Principle component analysis of ¹H-NMR spectra of unstressed and HOCl-stressed <i>E</i>. <i>coli</i> cell extracts.

<p>(A) PCA scores plot (on the left side) and loadings plot (on the right side) derived from ^<b>1</b>H-NMR data of methanol extracts of unstressed <i>E</i>. <i>coli</i> MG1655 cells (black dots) and cells stressed for 5 min (blue dots), 10 min (green dots), 20 min (red dots), 40 min (orange dots),) and 60 min (purple dots). Each dot in the scores plot represents a certain ^<b>1</b>H-NMR spectrum (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125823#pone.0125823.s003" target="_blank">S1 Table</a>). Each point in the loadings plot represents a spectral region (bin) on the scale of 0.05 ppm. Loadings highlighted in red boxes mark the ^<b>1</b>H-NMR peaks that were responsible for the greatest variance in the data and therefore for the group building in the scores plot. Scores and loadings for PC1 against PC2 are shown. Percentages in brackets specify the variance within all samples explained by a PC. (B) Recalculation of the PCA presented in Fig 2A by applying the same parameters and spectra; the bin at 2.67 ppm was not considered (see text for more details). (C) PCA scores plot (on the left side) and loadings plot (on the right side) derived from ^<b>1</b>H-NMR data of methanol extracts of unstressed <i>E</i>. <i>coli</i> MG1655 cells (black dots) and cells stressed for 5 min (blue dots), 10 min (green dots),) and 20 min (red dots). By focusing on the early stress response of <i>E</i>. <i>coli</i>, the loadings plot revealed additional outliers that contributed to the group formation in the scores plot.</p

Time-dependent GC/MS analysis of metabolites involved in various metabolic pathways.

<p>Relative concentrations of various metabolites involved in different metabolic pathways such as phosphate metabolism, organic acid metabolism and amine metabolism, identified by GC/MS analysis prior to HOCl stress (white bars) and in cells treated with 50 μM HOCl for 20 min (blue bars), 40 min (green bars), and 60 min (red bars). Concentrations are normalized to dry cell mass. Hit percentage of detected compounds compared with the NIST05 and NIST05s library is indicated in brackets after the compound name. Statistical significances are indicated as asterisks determined by Student’s t-test: *p < P<0.05; **p < P<0.01; and ***p < P<0.001. Shown is the mean ± standard deviation (n ≥ 3).</p

Time-dependent GC/MS analysis of metabolites involved in fatty acid metabolism and membrane integrity.

<p>Relative concentrations of metabolites involved in fatty acid metabolism and membrane integrity identified by GC/MS analysis prior to HOCl stress (white bars) and in cells treated with 50 μM HOCl for 20 min (blue bars), 40 min (green bars), and 60 min (red bars). Concentrations are normalized to dry cell mass. Hit percentage of detected compounds compared with the NIST05 and NIST05s library is indicated in brackets after the compound name. Statistical significances are indicated as asterisks determined by Student’s t-test: *p < P<0.05; **p < P<0.01; and ***p < P<0.001. Shown is the mean ± standard deviation (n ≥ 3).</p

Identification of a sub-lethal HOCl concentration.

<p>Viability of <i>E</i>. <i>coli</i> MG1655 cells was analyzed in M9 minimal medium in the presence of the indicated concentrations of HOCl. Samples were removed after 10 min (A) or the indicated time points (B), serially diluted to 1:30, and spotted onto LB agar plates (1st to 5th dilution). (A) The viability was analyzed in 25 μM HOCl concentration steps. Cells were 100% viable at 0 and 25 μM HOCl. At 50 μM HOCl, they showed first indications of cell stress. Nevertheless, the cell viability remained at a high level. At 75 μM HOCl, the cells are harshly stressed and show a very low viability. (B) Cells were treated with 50 μM HOCl for various time points. The survival assay indicates cell stress; however, the viability remains high even after 60 min of HOCl treatment. The figure shown is the result of one representative experiment.</p

Time-dependent GC/MS analysis of amino acid levels of unstressed and HOCl stressed cells.

<p>Relative concentrations of amino acids identified by GC/MS analysis prior to HOCl stress (white bars), and cells treated with 50 μM HOCl for 20 min (blue bars), 40 min (green bars), and 60 min (red bars). Concentrations are normalized to dry cell mass. Hit percentage of detected compounds compared with the NIST05 and NIST05s library is indicated in brackets after the compound name. Statistical significances are indicated as asterisks determined by Student’s t-test: *p < P<0.05; **p < P<0.01; and ***p < P<0.001. Shown is the mean ± standard deviation (n ≥ 3).</p

Model of metabolite alterations upon sub-lethal HOCl stress.

<p>Shown are the effects of HOCl stress within the first 20 min on the metabolism of <i>E</i>. <i>coli</i>. Identified metabolites are highlighted with a box. Metabolites that are downregulated are depicted in green, while upregulated metabolites are shown in red. Abbreviations: Ala, alanine; Asp, aspartate; Cit, citrate; Fum, fumarate; Glu, glutamate; GSH, glutathione; Iso-Cit, isocitrate; KG, α-ketoglutarate; Mal, malate; Met, methionine; Phe, phenylalanine; Pro, proline; Pyro-Glu, pyroglutamic acid; Succ, succinate; Succ-Coa, succinate-CoA; Ox-Succ, oxalosuccinate; Thr, threonine.</p

Stress-dependent alteration of metabolite concentrations after 20 min as determined by GC/MS and comparison with H₂O₂ exposure.

<p>Overview of the concentration alterations after HOCl exposure of metabolites included in the discussion of the main text. Comparison of metabolite levels of a previous study by Jozefczuk et al. who analyzed the metabolite alteration after H₂O₂ exposure. Alterations of the concentrations are indicated by arrows (↑ increase; ↓ decrease). For detailed information, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125823#pone.0125823.s004" target="_blank">S2 Table</a>.</p><p>Stress-dependent alteration of metabolite concentrations after 20 min as determined by GC/MS and comparison with H₂O₂ exposure.</p

Reference metabolites measured by ¹H-NMR spectroscopy using MeOD as solvent.

<p>s—singlet, d—doublet, dd—doublet of doublets, t—triplet, q—quartet, m—multiplet</p><p>Metabolites were measured under the same conditions as bacterial samples and referenced to TMS.</p><p>Reference metabolites measured by ¹H-NMR spectroscopy using MeOD as solvent.</p

Metabolic Response of <i>Escherichia coli</i> upon Treatment with Hypochlorite at Sub-Lethal Concentrations

<div><p>Hypochlorite is a reactive oxygen species that is worldwide as an antibacterial disinfectant. Hypochlorite exposure is known to cause oxidative damage to DNA and proteins. As a response to these effects, the metabolite profiles of organisms treated with sub-lethal doses of hypochlorite are assumed to be severely modified; however, the nature of these changes is hardly understood. Therefore, using nuclear magnetic resonance spectroscopy and gas chromatography-coupled mass spectrometry, we analyzed the time-dependent impact of hypochlorite exposure with a sub-lethal concentration (50 µM) on the metabolite profile of the <i>Escherichia coli</i> strain MG1655. Principle component analysis clearly distinguished between the metabolite profiles of bacteria treated for 0, 5,10, 20, 40, or 60 min. Major changes in the relative amounts of fatty acids, acetic acid, and formic acid occurred within the first 5 min. Comparative gas chromatography-coupled mass spectrometry analyses revealed that the amounts of free methionine and alanine were significantly decreased in the treated cells, demonstrating their susceptibility to hypochlorite exposure. The concentrations of succinate, urea, orotic acid, 2-aminobutyric acid, and 2-hydroxybutyric acid were also severely affected, indicating general changes in the metabolic network by hypochlorite. However, most metabolite levels relaxed to the reference values of untreated cells after 40–60 min, reflecting the capability of <i>E</i>. <i>coli</i> to rapidly adapt to environmental stress factors such as the presence of sub-lethal oxidant levels.</p></div