Expression profiles of protein spots.

<p>Protein spots PO2 (1), PO8 (2), PO9 (3), PO11 (4) and PO12 (5) of <i>Lb</i>. <i>paracasei</i> subsp. <i>paracasei</i> F19 in control (A) or under defined sublethal potassium chloride (1 M, 60 min) conditions (B).</p

MALDI-TOF Mass Spectrometry Enables a Comprehensive and Fast Analysis of Dynamics and Qualities of Stress Responses of <i>Lactobacillus paracasei</i> subsp. <i>paracasei</i> F19

<div><p>Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected <i>Lactobacillus</i> (<i>Lb</i>.) <i>paracasei</i> subsp. <i>paracasei</i> TMW 1.1434 (F19) to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress). We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS), which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC), it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses.</p></div

Stress qualities (SQ), stress treatments (ST) of various stress intensities (SI) subjected to <i>Lb</i>. <i>paracasei</i> subsp. <i>paracasei</i> F19 and identified sublethal stress condition (SSC).

<p>Stress qualities (SQ), stress treatments (ST) of various stress intensities (SI) subjected to <i>Lb</i>. <i>paracasei</i> subsp. <i>paracasei</i> F19 and identified sublethal stress condition (SSC).</p

Stacked spectra of eight different strains.

<p>30 single spectra of each strain were summarized to one mean spectra; y-axis represent the intensity (= Int) of the recorded peaks; x-axis show the mass to charges from 2000 to 12000 Da; the ID of each strain is placed on the right sight of every spectra with the code TMW (Technische Mikrobiologie Weihenstephan); beer types are written in brackets (WB = wheat beer; Alt = Alt-beer; Dias = <i>S</i>. <i>cerevisiae</i> var. <i>diastaticus</i>).</p

MALDI-TOF mass spectra obtained for <i>Lb</i>. <i>paracasei</i> subsp. <i>paracasei</i> F19 in the mass range from 2 000 Da to 15 000 Da.

<p>Control conditions (control) and sublethal potassium chloride at different sampling points are indicated. Dotted line 1 marks a single peak at approx. 6 991 m/z (1) with decreasing peak intensity. Dotted lines 2, 3, 4 and 5 mark peaks at 2 106 m/z (2), 2 122 m/z (3), 2 549 m/z (4) and 2 649 m/z (5) with increasing peak intensity.</p

Probable proteins identified from 2D gel electrophoresis.

<p>Displayed are the analysed protein spots from 2D gel electrophoresis (sample), the probable protein identifications (Protein name) including the protein identification probability (%) and sequence coverage (%) in accordance to the accession no., molecular weight (Mol wt) (kDa) and pI value of each protein.</p

The DAPC analysis performed with the ‘adegenet’ package for the R software, displayed as a scatterplot of the first 2 principle components.

<p>477 MALDI-TOF mass spectra were considered. (A) Optimal clusters defined by ‘find.clusters’. (B) Data labelled according to induced sublethal stress conditions.</p

2D electrophoresis analysis of silver stained whole cell proteome of <i>Lb</i>. <i>paracasei</i> subsp. <i>paracasei</i> F19.

<p>P, grown under sublethal potassium chloride stress. O, proteins overexpressed in potassium-stressed cells. R, proteins repressed in potassium-stressed cells.</p

Stacked spectra of four different strains.

<p>30 single spectra of each strain were summarized to one mean spectra; y-axis represent the intensity (= Int) of the recorded peaks; x-axis show the mass to charges from 2000 to 12000 Da; the ID of each strain is placed on the right sight of every spectra with the code TMW (Technische Mikrobiologie Weihenstephan); beer types are written in brackets (WB = wheat beer; Dias = <i>S</i>. <i>cerevisiae</i> var. <i>diastaticus</i>); blue boxes with dotted lines figure out peak differences.</p

Growth curve analysis of <i>Lb</i>. <i>paracasei</i> subsp. <i>paracasei</i> F19.

<p>(A) Growth in control conditions and in stress broth of different concentrations of potassium chloride. (B) Growth rate μ_stress plotted against concentration (M).</p