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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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Role of ClpB in the solubilization of thermo-mediated protein aggregates in Pseudomonas putida. Cells of P. putida strains were grown overnight at 30°C, and two aliquots were further cultured at 45°C for 30 min. One of the two aliquots that had been cultured at 45°C was further cultured at 30°C for 5 h. Insoluble proteins were prepared as described in Experimental Procedures. Fractions corresponding to identical cell masses (based on the optical density) were analyzed by SDS-PAGE (12% gel), and the proteins were visualized with Coomassie Brilliant Blue. The amount of protein loaded is shown below each lane. (A) ClpB was essential for the solibilization of thermo-mediated protein aggregates in P. putida. Proteins identified by time-of-flight mass spectrometry in the dnaJ mutant cells: 1, 50S ribosomal protein L2; 2, 30S ribosomal protein S2; 3, 30S ribosomal protein S3; 4, mixture of 50S ribosomal protein L3 and 30S ribosomal protein S4; 5, 50S ribosomal protein L5; 6, 50S ribosomal protein L16; and 7, 30S ribosomal protein S9. (B) The introduction of plasmid-borne clpB into the clpB mutant fully restored the disaggregation ability of the host cells. The clpB -mutant strain (KTΔclpB) carried an empty vector plasmid (pKT231).
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fig07: Role of ClpB in the solubilization of thermo-mediated protein aggregates in Pseudomonas putida. Cells of P. putida strains were grown overnight at 30°C, and two aliquots were further cultured at 45°C for 30 min. One of the two aliquots that had been cultured at 45°C was further cultured at 30°C for 5 h. Insoluble proteins were prepared as described in Experimental Procedures. Fractions corresponding to identical cell masses (based on the optical density) were analyzed by SDS-PAGE (12% gel), and the proteins were visualized with Coomassie Brilliant Blue. The amount of protein loaded is shown below each lane. (A) ClpB was essential for the solibilization of thermo-mediated protein aggregates in P. putida. Proteins identified by time-of-flight mass spectrometry in the dnaJ mutant cells: 1, 50S ribosomal protein L2; 2, 30S ribosomal protein S2; 3, 30S ribosomal protein S3; 4, mixture of 50S ribosomal protein L3 and 30S ribosomal protein S4; 5, 50S ribosomal protein L5; 6, 50S ribosomal protein L16; and 7, 30S ribosomal protein S9. (B) The introduction of plasmid-borne clpB into the clpB mutant fully restored the disaggregation ability of the host cells. The clpB -mutant strain (KTΔclpB) carried an empty vector plasmid (pKT231).

Mentions: The formation and disaggregation of heat-mediated protein aggregates in P. putida strains were examined in our study. Overnight-grown KT cells were exposed to various temperatures (from 30°C to 45°C) for 30 min to determine if thermal treatment could cause the formation of protein aggregates (Fig. S3). Compared with proteins in the insoluble fraction of KT cells grown at 30°C, which remained nonaggregated, some small-sized protein aggregates, (named hereafter as aggregated proteins) formed in the heat-stressed cultures, even at 37°C. The number and amount of aggregated proteins increased gradually upon temperature increase. A temperature shift-back to 30°C for less than 2 h allowed significant decreases of the thermo-mediated protein aggregates. The formation and decrease of protein aggregates in P. putida KTΔclpB and in the three J-domain protein gene mutants were also examined (Fig. 7). At 30°C, the KTΔdnaJ strain alone accumulated several unique insoluble proteins, which were subsequently found to be ribosomal proteins (Fig. 7A). Exposure to 45°C for 30 min resulted in protein aggregate formation in every strain. The amounts of protein aggregates formed in the wild-type, KTΔclpB, KTΔcbpA, and KTΔdjlA cells were quite similar; however, clearly more aggregates were formed in the dnaJ mutant, especially those with a high-molecular-mass (>200 kDa). Although a certain amount of protein aggregates that had formed in the dnaJ mutant cells had disappeared, those in the clpB mutant cells were virtually not solubilized during the recovery phase. A deficiency in cbpA or djlA had no effect on the formation and removal of protein aggregates. The introduction of pKT231-borne clpB into the clpB mutant cells (Fig. 7B), and that of pKT231-borne dnaJ into the dnaJ mutant cells (Fig. S4), allowed recovery of the protein aggregate clearance ability, indicating that these genes complemented the corresponding gene defects in the chromosome. Meanwhile, DnaK should also be essential for the solubilization of thermo-mediated protein aggregates, as aggregates that had formed in the dnaK point mutant R2 cells were not solubilized (Fig. S5). Essentially, the same result was obtained for R2ΔclpB cells, but a slight decrease of aggregated proteins was observed in the dnaJ mutant cells.


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

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

Role of ClpB in the solubilization of thermo-mediated protein aggregates in Pseudomonas putida. Cells of P. putida strains were grown overnight at 30°C, and two aliquots were further cultured at 45°C for 30 min. One of the two aliquots that had been cultured at 45°C was further cultured at 30°C for 5 h. Insoluble proteins were prepared as described in Experimental Procedures. Fractions corresponding to identical cell masses (based on the optical density) were analyzed by SDS-PAGE (12% gel), and the proteins were visualized with Coomassie Brilliant Blue. The amount of protein loaded is shown below each lane. (A) ClpB was essential for the solibilization of thermo-mediated protein aggregates in P. putida. Proteins identified by time-of-flight mass spectrometry in the dnaJ mutant cells: 1, 50S ribosomal protein L2; 2, 30S ribosomal protein S2; 3, 30S ribosomal protein S3; 4, mixture of 50S ribosomal protein L3 and 30S ribosomal protein S4; 5, 50S ribosomal protein L5; 6, 50S ribosomal protein L16; and 7, 30S ribosomal protein S9. (B) The introduction of plasmid-borne clpB into the clpB mutant fully restored the disaggregation ability of the host cells. The clpB -mutant strain (KTΔclpB) carried an empty vector plasmid (pKT231).
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fig07: Role of ClpB in the solubilization of thermo-mediated protein aggregates in Pseudomonas putida. Cells of P. putida strains were grown overnight at 30°C, and two aliquots were further cultured at 45°C for 30 min. One of the two aliquots that had been cultured at 45°C was further cultured at 30°C for 5 h. Insoluble proteins were prepared as described in Experimental Procedures. Fractions corresponding to identical cell masses (based on the optical density) were analyzed by SDS-PAGE (12% gel), and the proteins were visualized with Coomassie Brilliant Blue. The amount of protein loaded is shown below each lane. (A) ClpB was essential for the solibilization of thermo-mediated protein aggregates in P. putida. Proteins identified by time-of-flight mass spectrometry in the dnaJ mutant cells: 1, 50S ribosomal protein L2; 2, 30S ribosomal protein S2; 3, 30S ribosomal protein S3; 4, mixture of 50S ribosomal protein L3 and 30S ribosomal protein S4; 5, 50S ribosomal protein L5; 6, 50S ribosomal protein L16; and 7, 30S ribosomal protein S9. (B) The introduction of plasmid-borne clpB into the clpB mutant fully restored the disaggregation ability of the host cells. The clpB -mutant strain (KTΔclpB) carried an empty vector plasmid (pKT231).
Mentions: The formation and disaggregation of heat-mediated protein aggregates in P. putida strains were examined in our study. Overnight-grown KT cells were exposed to various temperatures (from 30°C to 45°C) for 30 min to determine if thermal treatment could cause the formation of protein aggregates (Fig. S3). Compared with proteins in the insoluble fraction of KT cells grown at 30°C, which remained nonaggregated, some small-sized protein aggregates, (named hereafter as aggregated proteins) formed in the heat-stressed cultures, even at 37°C. The number and amount of aggregated proteins increased gradually upon temperature increase. A temperature shift-back to 30°C for less than 2 h allowed significant decreases of the thermo-mediated protein aggregates. The formation and decrease of protein aggregates in P. putida KTΔclpB and in the three J-domain protein gene mutants were also examined (Fig. 7). At 30°C, the KTΔdnaJ strain alone accumulated several unique insoluble proteins, which were subsequently found to be ribosomal proteins (Fig. 7A). Exposure to 45°C for 30 min resulted in protein aggregate formation in every strain. The amounts of protein aggregates formed in the wild-type, KTΔclpB, KTΔcbpA, and KTΔdjlA cells were quite similar; however, clearly more aggregates were formed in the dnaJ mutant, especially those with a high-molecular-mass (>200 kDa). Although a certain amount of protein aggregates that had formed in the dnaJ mutant cells had disappeared, those in the clpB mutant cells were virtually not solubilized during the recovery phase. A deficiency in cbpA or djlA had no effect on the formation and removal of protein aggregates. The introduction of pKT231-borne clpB into the clpB mutant cells (Fig. 7B), and that of pKT231-borne dnaJ into the dnaJ mutant cells (Fig. S4), allowed recovery of the protein aggregate clearance ability, indicating that these genes complemented the corresponding gene defects in the chromosome. Meanwhile, DnaK should also be essential for the solubilization of thermo-mediated protein aggregates, as aggregates that had formed in the dnaK point mutant R2 cells were not solubilized (Fig. S5). Essentially, the same result was obtained for R2ΔclpB cells, but a slight decrease of aggregated proteins was observed in the dnaJ mutant cells.

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