- Research article
- Open Access
Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli
© Jana et al; licensee BioMed Central Ltd. 2009
Received: 16 September 2008
Accepted: 29 January 2009
Published: 29 January 2009
Protonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in E. coli cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in E. coli were correlated.
Induction of heat-shock-like response in E. coli attained the maximum level after about 20 minutes of cell growth in the presence of a protonophore like carbonyl cyanide m-chloro phenylhydrazone (CCCP) or 2, 4-dinitrophenol (DNP). With induction, cellular level of the heat-shock regulator protein sigma-32 also increased. The increase in sigma-32 level was resulted solely from its stabilization, not from its increased synthesis. On the other hand, the protonophores inhibited the translocation of the periplasmic protein alkaline phosphatase (AP), resulting its accumulation in cell cytosol partly in aggregated and partly in dispersed form. On further cell growth, after withdrawal of the protonophores, the previously accumulated AP could not be translocated out; instead the AP-aggregate had been degraded perhaps by an induced heat-shock protease ClpP. Moreover, the non-translocated AP formed binary complex with the induced heat-shock chaperone DnaK and the excess cellular concentration of DnaK disallowed the induction of heat-shock response by the protonophores.
Our experimental results suggested that the protonophores-mediated accumulation and aggregation of membrane proteins (like AP) in cell cytosol had signaled the induction of heat-shock proteins in E. coli and the non-translocated protein aggregates were possibly degraded by an induced heat-shock protease ClpP. Moreover, the induction of heat-shock response occurred by the stabilization of sigma-32. As, normally the DnaK-bound sigma-32 was known to be degraded by the heat-shock protease FtsH, our experimental results further suggested that the engagement of DnaK with the non-translocated proteins (like AP) had made the sigma-32 free and stable.
The heat-shock response is a universal reaction in nature to defend cells against the temperature-induced damage. Cells of bacteria or almost any organism respond to sudden increase in temperature by synthesizing a set of proteins called the heat-shock proteins (hsps). In E. coli, heat-shock regulon includes genes for about 30 proteins and is induced after a temperature up-shift from 30 to 45°C. The hsps counter the effects of heat by serving as 1) molecular chaperones (e.g., GroEL, GroES, DnaK, DnaJ, ClpB etc.) that assist in the refolding of the partially denatured proteins and 2) proteases (e.g., Lon, ClpP, FtsH etc.) that degrade and remove the permanently denatured proteins . Not only important during heat stress, many hsps are present at the basal level in cells to assist protein folding . Transcription of the heat shock genes is known to be initiated by RNA polymerase, which contains the alternative sigma factor sigma-32 . At normal growth condition, cellular concentration of sigma-32 is very low (10–30 copies/cell at 30°C) and increases up to 12–15 folds with the temperature up-shift .
Instead of heat, cytoplasmic accumulation of the membrane or periplasmic proteins elevates the syntheses of hsps in E. coli. Any membrane or periplasmic protein of E. coli is known to be synthesized initially in cell cytoplasm as precursor form, which contains an N-terminal signal-sequence . The signal sequence targets the precursor towards the plasma membrane translocase that transports the precursor across the membrane . The signal peptide is then cleaved by a signal peptidase, an integral membrane protein with active site facing the periplasm . The matured protein is then positioned at its membrane or periplasmic location with functionally correct orientation. The PMF across E. coli plasma membrane acts as an energy source for protein translocation [8, 9]. The inhibition of translocation and consequent storage of membrane proteins in cell cytosol is found to induce hsps in export deficient mutants (where the multi-subunit translocase is nonfunctional) [10, 11], in signal sequence mutants (where the precursor proteins cannot be targeted to the translocase) [12, 13], and in wild type cells treated with protonophores like CCCP or DNP [14, 15]. However, it is still obscure how the inhibition of protein translocation phenomenon is related to the induction of cellular heat-shock response at the molecular level. Therefore, in the present study, we target to investigate 1) how the cellular level of the heat-shock regulator protein sigma-32 is modulated under the condition of inhibition of protein translocation by the protonophores like CCCP/DNP, 2) what is the final fate of the non-translocated proteins, stored in cell cytoplasm and 3) how the induced hsps do interact with the non-translocated proteins.
Bacterial strains and plasmid
The E. coli strain Mph42 , mostly used in this study, was a generous gift from Dr. Jonathan Beckwith, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, USA. The E. coli strains JT4000 (∇ lon-510)  and SG22159 (clpP:: kan) , mutants of the Lon and ClpP protease respectively, and their wild type strain SG20250 (MC4100, clp+, lon+)  were kindly gifted by Dr. Susan Gottesman, Laboratory of Molecular Biology, NCI, NIH Bethesda, USA. Sigma-32 was isolated from E. coli strain BB2012 (a His-tagged clone), a kind gift from Dr. Matthias P. Mayer, Institute for Biochemistry and Molecular Biology, University of Freidburg, Germany. The plasmid pET vector containing dnaK gene was obtained from Prof. C. K. Dasgupta, Department of Biophysics, Molecular Biology & Genetics, University of Calcutta, Kolkata, India.
Media, Reagents and Chemicals
All the components of growth medium, the required antibiotics, sucrose, lysozyme, NONIDET-P40 (NP40) and the electrophoresis reagents were purchased from Pharmacia Biotech., Sweden; purified E. coli AP, DNP, CCCP, antibody to GroEL, 4-chloro-1-napthol and Freunds adjuvant from Sigma-Aldrich, USA; Ni-NTA Agarose from QIAGEN, Germany; HRP-conjugated goat anti-rabbit IgG (secondary antibody) and proteinA-CL agarose from Genei, India; the Nitrocellulose transfer membrane from BioRad Laboratories, USA; 35S-methionine from Board of Radiation and Isotope Technology, India; H2O2, Tween-20 and anti-DnaK antibody from Merck, India; Isopropyl β-D-thiogalacto pyranoside (IPTG) and p-nitrophenyl phosphate (PNPP) from Sisco Research Laboratories, India.
Western blot experiment
This experiment was performed according to the method described in . Interested specific protein on the blotted membrane was identified by using the antiserum of the protein (raised in rabbit) as the primary antibody, HRP-conjugated goat anti-rabbit IgG as the secondary antibody and 4-chloro-1-napthol and H2O2 as the HRP substrates.
Pulse-label/Pulse-chase and immunoprecipitation experiments
Cells of E. coli Mph42 were initially grown to the log phase (up to [OD]600 nm ≈ 0.3, i.e., 1.5 × 108 cells/ml) at 30°C in MOPS medium (where the methionine concentration was 1/10th of the normal MOPS medium ) and were subsequently transferred to the methionine-free MOPS medium.
For pulse-label and immunoprecipitation experiment, log phase grown cells in methionine-free MOPS medium were allowed to grow further at 30°C. At different instants of growth, 1 ml cell aliquot was withdrawn to label with 35S-methionine (100 μCi/ml) for 1 min. The labeled cells were treated with 5% Trichloroacitic acid. The protein precipitate was washed with 80% cold acetone. The air dried precipitate was suspended in 50 μl of 50 mM Tris buffer (pH 8.0) containing 1% SDS and 1 mM EDTA. It was then heated at 100°C for 3 min; 30 μl of this sample was diluted with 1 ml of Triton X-100 buffer [2% Triton X-100, 50 mM Tris, pH 8.0, 150 mM NaCl and 1 mM EDTA] and centrifuged to remove nonspecific precipitates. From the supernatant, for immunoprecipitation of any protein, requisite amount of the antibody to that protein was added and subsequently incubated overnight at 0°C. To this incubated sample, 50 μl of proteinA-CL agarose was added and incubated further at 0°C for 20 min. The immunocomplex was washed and finally suspended in 50 μl of 2× sample buffer , heated at 100°C for 3 min prior to loading on 12% SDS-polyacrylamide gel for electrophoresis; finally phosphorimaging of the gel was performed in Typhoon 9210 (GE Health Care).
For pulse-chase and immunoprecipitation experiment, log phase grown cells in methionine-free MOPS medium were radio-labeled with 35S-methionine (at a concentration of 30 μCi/ml of cell culture) for the required time and the label was subsequently chased by 0.2 M cold methionine. At different instants of chasing, cell aliquot was withdrawn to extract proteins by the method of Oliver and Beckwith . Subsequent steps of immunoprecipitation from the cell extract with requisite amount of an antibody, gel electrophoresis and phosphorimaging were done as described above.
Induction, activity assay and determination of location of AP
For the induction of AP, E. coli MPh42 cells were grown in the phosphate-less MOPS medium at 30°C, as described in . At different instants of induction, an aliquot of 1.0 ml cell suspension was collected over 0.2 ml toluene and the activity of AP was assayed as described in , using PNPP as the substrate. The amount of AP, which led to a change of absorbance of p-nitrophenol by 0.1 per 6 min of enzyme-substrate reaction, had been considered as one unit of the enzyme .
For determination of the location of AP, the periplasmic, cytoplasmic and membrane fractions of cells were isolated from 1.0 ml of AP induced cell culture, as described in . After electrophoresis of the fractions in 12% SDS-polyacrylamide gel, 'western blot' experiment with anti-AP antibody was performed.
Isolation of aggregated proteins
Isolation of total soluble (containing dispersed protein pool) and insoluble (containing aggregated protein pool) cell fractions was based on the method described in . Cells were allowed to grow at 30°C in MOPS medium up to bacterial OD600 nm ~0.5. 25.0 ml of grown culture was rapidly cooled to 0°C and centrifuged at 4°C for 10 min at 6000 rpm. The cell pellet was re-suspended in 80 μl of buffer A [10 mM potassium phosphate buffer (pH-6.5); 1.0 mM EDTA; 20% (w/v) sucrose and 1.0 mg/ml lysozyme] and incubated for 30 min on ice. To the cell suspension, 720 μl of buffer B [10 mM potassium phosphate buffer (pH-6.5); 1 mM EDTA] was added and the cells were dipped in ice to sonicate by microtip ultrasonicator (using level 2, 1 min, 50% duty, three cycles). Intact cells were removed by centrifugation at 2000 g for 15 min at 4°C. The supernatant was further centrifuged at 15000 g for 20 min at 4°C and the pellet was collected. The pellet, which contained membrane and aggregated proteins, was washed with and finally re-suspended by brief sonication in 320 μl of buffer B. 80 μl of 10% (v/v) NP40 was then added to the suspension, mixed well and centrifuged at 15000 g for 30 min at 4°C to isolate the aggregated proteins as the pellet and to remove the membrane proteins as supernatant. The steps of re-suspension in buffer B, addition of NP40 and subsequent centrifugation were repeated three times. NP40-insoluble aggregated protein pellets were washed with 400 μl buffer B and finally re-suspended in 200 μl of buffer B.
Isolation and purification of sigma-32
The isolation and purification of the His-tagged sigma-32 from E. coli strain BB2012, using the Ni2+-NTA agarose column, were carried out according to .
Results and discussion
Under heat stress, the increase in sigma-32 was known to be caused by two means – by the increase in sigma-32 translation and by the stabilization of normally unstable sigma-32. Control of sigma-32 translation was mainly mediated by two cis-acting elements on sigma-32 mRNA; extensive base pairing between the elements formed secondary structure in sigma-32 mRNA, which had prevented its entry into the ribosome and consequently the translation initiation. The thermal induction of translation resulted from melting of the mRNA secondary structure at increased temperature . Again, control of sigma-32 stabilization is mediated by the hsps like DnaK/J and FtsH; normally at 30°C, the DnaK/J chaperone system binds with sigma-32, limiting its binding to core RNA polymerase  and the FtsH, an ATP-dependent metalloprotease, degrades sigma-32 (bound with DnaK/J) [25, 26]. Upon heat stress, the chaperone system DnaK/J becomes engaged with the increased cellular level of unfolded proteins and thus makes the sigma-32 free and stable .
The presence of aggregated proteins in cells was reported to elicit induction of hsps for cell survival . Therefore, in the following experiments, focus was made on the ultimate fate of the AP-aggregates in cytoplasm of the protonophores-treated cells, with a view to observe the role of induced hsps on the aggregates. The result of the following 'pulse-chase and immunoprecipitation' experiment on the E. coli strain SG20250 showed degradation of the AP-aggregate with time. For this study, log phase grown cells, in phosphate- and methionine-free MOPS medium, were allowed to label with 35S-methionine (30 μCi/ml) at 30°C in the presence of 50 μM CCCP. After 30 min of labeling, cells were transferred to fresh phosphate-free MOPS medium containing 1 mM BSA and 150 μg/ml spectinomycin and allowed to incubate further for 3 hr without CCCP. At 0 and 3 hr of chasing, equal volume of cell aliquot was withdrawn on ice, centrifuged and subjected to isolation of aggregated proteins. The isolated aggregates were immunoprecipitated with anti-AP antibody. The immunocomplex was run on 12% SDS-polyacrylamide gel, the gel was dried and subsequently set to autoradiography. The autoradiograph (Fig. 6B) of the electrophoresed immunoprecipitates indicated that the amount of AP-aggregate, after 3 hr of chasing (lane b), was about 66% less than its initial amount at 0 hr of chasing (lane a). This signified that the AP-aggregate had been degraded finally with time. It seemed that the degradation of AP-aggregate had been possibly caused by some induced heat-shock protease(s). When the degradation of the CCCP-mediated AP-aggregate was checked, by the same 'pulse-chase and immunoprecipitation' experiment in two different E. coli mutants for the heat-shock proteases Lon (JT4000) and ClpP (SG22159), it was observed that in the clpP mutant, no degradation of the AP-aggregate took place (lanes c and d, Fig. 6B); whereas in the lon mutant, degradation occurred (lanes e and f, Fig. 6B). This result clearly implied that not the major heat-shock protease Lon, rather a minor protease ClpP was responsible for the degradation phenomenon. Such degradation removed the translocation-incompetent, non-functional AP and thus was essential for cell survival; this was supplemented from the fact that the clpP mutant (SG22159) was more sensitive to CCCP than wild type strain SG20250. In the presence of 25 μM CCCP, where the wild type cells had some growth, the mutant cells became bacteriostatic, and by the treatment of 50 μM CCCP for 90 min, where there was no killing of E. coli SG20250 cells, about 90% cell-killing occurred in case of E. coli SG22159 strain (data not shown).
The whole study can, therefore, be concluded as: the protonophores like CCCP and DNP, by blocking the translocation of membrane and periplasmic proteins in E. coli, caused cytoplasmic accumulation of those proteins – partly in the insoluble aggregated form and partly in the soluble dispersed form; such stored proteins, which could never be translocated out even after removal of the protonophores, had induced cellular heat-shock response enhancing the syntheses of a few heat-shock chaperones and proteases – perhaps, the heat-shock protease ClpP ultimately degraded the non-translocated protein-aggregates to remove them from the cell. Moreover, the induction of hsps had taken place mainly due to stabilization of the normally unstable heat-shock regulator protein sigma-32; the stabilization had occurred due to titration of the chaperone system DnaK/J by the non-translocated, inactive periplasmic and membrane proteins stored in the cytoplasm of the CCCP-treated cells, because the titration consequently made the sigma-32 free of DnaK/J and so prevented its cleavage by the FtsH protease.
The Department of Science and Technology, Govt. of India is acknowledged for the financial assistance (Project No. SR/SO/BB-51/2006) and also for its 'FIST Programme – 2001-2011', going on in our department to provide different instrumental and infrastructural support.
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