GroES/GroEL and DnaK/DnaJ have distinct roles in stress responses and during cell cycle progression in Caulobacter crescentus

Abstract

Misfolding and aggregation of protein molecules are major threats to all living organisms. Therefore, cells have evolved quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins. DnaK/DnaJ and GroES/GroEL are the best-characterized molecular chaperone systems in bacteria. In Caulobacter crescentus these chaperone machines are the products of essential genes, which are both induced by heat shock and cell cycle regulated. In this work, we characterized the viabilities of conditional dnaKJ and groESL mutants under different types of environmental stress, as well as under normal physiological conditions. We observed that C. crescentus cells with GroES/EL depleted are quite resistant to heat shock, ethanol, and freezing but are sensitive to oxidative, saline, and osmotic stresses. In contrast, cells with DnaK/J depleted are not affected by the presence of high concentrations of hydrogen peroxide, NaCl, and sucrose but have a lower survival rate after heat shock, exposure to ethanol, and freezing and are unable to acquire thermotolerance. Cells lacking these chaperones also have morphological defects under normal growth conditions. The absence of GroE proteins results in long, pinched filamentous cells with several Z-rings, whereas cells lacking DnaK/J are only somewhat more elongated than normal predivisional cells, and most of them do not have Z-rings. These findings indicate that there is cell division arrest, which occurs at different stages depending on the chaperone machine affected. Thus, the two chaperone systems have distinct roles in stress responses and during cell cycle progression in C. crescentus.

FIG. 1.

GroEL and DnaK levels in groESL and dnaKJ conditional mutants. Cells from parental strain NA1000 and from conditional mutants SG300 and SG400, in which the groESL and dnaKJ operons, respectively, are under control of promoter P_xylX, were grown at 30°C in the presence or absence of xylose for 6 h. At different times, samples were collected and proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using antisera against C. crescentus GroEL and DnaK, as described in Material and Methods. Equal amounts of protein were applied to all lanes.

FIG. 2.

Induction of DnaK/J synthesis but not induction of GroES/EL synthesis is important for survival after heat shock. Overnight SG300 and SG400 cultures growing in PYEX at 30°C were washed, diluted in PYEX or PYEG, and incubated for 6 h at 30°C. Cells were then transferred to a 42°C water bath with agitation, and samples were taken at different times and plated on PYEX. The survival values are the averages of three independent experiments, and the error bars indicate the standard deviations. Light gray bars, SG400 diluted in PYEX; open bars, SG400 diluted in PYEG; black bars, SG300 diluted in PYEX; dark gray bars, SG300 diluted in PYEG.

FIG. 3.

Absence of DnaK/J makes SG400 cells incapable of acquiring thermotolerance. Cultures of strains SG300 and SG400 were incubated at 30°C for 12 h in PYEX, and cells were subsequently washed, diluted in PYEX or PYEG, and incubated for 6 h at 30°C. A similar procedure was used for parental strain NA1000, except that only PYE was used. One half of the cultures were preconditioned for 30 min at the nonlethal temperature (40°C) (gray bars), while the other half of the cultures were preincubated at 30°C (open bars). The cultures were then exposed to 48°C for 30 min, and cell survival was evaluated after dilution and plating on PYEX agar. The values are the levels of cell survival (averages of three independent experiments) compared with the survival of control cultures grown in the absence of stress. The error bars indicate standard deviations.

FIG. 4.

Depletion of DnaK/J makes cells more sensitive to ethanol and freezing. (A) Overnight cultures of SG400 or SG300 in PYEX were washed, diluted in PYEX or PYEG, and incubated for 6 h at 30°C for depletion of DnaK/J or GroES/EL. The cultures were then exposed to 15% (vol/vol) ethanol, and samples were collected at different times and plated on PYEX. (B) Samples of the 6-h cultures were frozen at −80°C without any supplement. At different times, aliquots were transferred to room temperature, allowed to defrost, diluted, and plated on PYEX. The bars indicate the averages of three independent experiments, and the error bars indicate standard deviations. Light gray bars, SG400 diluted in PYEX; open bars, SG400 diluted in PYEG; black bars, SG300 diluted in PYEX; dark gray bars, SG300 diluted in PYEG.

FIG. 5.

Depletion of GroES/EL results in increased sensitivity to oxidative, osmotic, and saline stresses. Overnight cultures of SG400 or SG300 in PYEX were washed, diluted in PYEX or PYEG, and incubated for 6 h at 30°C. The cultures were divided and exposed to 2.5 mM H₂O₂ (A), 85 mM NaCl (B), or 150 mM sucrose (C), and samples were collected at different times and plated on PYEX. The values are the levels of cell survival (averages of three independent experiments) compared with the survival of control cultures grown in the absence of stress. The error bars indicate standard deviations. Light gray bars, SG400 diluted in PYEX; open bars, SG400 diluted in PYEG; black bars, SG300 diluted in PYEX; dark gray bars, SG300 diluted in PYEG.

FIG. 6.

Survival of mutant strains SG300 and SG400 under restrictive or permissive growth conditions. Cultures of strains NA1000 (A and B), SG300 (A), and SG400 (B) were grown in PYEX or PYEG liquid medium at 30°C and monitored through the logarithmic and stationary growth phases. The numbers of CFU were determined at different times by plating cell samples on solid medium (PYEX). The values are the averages of three independent experiments. •, SG300 grown in PYEX; ○, SG300 grown in PYEG; ▴, SG400 grown in PYEX; ▵, SG400 grown in PYEG; ▪, NA1000.

FIG. 7.

Altered morphology of SG300 and SG400 cells lacking GroES/L or DnaK/J. Strains SG300 (A and B) and SG400 (C to F) were grown in PYEX (A, C, and E) or PYEG (B, D, and F). Cells were visualized by light microscopy using a 100× objective and differential interference contrast optics after 10 h (A to D) and 24 h (E and F) of incubation at 30°C.

FIG. 8.

Septum formation in SG300 and SG400 cells grown under restrictive conditions. After incubation for 10 h in PYEX (A and C) or PYEG (B and D), SG300 (A and B) and SG400 (C and D) cells were stained with the membrane dye FM1-43, and images were captured using a 100× objective and an FITC filter. Arrowheads indicate invagination of the cytoplasmic membrane.

FIG. 9.

Immunolocalization of FtsZ in SG300 and SG400 cells. After 10 h of incubation in PYEX (A and C) or PYEG (B and D), SG300 (A and B) and SG400 (C and D) cells were collected and prepared for immunofluorescence microscopy using the anti-FtsZ antibody and an FITC-labeled goat anti-rabbit secondary antibody. Images were captured with a Roper CoolSnap HQ cooled charge-coupled device camera using a 100× objective and an FITC filter. Arrowheads indicate the Z ring.