The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

Abstract Background The aminoglycoside antibiotic gentamicin was supposed to induce a crosstalk between the Cpx- and the Arc-two-component systems (TCS). Here, we investigated the physical interaction of the respective TCS components and compared the results with their respective gene expression and protein abundance. The findings were interpreted in relation to the global proteome profile upon gentamicin treatment. Results We observed specific interaction between CpxA and ArcA upon treatment with the aminoglycoside gentamicin using Membrane-Strep-tagged protein interaction experiments (mSPINE). This interaction was neither accompanied by detectable phosphorylation of ArcA nor by activation of the Arc system via CpxA. Furthermore, no changes in absolute amounts of the Cpx- and Arc-TCS could be determined with the sensitive single reaction monitoring (SRM) in presence of gentamicin. Nevertheless, upon applying shotgun mass spectrometry analysis after treatment with gentamicin, we observed a reduction of ArcA ~ P-dependent protein synthesis and a significant Cpx-dependent alteration in the global proteome profile of E. coli. Conclusions This study points to the importance of the Cpx-TCS within the complex regulatory network in the E. coli response to aminoglycoside-caused stress

Interaction Analysis of a Two-Component System Using Nanodiscs.

Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems

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

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

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

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

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