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Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida.

Ito F, Tamiya T, Ohtsu I, Fujimura M, Fukumori F - Microbiologyopen (2014)

Bottom Line: Molecular chaperones function in various important physiological processes.Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles.P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of σ(32) activity and quantity, as well as the mRNA levels of hsp genes.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life Sciences, Toyo University, Gunma.

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Time course of mRNAs of relevant genes upon heat shock. Cells of Pseudomonas putida strains were grown in LB broth to the late logarithmic phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture was shifted to various temperatures, and samples were taken at every 10 min three times (10, 20, and 30 min). Total RNAs were prepared as described in Experimental Procedures. One-μg of total RNA was used to quantify the mRNA level of relevant genes. The relative amount of mRNA (in-fold) was calculated by assuming that one cycle of polymerase chain reaction doubles the amount, and that time 0 is taken as 1 for each gene. Data from at least three replicates are presented. Error bar indicates SD and some of error bars are covered by plot symbols. The raw data are listed in Table S3. The samples were the same as those used for the SDS-PAGE analysis shown in Figure 5. Symbols: dark blue (33°C), blue (35°C), pale blue (37°C), green (40°C), yellow (42°C) and red (45°C).
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fig06: Time course of mRNAs of relevant genes upon heat shock. Cells of Pseudomonas putida strains were grown in LB broth to the late logarithmic phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture was shifted to various temperatures, and samples were taken at every 10 min three times (10, 20, and 30 min). Total RNAs were prepared as described in Experimental Procedures. One-μg of total RNA was used to quantify the mRNA level of relevant genes. The relative amount of mRNA (in-fold) was calculated by assuming that one cycle of polymerase chain reaction doubles the amount, and that time 0 is taken as 1 for each gene. Data from at least three replicates are presented. Error bar indicates SD and some of error bars are covered by plot symbols. The raw data are listed in Table S3. The samples were the same as those used for the SDS-PAGE analysis shown in Figure 5. Symbols: dark blue (33°C), blue (35°C), pale blue (37°C), green (40°C), yellow (42°C) and red (45°C).

Mentions: The temperature-dependent transcriptional responses of four hsp genes (clpB, dnaK, htpG, and groEL) and two σ-factor genes (rpoD, which encodes σ70, and rpoH, which encodes σ32) were examined in parallel by quantifying their mRNA levels in the heat-treated KT cells by qRT-PCR (Fig. 6, Table S3). Increased expressions of the hsp genes occurred within 10 min and seemed to be correlated with the level of σ32 in the cell. Treatments at 40°C, 42°C, and 45°C resulted in the same induction pattern for the hsp genes, and their mRNA levels remained high after 30 min. At 33°C, clpB, dnaK, and htpG mRNAs were induced for the first 10 min and decreased rapidly in the next 10 min but then increased again after 30 min; however, this fluctuation was not observed for groEL. Higher temperature shifts caused less fluctuation. It should be noted that the fluctuation, which was also seen in the clpB mutant (data not shown), was compromised with a depletion of DnaJ (Table S3). The rpoD gene was induced during the first 10 min, but thereafter returned to the steady-state level. In contrast, rpoH mRNA continued to increase at every temperature, even though the level of σ32 decreased after transient induction at lower temperatures (Fig. 5B). Levels of the relevant mRNAs in KTΔclpB, KTΔdnaJ, KTΔalgU, and R2 cells were also measured at 42°C and 45°C (Fig. S 1, Table S3). Since P. putida AlgU belongs to the σ24 family and is known to be involved in rpoH expression (Aramaki et al. 2001), the HSR of KTΔalgU was examined. The induction ratios of the hsp genes in R2 seemed to be lower due to its high basal expression, but they were generally induced to levels equivalent to or higher than the other strains (Table S3).The expression profiles of the relevant genes were essentially the same in the tested strains at high temperatures, besides that of rpoH in KTΔalgU. The significant decrease of the rpoH mRNA level after prolonged treatment at 45°C indicated that rpoH was primary controlled by AlgU at this temperature (Fig. S1, Table S3).


Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida.

Ito F, Tamiya T, Ohtsu I, Fujimura M, Fukumori F - Microbiologyopen (2014)

Time course of mRNAs of relevant genes upon heat shock. Cells of Pseudomonas putida strains were grown in LB broth to the late logarithmic phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture was shifted to various temperatures, and samples were taken at every 10 min three times (10, 20, and 30 min). Total RNAs were prepared as described in Experimental Procedures. One-μg of total RNA was used to quantify the mRNA level of relevant genes. The relative amount of mRNA (in-fold) was calculated by assuming that one cycle of polymerase chain reaction doubles the amount, and that time 0 is taken as 1 for each gene. Data from at least three replicates are presented. Error bar indicates SD and some of error bars are covered by plot symbols. The raw data are listed in Table S3. The samples were the same as those used for the SDS-PAGE analysis shown in Figure 5. Symbols: dark blue (33°C), blue (35°C), pale blue (37°C), green (40°C), yellow (42°C) and red (45°C).
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fig06: Time course of mRNAs of relevant genes upon heat shock. Cells of Pseudomonas putida strains were grown in LB broth to the late logarithmic phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture was shifted to various temperatures, and samples were taken at every 10 min three times (10, 20, and 30 min). Total RNAs were prepared as described in Experimental Procedures. One-μg of total RNA was used to quantify the mRNA level of relevant genes. The relative amount of mRNA (in-fold) was calculated by assuming that one cycle of polymerase chain reaction doubles the amount, and that time 0 is taken as 1 for each gene. Data from at least three replicates are presented. Error bar indicates SD and some of error bars are covered by plot symbols. The raw data are listed in Table S3. The samples were the same as those used for the SDS-PAGE analysis shown in Figure 5. Symbols: dark blue (33°C), blue (35°C), pale blue (37°C), green (40°C), yellow (42°C) and red (45°C).
Mentions: The temperature-dependent transcriptional responses of four hsp genes (clpB, dnaK, htpG, and groEL) and two σ-factor genes (rpoD, which encodes σ70, and rpoH, which encodes σ32) were examined in parallel by quantifying their mRNA levels in the heat-treated KT cells by qRT-PCR (Fig. 6, Table S3). Increased expressions of the hsp genes occurred within 10 min and seemed to be correlated with the level of σ32 in the cell. Treatments at 40°C, 42°C, and 45°C resulted in the same induction pattern for the hsp genes, and their mRNA levels remained high after 30 min. At 33°C, clpB, dnaK, and htpG mRNAs were induced for the first 10 min and decreased rapidly in the next 10 min but then increased again after 30 min; however, this fluctuation was not observed for groEL. Higher temperature shifts caused less fluctuation. It should be noted that the fluctuation, which was also seen in the clpB mutant (data not shown), was compromised with a depletion of DnaJ (Table S3). The rpoD gene was induced during the first 10 min, but thereafter returned to the steady-state level. In contrast, rpoH mRNA continued to increase at every temperature, even though the level of σ32 decreased after transient induction at lower temperatures (Fig. 5B). Levels of the relevant mRNAs in KTΔclpB, KTΔdnaJ, KTΔalgU, and R2 cells were also measured at 42°C and 45°C (Fig. S 1, Table S3). Since P. putida AlgU belongs to the σ24 family and is known to be involved in rpoH expression (Aramaki et al. 2001), the HSR of KTΔalgU was examined. The induction ratios of the hsp genes in R2 seemed to be lower due to its high basal expression, but they were generally induced to levels equivalent to or higher than the other strains (Table S3).The expression profiles of the relevant genes were essentially the same in the tested strains at high temperatures, besides that of rpoH in KTΔalgU. The significant decrease of the rpoH mRNA level after prolonged treatment at 45°C indicated that rpoH was primary controlled by AlgU at this temperature (Fig. S1, Table S3).

Bottom Line: Molecular chaperones function in various important physiological processes.Null mutants of genes for the molecular chaperone ClpB (Hsp104), and those that encode J-domain proteins (DnaJ, CbpA, and DjlA), which may act as Hsp40 co-chaperones of DnaK (Hsp70), were constructed from Pseudomonas putida KT2442 (KT) to elucidate their roles.P. putida CbpA, a probable Hsp, partially substituted the functions of DnaJ in cell growth and solubilization of thermo-mediated protein aggregates, and might be involved in the HSR which was regulated by a fine-tuning system(s) that could sense subtle changes in the ambient temperature and control the levels of σ(32) activity and quantity, as well as the mRNA levels of hsp genes.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life Sciences, Toyo University, Gunma.

Show MeSH
Related in: MedlinePlus