Model for GSR regulation in <i>S</i>. <i>melonis</i> Fr1, which involves SdrG and PhyT.

<p>Upon stress induction, the Paks autophosphorylate and transfer the phosphoryl group to the SDRR SdrG. Direct phosphorylation of PhyR by the Paks is inhibited due to PhyT-PhyR complex formation. PhyT transfers the phosphoryl group from SdrG~P to PhyR. PhyR~P dissociates from PhyT to bind the anti-sigma factor NepR and thereby releases the alternative sigma-factor EcfG, which initiates transcription of the GSR regulon by binding to RNA polymerase. For further details on the PhyR-NepR-EcfG cascade and Paks see [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.ref019" target="_blank">19</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.ref021" target="_blank">21</a>].</p

PhyT is a negative regulator of GSR <i>in vivo</i>.

<p>β-galactosidase activity of the EcfG-dependent <i>nhaA2p-lacZ</i> fusion in indicated <i>S</i>. <i>melonis</i> Fr1 mutant backgrounds upon overnight overexpression of <i>phyT</i> from the cumate-inducible pQH vector with 25 μM cumate. Empty pQH vector was used as a negative control. Black bars and gray bars represent β-galactosidase activity pre- and 1 h post-induction with the chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP). Values are given as mean ±SD of three independent experiments.</p

Membrane localization of PhyR depends on stress level in a H341-PhyT dependent fashion.

<p>Spinning-disc confocal images of different <i>S</i>. <i>melonis</i> Fr1 knockout mutants (A) upon production of sfGFP-PhyR, which was induced by addition of 25 μM cumate for 12 min. The chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP) was applied for 60 min. (B) Bacteria were imaged under unstressed conditions upon production of sfGFP-PhyR, which was induced by addition of 25 μM cumate for 12 min. (C) Bacteria were imaged under unstressed conditions upon overnight production of PhyT or the PhyT (H341A) derivative, which was induced by addition of 25 μM cumate and production of sfGFP-PhyR, induced by addition of 250 μM vanillate for 12 min. Scale bar, 5 μm. Comparable production of PhyT and the PhyT (H341A) derivative was confirmed using Western blot analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.s002" target="_blank">S2C Fig</a>).</p

Phosphorylatable SdrG is essential for its positive regulatory role.

<p>β-galactosidase activity of the EcfG-dependent <i>nhaA2p-lacZ</i> fusion in indicated <i>S</i>. <i>melonis</i> Fr1 mutant backgrounds upon overnight overexpression of <i>sdrG</i> and variants from the vanillate-inducible pVH vector with 250 μM vanillate. pVH was used as empty-vector control. Black bars and gray bars represent β-galactosidase activity pre- and 1 h post-induction with the chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP), respectively. Values are given as mean ±SD of three independent experiments.</p

PhyT forms a complex with PhyR.

<p>(A) BACTH assay with bacteria spotted onto LB plates containing X-Gal (40 μg/mL), IPTG (0.5 mM) and antibiotics for selection. Wild-type PhyR and PhyR derivatives carrying either a D194A, a E235A or a combination of both mutations, were analyzed as C-terminal T18 fusions, while wild-type NepR was fused N-terminally and PhyT C-terminally to the T25 fragment of the <i>B</i>. <i>pertussis</i> CyaA protein to investigate interaction. Pictures were taken after 24 h of incubation at 30°C. Blue colonies indicate protein interaction. This image is a representative of three independent experiments. (B) β-galactosidase assays were performed for quantification in three biological replicates. Overnight cultures containing 0.5 mM IPTG and antibiotics for selection, were inoculated from single colonies of the co-transformed bacteria and incubated at 30°C. (C) Adenylate cyclase T18 fusions to PhyR proteins were detected in the samples used for quantitative analysis with Western blot analysis with CyaA monoclonal antibody (3D1) (1:2.000) (Santa Cruz Biotechnology) and a goat α-mouse antibody (1:3.000) with an exposure time of 2 min.</p

PhyT (formerly PhyP) transfers phosphoryl groups from SdrG~P to PhyR.

<p><i>In vitro</i> phosphotransfer from SdrG~P to PhyT and further to PhyR in absence and presence of NepR over time. SdrG (10 μM) was phosphorylated using PakF (autophosphorylated with [γ-32P] ATP) on Ni-NTA columns. PhyR (5 μM), NepR (7.5 μM) and <i>E</i>. <i>coli</i> membrane particles (5 mg membrane fraction/mL) harboring either wild-type PhyT or the PhyT (H341A) derivative as a control were added as indicated. For confirmation of comparable amounts of PhyT and PhyT (H341A), Western blot analysis was conducted (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.s002" target="_blank">S2A Fig</a>).</p