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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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Sensitivity of Pseudomonas putida mutants to heat stress. Overnight-grown cells equivalent to an OD600 unit of 0.6 were mixed with LB broth in a total volume of 2 mL and incubated at 30°C for five doubling times of the tested strain, to reach the stationary phase. Then, 0.5 mL of the culture was transferred to a 16-mmφ glass tube and incubated for 5 min at 50°C. Viable counts were measured after plating serial dilutions of the culture onto LB solid medium at 30°C. The data are the means of three independent experiments. Error bar indicates SD.
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fig04: Sensitivity of Pseudomonas putida mutants to heat stress. Overnight-grown cells equivalent to an OD600 unit of 0.6 were mixed with LB broth in a total volume of 2 mL and incubated at 30°C for five doubling times of the tested strain, to reach the stationary phase. Then, 0.5 mL of the culture was transferred to a 16-mmφ glass tube and incubated for 5 min at 50°C. Viable counts were measured after plating serial dilutions of the culture onto LB solid medium at 30°C. The data are the means of three independent experiments. Error bar indicates SD.

Mentions: We next examined various P. putida strains for their sensitivity to high temperatures. For the strains of KT background, about one-tenth of stationary phase cells of the wild-type (KT) and all mutant strains obtained (except for KTΔclpB) retained their colony-forming ability after exposure to 50°C for 5 min (Fig. 4). Deletion of dnaJ, which caused growth defect, did not affect the survival rate. For the strains of R2 background, R2, and the R2ΔdjlA mutant showed survival rates that were similar to that of KT, but the loss of dnaJ or cbpA reduced the thermotolerance in the strain. Notably, the survival rates of clpB mutants isolated from both the KT and R2 strains were much lower than that of the others (Fig. 4). Introduction of plasmid-borne clpB recovered their survival rate, indicating that the exogenous clpB complemented the gene loss on the chromosome. Preliminary experiments revealed that logarithmically growing cells were considerably more sensitive to the thermal stress than were stationary phase cells, as was observed in Pseudomonas aeruginosa (Jørgensen et al. 1999). The thermal sensitivity shown by logarithmically growing cells examined at 45°C again indicated that the clpB mutants were quite sensitive to thermal stress (data not shown).


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

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

Sensitivity of Pseudomonas putida mutants to heat stress. Overnight-grown cells equivalent to an OD600 unit of 0.6 were mixed with LB broth in a total volume of 2 mL and incubated at 30°C for five doubling times of the tested strain, to reach the stationary phase. Then, 0.5 mL of the culture was transferred to a 16-mmφ glass tube and incubated for 5 min at 50°C. Viable counts were measured after plating serial dilutions of the culture onto LB solid medium at 30°C. The data are the means of three independent experiments. Error bar indicates SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263515&req=5

fig04: Sensitivity of Pseudomonas putida mutants to heat stress. Overnight-grown cells equivalent to an OD600 unit of 0.6 were mixed with LB broth in a total volume of 2 mL and incubated at 30°C for five doubling times of the tested strain, to reach the stationary phase. Then, 0.5 mL of the culture was transferred to a 16-mmφ glass tube and incubated for 5 min at 50°C. Viable counts were measured after plating serial dilutions of the culture onto LB solid medium at 30°C. The data are the means of three independent experiments. Error bar indicates SD.
Mentions: We next examined various P. putida strains for their sensitivity to high temperatures. For the strains of KT background, about one-tenth of stationary phase cells of the wild-type (KT) and all mutant strains obtained (except for KTΔclpB) retained their colony-forming ability after exposure to 50°C for 5 min (Fig. 4). Deletion of dnaJ, which caused growth defect, did not affect the survival rate. For the strains of R2 background, R2, and the R2ΔdjlA mutant showed survival rates that were similar to that of KT, but the loss of dnaJ or cbpA reduced the thermotolerance in the strain. Notably, the survival rates of clpB mutants isolated from both the KT and R2 strains were much lower than that of the others (Fig. 4). Introduction of plasmid-borne clpB recovered their survival rate, indicating that the exogenous clpB complemented the gene loss on the chromosome. Preliminary experiments revealed that logarithmically growing cells were considerably more sensitive to the thermal stress than were stationary phase cells, as was observed in Pseudomonas aeruginosa (Jørgensen et al. 1999). The thermal sensitivity shown by logarithmically growing cells examined at 45°C again indicated that the clpB mutants were quite sensitive to thermal stress (data not shown).

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