PapE induces the release of CpxP from CpxA.

<p>A) mSPINE experiments were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107383#pone-0107383-g002" target="_blank">Fig. 2A</a> with <i>E. coli</i> TG1 producing CpxA-Strep and either CpxP (WT) or the cleft mutant CpxPA108V (A108V) without or with PapE co-expression. Due to reduced CpxA-Strep level, mSPINE samples co-expressing PapE were five-fold stronger concentrated than samples without PapE. To allow comparison, immunoblots were cut into an upper and lower part. The upper part was probed with antiserum against CpxA and the lower part with antiserum against CpxP. Immunodetection was carried out for both parts simultaneously. Black triangles show specific and the white triangle unspecific reactions. Shown are representatives of three biological replicates. B) To visualize protein level in each mSPINE experiment, whole cells from (A) were collected after formaldehyde treatment, subjected to immunological determination using antiserum to CpxA, CpxP, and MalE (loading control). Purified CpxA-His6, His6-CpxP and MalE served as controls for antibody specificity (C). Black triangles show specific and white triangles unspecific reactions.</p

Model depicting CpxP-dependent signal integration by the Cpx-two-component system.

<p>(A) Polar interaction between the inner cavity of the CpxP dimer and CpxA keeps the sensor kinase in an “Off” mode. The release of CpxP from CpxA switches CpxA to the “On” mode (B–C). Release of CpxP from CpxA results from a high salt concentration that disturbs the polar interaction between the two proteins (B), or by competing interaction of CpxP with misfolded P-pilus subunits (C) (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107383#pone.0107383-Hunke1" target="_blank">[7]</a>).</p

Membrane-SPINE demonstrates physical interaction between CpxP and CpxA in vivo.

<p>A) <i>E. coli</i> TG1 producing CpxA-Strep (pKT01E) was grown in LB to OD600 = 1.3 and crosslinking was performed for 20 min with 0.6% formaldehyde (CH₂O). TG1 carrying the CpxA-Strep producing plasmid pKT01E without formaldehyde treatment served as a control. Cytosolic membranes were prepared, membrane proteins were solubilized by detergent treatment and CpxA-Strep was purified according to our established protocol for mSPINE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107383#pone.0107383-Mller1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107383#pone.0107383-Mller2" target="_blank">[32]</a>. Aliquots of each sample were incubated at 95°C for 20 min to separate cross-linked proteins from CpxA-Strep and subjected to immunological detection using antiserum to the CpxA, the CpxR and the CpxP protein, respectively. Purified CpxA-His6, His6-CpxR and His6-CpxP served as controls for antibody specificity (C). Black triangles show specific and the white triangle unspecific reactions. Shown are representatives of two biological replicates. B) To check the protein level of CpxP, cell fractionation assays were performed. <i>E. coli</i> TG1 cells producing CpxA-Strep (pKT01E) and CpxP from different vectors (pTcpxP, pBcpxP) were grown in LB to OD600 = 0.6. Periplasmic fractions and membrane fractions were prepared and subjected to immunological detection using antiserum to the CpxP protein, the Strep-tag and the MalE protein (loading control), respectively. Purified His6-CpxP, CpxA-Strep and MalE served as controls for antibody specificity (C). C) mSPINE experiments were performed as described in (A) with <i>E. coli</i> TG1 producing CpxA-Strep (pKT01E) and CpxP (pBcpxP) grown in LB supplemented with 0.5 mM IPTG and 0.002% arabinose. Cells expressing <i>cpxA</i> without a Strep-tag (pEC01E) and <i>cpxP</i> (pBcpxP) with formaldehyde treatment (lanes 1 and 2) and cells carrying the CpxA-Strep producing plasmid pKT01E without formaldehyde treatment (lanes 3 and 4) served as controls. Purified CpxA-His6 and His6-CpxP served as controls for antibody specificity (C). Black triangles show specific and the white triangle unspecific reactions. Shown are representatives of two biological replicates. D) To verify similar CpxP protein level in each mSPINE experiment, whole cells from (C) were collected after formaldehyde treatment, and subjected to immunological determination using antiserum to the CpxP protein, and the MalE protein (loading control), respectively. Purified His6-CpxP and MalE served as controls for antibody specificity (C). Black triangles show specific and white triangles unspecific reactions.</p

Variant CpxPR56Q is not co-purified with CpxA.

<p>A) Substitution of amino acid residue R56 to Q of CpxP results in a stable protein that does not inhibit the Cpx-pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107383#pone.0107383-Zhou1" target="_blank">[27]</a>. mSPINE experiments were performed as described in Fig. 2A with <i>E. coli</i> TG1 producing CpxA-Strep (pKT01E) and CpxPR56Q (pBcpxPR56Q/R56Q) or CpxP (pBcpxP/WT) grown in LB supplemented with 0.002% arabinose. Black triangles show specific and the white triangle unspecific reactions. Shown are representatives of two biological replicates. B) To verify similar CpxP protein level in each mSPINE experiment, whole cells from (A) were collected after formaldehyde treatment, and subjected to immunological determination using antiserum to the CpxP protein, and the MalE protein (loading control), respectively. Purified His6-CpxP and MalE served as controls for antibody specificity (C). Black triangles show specific and white triangles unspecific reactions.</p

Dynamic Interaction between the CpxA Sensor Kinase and the Periplasmic Accessory Protein CpxP Mediates Signal Recognition in <i>E. coli</i>

<div><p>Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in <i>Escherichia coli</i> in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA <i>in</i><i>vivo.</i> Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.</p></div

BACTH demonstrates physical interaction between CpxP and the periplasmic sensor domain of CpxA.

<p>For protein–protein interaction analysis using a bacterial two-hybrid system (BACTH) CpxP and the sensor domain of CpxA (CpxA-SD) were fused to the N- or C-terminal ends of the T25 and T18 fragments of <i>B. pertussis</i> adenylate cyclase as indicated. Strain BTH101 was co-transformed with plasmids encoding the different T25- and T18-hybrid proteins. T25- and T18-fragments fused to the leucine zipper of transcription factor GCN4 and the empty vectors served as positive (+) and negative (−) controls. (A) Illustration of functional complementation of CyaA fragments by BACTH. Interaction between two hybrid proteins in the cytosol results in functional complementation between the T25 and T18 fragments, resulting in cAMP synthesis. cAMP together with the catabolite activator protein (CAP) induces the expression of <i>E. coli</i> sugar catabolic operons, such as lactose and maltose. (B) 3 µl of a LB overnight culture were spotted on a MacConkey-Lactose plate and incubated for 24 h at 30°C. (C) The degree of functional complementation between the indicated hybrid proteins was quantified by measuring ß-galactosidase activities in suspensions of toluene-treated <i>E. coli</i> BTH101 cells harboring the corresponding plasmids. The activity of the negative control (pKT25, pUT18C) represents the background (dashed line). Shown are the averages ± S.E.M. of three biological replicates each in technical triplicates (t test). Numbers above bars give percentage of ß-galactosidase activity relative to the positive control.</p

<i>E. coli</i> strains and plasmids used in this study.

<p><i>E. coli</i> strains and plasmids used in this study.</p