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Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli.

Jana B, Panja S, Saha S, Basu T - BMC Microbiol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani - 741 235, West Bengal, India. bimal_edu@rediffmail.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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A. Rate of synthesis of GroEL in E. coli MPh42 cells at different instants of growth in the presence of 50 μM CCCP. Pulse-label at 0, 5, 10, 15, 20, 30, 40 and 50 minutes of cell growth and subsequent immunoprecipitation experiment using anti-GroEL antibody was performed as described in 'Methods'. B. The level of sigma-32 in the CCCP-treated E. coli MPh42 cells. Log phase grown cells were divided into three parts. One part was grown at 30°C, one part was grown at 50°C and the other part was grown in the presence of 50 μM CCCP at 30°C. After 20 min of growth, 1 ml cell aliquot was withdrawn from each set. Cellular proteins were extracted by boiling the cells with SDBME buffer [18] and equal amount of protein from each extract, estimated by Bradford method [37], was electrophoresed on 12% SDS-polyacrylamide gel and subsequently the western blot study was performed using anti-sigma-32 antibody. Lane a: cells grown at 30°C; lane b: cells grown at 50°C and lane c: cells grown at 30°C in the presence of 50 μM CCCP.
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Figure 1: A. Rate of synthesis of GroEL in E. coli MPh42 cells at different instants of growth in the presence of 50 μM CCCP. Pulse-label at 0, 5, 10, 15, 20, 30, 40 and 50 minutes of cell growth and subsequent immunoprecipitation experiment using anti-GroEL antibody was performed as described in 'Methods'. B. The level of sigma-32 in the CCCP-treated E. coli MPh42 cells. Log phase grown cells were divided into three parts. One part was grown at 30°C, one part was grown at 50°C and the other part was grown in the presence of 50 μM CCCP at 30°C. After 20 min of growth, 1 ml cell aliquot was withdrawn from each set. Cellular proteins were extracted by boiling the cells with SDBME buffer [18] and equal amount of protein from each extract, estimated by Bradford method [37], was electrophoresed on 12% SDS-polyacrylamide gel and subsequently the western blot study was performed using anti-sigma-32 antibody. Lane a: cells grown at 30°C; lane b: cells grown at 50°C and lane c: cells grown at 30°C in the presence of 50 μM CCCP.

Mentions: In this study, investigations were carried out to establish the correlation in molecular detail between the phenomena of inhibition of protein translocation and induction of hsps in E. coli, grown in the presence of protonophores like CCCP and DNP. Therefore, growth of E. coli cells in the presence of different concentrations of the protonophores was studied first and the results indicated that the increasing concentrations of CCCP (0 – 50 μM) or DNP (0 – 1.5 mM) in the growth medium had gradually slowed down the cell growth, causing bacteriostatic condition at 50 μM CCCP or 1.5 mM DNP (data not shown). When checked using 2-D gel electrophoresis technique, cell growth in the presence of CCCP (50 μM) or DNP(1.5 mM) was found to induce the hsps like ClpB, DnaK, GroEL, GrpE, ClpP, and GroES in E. coli cell (results not shown); protonophores-mediated induction of hsps were reported earlier (14, 15). As, in all the following experiments, the results for CCCP (50 μM) and DNP (1.5 mM) separately were qualitatively similar, the results for the CCCP only have been presented here. At different intervals of growth in the presence of CCCP, when the rate of GroEL synthesis was investigated by the pulse-label and immunoprecipitation experiment using anti-GroEL antibody, the result showed that the rate had increased with time up to 20 min (fig. 1A), beyond which it had declined. This implied that the maximum induction of hsps had taken place after 20 minutes of cell growth in the presence of 50 μM CCCP. After 20 min of cell growth, when the western blot experiment of cell extract was performed using anti-sigma-32 antibody, the result (fig. 1B) showed that the cellular level of the heat-shock regulator protein sigma-32 had also been increased (lane c) by the CCCP treatment. Fig. 1B also showed that the level of sigma-32 in normal cells was so low in amount that it had no trace (lane a) in the western blot. Similar enhancement of cellular sigma-32 level was found to take place in cells grown at 50°C (lane b).


Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli.

Jana B, Panja S, Saha S, Basu T - BMC Microbiol. (2009)

A. Rate of synthesis of GroEL in E. coli MPh42 cells at different instants of growth in the presence of 50 μM CCCP. Pulse-label at 0, 5, 10, 15, 20, 30, 40 and 50 minutes of cell growth and subsequent immunoprecipitation experiment using anti-GroEL antibody was performed as described in 'Methods'. B. The level of sigma-32 in the CCCP-treated E. coli MPh42 cells. Log phase grown cells were divided into three parts. One part was grown at 30°C, one part was grown at 50°C and the other part was grown in the presence of 50 μM CCCP at 30°C. After 20 min of growth, 1 ml cell aliquot was withdrawn from each set. Cellular proteins were extracted by boiling the cells with SDBME buffer [18] and equal amount of protein from each extract, estimated by Bradford method [37], was electrophoresed on 12% SDS-polyacrylamide gel and subsequently the western blot study was performed using anti-sigma-32 antibody. Lane a: cells grown at 30°C; lane b: cells grown at 50°C and lane c: cells grown at 30°C in the presence of 50 μM CCCP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: A. Rate of synthesis of GroEL in E. coli MPh42 cells at different instants of growth in the presence of 50 μM CCCP. Pulse-label at 0, 5, 10, 15, 20, 30, 40 and 50 minutes of cell growth and subsequent immunoprecipitation experiment using anti-GroEL antibody was performed as described in 'Methods'. B. The level of sigma-32 in the CCCP-treated E. coli MPh42 cells. Log phase grown cells were divided into three parts. One part was grown at 30°C, one part was grown at 50°C and the other part was grown in the presence of 50 μM CCCP at 30°C. After 20 min of growth, 1 ml cell aliquot was withdrawn from each set. Cellular proteins were extracted by boiling the cells with SDBME buffer [18] and equal amount of protein from each extract, estimated by Bradford method [37], was electrophoresed on 12% SDS-polyacrylamide gel and subsequently the western blot study was performed using anti-sigma-32 antibody. Lane a: cells grown at 30°C; lane b: cells grown at 50°C and lane c: cells grown at 30°C in the presence of 50 μM CCCP.
Mentions: In this study, investigations were carried out to establish the correlation in molecular detail between the phenomena of inhibition of protein translocation and induction of hsps in E. coli, grown in the presence of protonophores like CCCP and DNP. Therefore, growth of E. coli cells in the presence of different concentrations of the protonophores was studied first and the results indicated that the increasing concentrations of CCCP (0 – 50 μM) or DNP (0 – 1.5 mM) in the growth medium had gradually slowed down the cell growth, causing bacteriostatic condition at 50 μM CCCP or 1.5 mM DNP (data not shown). When checked using 2-D gel electrophoresis technique, cell growth in the presence of CCCP (50 μM) or DNP(1.5 mM) was found to induce the hsps like ClpB, DnaK, GroEL, GrpE, ClpP, and GroES in E. coli cell (results not shown); protonophores-mediated induction of hsps were reported earlier (14, 15). As, in all the following experiments, the results for CCCP (50 μM) and DNP (1.5 mM) separately were qualitatively similar, the results for the CCCP only have been presented here. At different intervals of growth in the presence of CCCP, when the rate of GroEL synthesis was investigated by the pulse-label and immunoprecipitation experiment using anti-GroEL antibody, the result showed that the rate had increased with time up to 20 min (fig. 1A), beyond which it had declined. This implied that the maximum induction of hsps had taken place after 20 minutes of cell growth in the presence of 50 μM CCCP. After 20 min of cell growth, when the western blot experiment of cell extract was performed using anti-sigma-32 antibody, the result (fig. 1B) showed that the cellular level of the heat-shock regulator protein sigma-32 had also been increased (lane c) by the CCCP treatment. Fig. 1B also showed that the level of sigma-32 in normal cells was so low in amount that it had no trace (lane a) in the western blot. Similar enhancement of cellular sigma-32 level was found to take place in cells grown at 50°C (lane b).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani - 741 235, West Bengal, India. bimal_edu@rediffmail.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus