Induction of<i>qsdA</i> gene transcription, NAHSL-breakdown and biocontrol activity of <b><i>R. erythropolis</i></b> in potato tubers.

<p>(A) <i>qsdA</i> gene transcription and biocontrol activity of the <i>R. erythropolis</i> BCA-<i>qsdA::gfp</i> against <i>P. atrosepticum</i> 6276 defective (<i>Pa</i>-QS<b>–</b>) or not (<i>Pa</i>-QS<b>+</b>) for NAHSL production were analyzed at 1, 2, 3 and 7 days after inoculation of <i>S. tuberosum</i> var. Allians tubers. For the controls, one of the two strains was replaced in the inoculum with a 0.9% NaCl solution. Asterisks indicate significantly less severe maceration symptoms in the presence of the BCA-<i>qsdA::gfp</i>, as assessed with the Mann and Whitney test (<i>α</i> = 0.05). The fluorescence of the BCA-<i>qsdA::gfp</i> was analyzed by confocal laser scanning microscopy. (B) The numbers of <i>P. atrosepticum</i> (black lines) and <i>R. erythropolis</i> (red lines) bacteria per unit weight (CFU/g fresh weight of potato tubers), and NAHSL concentration (ng/g of potato tubers; black and white bars) were determined for each condition in potato tubers. For lines and bars, each value is the mean of three replicates with the standard deviation indicated. NS, non-significant; NAHSL, <i>N</i>-acyl homoserine lactone.</p

NAHSL-breakdown and biocontrol activity of the QsdA-expressing<i>E. coli</i> strain in potato tubers.

<p>(A) <i>E. coli</i> DH5α(pUC19) (<i>Ec</i>) and <i>E. coli</i> DH5α(pUC19-<i>qsdA</i>) (<i>Ec-qsdA</i>) were compared for biocontrol activity against <i>P. atrosepticum</i> 6276 (<i>Pa</i>-QS+) 1, 2, 3 and 7 days after inoculation of potato tubers. For the controls, one or both strains were replaced in the inoculum with a 0.9% NaCl solution. Significant differences (Mann and Whitney test; <i>α</i> = 0.05) in maceration symptoms between infected tubers inoculated with the <i>Ec</i> or the <i>Ec-qsdA</i> strains are indicated with an asterisk. (B) Population dynamics of <i>P. atrosepticum</i> and <i>E. coli</i> bacteria (CFU/g fresh weight of potato tubers; black and blue lines respectively), and NAHSL concentration (ng/g of potato tubers; black and white bars), were determined for each condition in potato tubers. For lines and bars, each value is the mean of three replicates with the standard deviation indicated. NS, non-significant; NAHSL, <i>N</i>-acyl homoserine lactone.</p

NAHSL-breakdown and biocontrol activity of the<i>R.erythropolis qsdA</i> deletion mutant in potato tubers.

<p>(A) The <i>R. erythropolis</i> R138 wild-type (BCA) and the <i>R. erythropolis</i> R138 Δ<i>qsdA</i> (BCA-Δ<i>qsdA</i>) strains were compared for biocontrol activity against <i>P. atrosepticum</i> 6276 (<i>Pa</i>-QS+) 1, 2, 3 and 7 days after inoculation of potato tubers. For the controls, one or both strains were replaced in the inoculum with a 0.9% NaCl solution. Significant differences (Mann and Whitney test; <i>α</i> = 0.05) in maceration symptoms between infected tubers inoculated with the BCA or the BCA-Δ<i>qsdA</i> are indicated with an asterisk. (B) Population dynamics of <i>P. atrosepticum</i> and <i>R. erythropolis</i> bacteria (CFU/g fresh weight of potato tubers; black and red lines respectively), and NAHSL concentrations (ng/g of potato tubers; black and white bars) were determined for each condition in potato tubers. For lines and bars, each value is the mean of three replicates with the standard deviation indicated. NS, non-significant; NAHSL, <i>N</i>-acyl homoserine lactone.</p

Bacterial strains and plasmids.

<p>Km^R, Ap^R, Gm^R and Tc^R indicate resistance to kanamycin, ampicillin, gentamicin and tetracycline, respectively. NAHSL, <i>N</i>-acyl homoserine lactone; CFBP, Collection Française de Bactéries associées aux Plantes, Institut National de la Recherche Agronomique (INRA), Angers, France.</p

Effect of Substance P reverse (SPrev) and Substance P (SP) (10⁻⁶ M) on the biofilm formation activity of <i>Bacillus cereus</i>.

<p>The biofilm formation was observed after 2, 5 and 24 h. (A) Two dimensions (2D) and reconstructed three-dimensions (3D) and ortho cuts (3D/z) images showed that the density of the biofilm was essentially unchanged. (B) In contrast, the thickness of the biofilm was significantly increased after 5 and 24 h inclubation with SP (NS = <i>non significant</i>;  = <i>p<0.01</i>;  = <i>p<0.001</i>).</p

Bidimensional electrophoresis analysis of secreted proteins by <i>Bacillus cereus</i> after a 13 h exposure to Substance P reverse (Control) or Substance P (SP) (10⁻⁶ M).

<p>(<b>A</b>) Twelve spots were modified after exposure of the bacteria to SP. (<b>B</b>) Two proteins (1 and 2) were over produced whereas the expression of 10 others was down regulated. Identified proteins are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078773#pone-0078773-t001" target="_blank">Table 1</a>. Results are representative of three independent experiments.</p

Effect of Substance P treated <i>Bacillus cereus</i> on caspase 1 induction and on the morphology of HaCaT cells.

<p>(<b>A</b>) Cells exposed to SP treated bacteria showed an increase of caspase 1 production ( = <i>p<0.05</i>). (<b>B</b>) After a 30 min infection with control bacteria the morphology of HaCaT cells remained unchanged but when the cells were exposed to SP treated <i>B. cereus</i> we observed a complete disorganization of the actin cytoskeleton (□) and the formation of multiple cytoplasmic vacuoles (γ). Bar = 20 µm.</p

Inhibition of the effects of Substance P (SP) on the cytotoxic activity of <i>Bacillus cereus</i>, <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i>.

<p>(A) Before infection, bacteria were exposed to SP in the presence of thermal water from Uriage-les-Bains (UTW) or (B) Teflose® (TF) ( = <i>p<0.05</i>,  = <i>p<0.01</i>,  = <i>p<0.001</i>).</p

SDS page analysis of <i>Bacillus cereus</i> Substance P (SP) binding proteins separated by immunoprecipitation using SP antibody-associated beads.

<p>SP was found able to bind on a 43-TOF/TOF as the Thermo Unstable ribosomal Elongation factor Ef-Tu (arrow: 43 kDa). SPrev was also capable of binding to this protein but with a reduced effectiveness. Results are representative of three independent experiments.</p

SDS page analysis of secreted Bacillus cereus proteins after 1 or 13(SP) or Substance P reverse (SPrev) (10⁻⁶ M).

<p>Bacteria exposed for 1 h to SP showed an increase in the production of the collagenase (ColT) and of superoxide dismutase (SOD). Bacteria treated for 13 h with SP presented in contrast an increase in the S-layer crystal protein (SL). Results are representative of three independent experiments.</p