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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.

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

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Temperature sensitivity of Pseudomonas putida mutant strains. Overnight-grown cells cultured in LB broth were serially diluted 10-fold with LB, and 5 μL of each dilution was then spotted onto prewarmed LB plates-containing streptomycin and incubated at the indicated temperatures. (A) Colony forming ratio at 35°C or 37°C. The wild-type (KT) strain and the dnaJ -mutant strain (KTΔdnaJ) carried either an empty vector plasmid (pKT231) or a plasmid carrying dnaJ, cbpA, or djlA. Colony numbers formed at 30°C is taken as 1 for each strain. Error bar indicates SD. (B) Photographs of the colonies. Most of the P. putida KT2442 dnaJ mutant cells could not form colonies on the plate at 35°C or 37°C, and visible colony formation required a few days. The plasmid-borne dnaJ efficiently restored the colony-forming ability of the mutant cells at both temperatures. Most of the P. putida dnaJ mutant cells carrying a plasmid-borne cbpA gene were able to form colonies at 35°C but grew slower than those carrying plasmid-borne dnaJ. Essentially the same result was obtained from three independent experiments. Representative photographs, taken after 1 day (not labeled), 2 days (Day2), or 3 days (Day3), are shown.
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fig03: Temperature sensitivity of Pseudomonas putida mutant strains. Overnight-grown cells cultured in LB broth were serially diluted 10-fold with LB, and 5 μL of each dilution was then spotted onto prewarmed LB plates-containing streptomycin and incubated at the indicated temperatures. (A) Colony forming ratio at 35°C or 37°C. The wild-type (KT) strain and the dnaJ -mutant strain (KTΔdnaJ) carried either an empty vector plasmid (pKT231) or a plasmid carrying dnaJ, cbpA, or djlA. Colony numbers formed at 30°C is taken as 1 for each strain. Error bar indicates SD. (B) Photographs of the colonies. Most of the P. putida KT2442 dnaJ mutant cells could not form colonies on the plate at 35°C or 37°C, and visible colony formation required a few days. The plasmid-borne dnaJ efficiently restored the colony-forming ability of the mutant cells at both temperatures. Most of the P. putida dnaJ mutant cells carrying a plasmid-borne cbpA gene were able to form colonies at 35°C but grew slower than those carrying plasmid-borne dnaJ. Essentially the same result was obtained from three independent experiments. Representative photographs, taken after 1 day (not labeled), 2 days (Day2), or 3 days (Day3), are shown.

Mentions: The E. coli DnaK system functions in the disaggregation of thermo-mediated protein aggregates, in cooperation with ClpB. We attempted to construct mutants of clpB and the J-domain protein genes from P. putida strain KT and its dnaK point mutant strain KT-R2 (R2) to assess their physiological roles. The acquisition of strains that each lacks clpB and one of the J-domain protein genes (dnaJ, cbpA, and djlA) would suggest that these genes are dispensable for P. putida under certain conditions (Table S1). A clpB mutant isolated from KT showed similar growth rates with the parental strain up to 37°C; however, that from R2 showed considerably slower growth at 33°C. Notably, dnaJ mutants from both KT and R2 showed retardation of growth at all the temperatures tested. Moreover, KTΔdnaJ was temperature sensitive. Large portions of the population failed to form colonies at 35°C and much less formed at 37°C (Fig. 3). The other J-domain protein gene mutants (KTΔcbpA and KTΔdjlA) were not temperature sensitive. The loss of dnaJ did not further affect the upper limit of growth temperature in strain R2 (Table S1). On the other hand, deletion of clpB and the J-domain protein genes caused marginal effects on their oxidative chemical tolerance (Table S2).


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

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

Temperature sensitivity of Pseudomonas putida mutant strains. Overnight-grown cells cultured in LB broth were serially diluted 10-fold with LB, and 5 μL of each dilution was then spotted onto prewarmed LB plates-containing streptomycin and incubated at the indicated temperatures. (A) Colony forming ratio at 35°C or 37°C. The wild-type (KT) strain and the dnaJ -mutant strain (KTΔdnaJ) carried either an empty vector plasmid (pKT231) or a plasmid carrying dnaJ, cbpA, or djlA. Colony numbers formed at 30°C is taken as 1 for each strain. Error bar indicates SD. (B) Photographs of the colonies. Most of the P. putida KT2442 dnaJ mutant cells could not form colonies on the plate at 35°C or 37°C, and visible colony formation required a few days. The plasmid-borne dnaJ efficiently restored the colony-forming ability of the mutant cells at both temperatures. Most of the P. putida dnaJ mutant cells carrying a plasmid-borne cbpA gene were able to form colonies at 35°C but grew slower than those carrying plasmid-borne dnaJ. Essentially the same result was obtained from three independent experiments. Representative photographs, taken after 1 day (not labeled), 2 days (Day2), or 3 days (Day3), are shown.
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Related In: Results  -  Collection

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fig03: Temperature sensitivity of Pseudomonas putida mutant strains. Overnight-grown cells cultured in LB broth were serially diluted 10-fold with LB, and 5 μL of each dilution was then spotted onto prewarmed LB plates-containing streptomycin and incubated at the indicated temperatures. (A) Colony forming ratio at 35°C or 37°C. The wild-type (KT) strain and the dnaJ -mutant strain (KTΔdnaJ) carried either an empty vector plasmid (pKT231) or a plasmid carrying dnaJ, cbpA, or djlA. Colony numbers formed at 30°C is taken as 1 for each strain. Error bar indicates SD. (B) Photographs of the colonies. Most of the P. putida KT2442 dnaJ mutant cells could not form colonies on the plate at 35°C or 37°C, and visible colony formation required a few days. The plasmid-borne dnaJ efficiently restored the colony-forming ability of the mutant cells at both temperatures. Most of the P. putida dnaJ mutant cells carrying a plasmid-borne cbpA gene were able to form colonies at 35°C but grew slower than those carrying plasmid-borne dnaJ. Essentially the same result was obtained from three independent experiments. Representative photographs, taken after 1 day (not labeled), 2 days (Day2), or 3 days (Day3), are shown.
Mentions: The E. coli DnaK system functions in the disaggregation of thermo-mediated protein aggregates, in cooperation with ClpB. We attempted to construct mutants of clpB and the J-domain protein genes from P. putida strain KT and its dnaK point mutant strain KT-R2 (R2) to assess their physiological roles. The acquisition of strains that each lacks clpB and one of the J-domain protein genes (dnaJ, cbpA, and djlA) would suggest that these genes are dispensable for P. putida under certain conditions (Table S1). A clpB mutant isolated from KT showed similar growth rates with the parental strain up to 37°C; however, that from R2 showed considerably slower growth at 33°C. Notably, dnaJ mutants from both KT and R2 showed retardation of growth at all the temperatures tested. Moreover, KTΔdnaJ was temperature sensitive. Large portions of the population failed to form colonies at 35°C and much less formed at 37°C (Fig. 3). The other J-domain protein gene mutants (KTΔcbpA and KTΔdjlA) were not temperature sensitive. The loss of dnaJ did not further affect the upper limit of growth temperature in strain R2 (Table S1). On the other hand, deletion of clpB and the J-domain protein genes caused marginal effects on their oxidative chemical tolerance (Table S2).

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