold-change with salt tolerance for selected compounds in lower glycolysis; (D) in cysteine and methionine metabolism; (E) in branched-chain amino acid metabolism; and (F) in heme biosynthesis. In panels C to F mean and standard deviation of four (microbes) or three (human cell lines) replicates are shown. Note that metabolite annotations are based on accurate mass and can be ambiguous; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.s001" target="_blank">S1 Data</a> for complete annotations.</p

Individual metabolite responses are influenced by species taxonomy, habitat, cell wall thickness and salt tolerance.

<p>Four-way analysis of variance (ANOVA) was performed on log₂ metabolite fold-changes in the different species upon high salt stress (IC₅₀). Shown are the mean and standard errors of the 40 most significant metabolites of each factor (all with ANOVA <i>P</i> < 0.01 and |log₂ fold-change| > 1 in at least one group). Metabolites are sorted from left to right by ascending <i>P</i>-value. (A) Grouping of species according to taxonomic classification. (B) Grouping according to habitat. (C) Grouping according to cell wall thickness. (D) Grouping according to salt tolerance (IC₅₀ < 500 mM NaCl = low; 500 ≤ IC₅₀ ≤ 1,000 mM = medium; IC₅₀ > 1,000 mM = high). Note that metabolite annotations are based on accurate mass and can be ambiguous; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.s001" target="_blank">S1 Data</a> for complete annotations.</p

Responses of known osmoprotectants to sustained hyperosmotic salt stress.

<p>(A) Fold changes relative to unstressed control conditions of 54 detected metabolites with confirmed osmoprotective activity listed in the DEOP database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.ref036" target="_blank">36</a>], as well as of additional relevant polyprenyl quinones. For each species, only osmoprotectants showing a significant response (|log₂ fold-change| ≥ 1, <i>P</i> < 0.05, multiple-testing corrected two-sided unpaired <i>t</i>-tests) in at least one stress intensity were considered. Duplicate names represent different ions of the same metabolite because accurate mass annotation was ambiguous; refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.s001" target="_blank">S1 Data</a> for complete annotations. Osmoprotectants were grouped based on their main chemical characteristics. (B) Baseline abundances of osmoprotectants at IC₀ (unstressed control condition). Data are represented as Z-scores to compare relative abundances between species. Small Z-scores (|Z| < 1) are not colored.</p

Analysis of salt tolerance in fifteen diverse species.

<p>(A) Phylogenetic tree of analyzed species. Jukes-Cantor distances between bacteria are drawn to scale based on aligned 16S small ribosomal subunit RNA sequences. Distances between eukaryotes are not drawn to scale for visualization purposes. Organisms are colored based on taxonomic classification, and cell wall strengths and typical habitats are indicated. Further information about strains and cell lines is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148888#pone.0148888.s007" target="_blank">S1 Table</a>. (B) Sustained hyperosmotic salt tolerance based on growth inhibition experiments. Salt tolerance is expressed as mean and standard deviation (n = 2) of concentrations inhibiting growth rates by 10% (IC₁₀), 25% (IC₂₅) and 50% (IC₅₀) compared to unstressed conditions. Species are grouped according to taxonomic classification, and the colored horizontal bars indicate the average IC₅₀ of each taxonomic group. (C) Comparison of salt tolerance between species colonizing different habitats. (D) Comparison of salt tolerance between species with different cell wall strengths. Differences between groups in panels B to D were not statistically significant (P > 0.05, unpaired two-tailed <i>t</i>-tests) for all comparisons except those with human cell lines.</p