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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 dependence of the heat shock response. Cells of Pseudomonas putida strains were grown in LB broth to the logarithmic growth phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture temperature was shifted to 42°C and samples were taken at every 10 min three times (10, 20, and 30 min). The solubilized proteins were analyzed on 10% SDS-polyacrylamide gels and used for protein staining (prepared from cells in a culture of 100 μL, A) and immunoblotting (prepared from cells in a culture of 50 μL, B). (A) Heat shock response (HSR) in terms of protein levels. Coomassie Brilliant Blue staining of gels showed that the temperature shift induced representative heat shock proteins (ClpB, DnaK, HtpG, and GroEL), which were identified by mass spectrometry. The positions of relevant proteins are marked in the right margin. (B) HSR in terms of σ32 levels. The cellular level of σ32 during the temperature up-shift was examined by western blotting with an antiserum against Serratia marcescens σ32. (C) Effect of gene disruptions on HSR in terms of protein levels. The representative heat shock proteins (Hsps) were induced at 42°C, but not significantly at 45°C. Temperature up-shifts downregulated levels of elongation-factor (EF) G and ribosomal protein S1. The positions of Hsps are shown in the middle margin, and those of EF-G (EF-G, ○) and ribosomal protein S1 (RpsA, •) are shown in the left margin.
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fig05: Temperature dependence of the heat shock response. Cells of Pseudomonas putida strains were grown in LB broth to the logarithmic growth phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture temperature was shifted to 42°C and samples were taken at every 10 min three times (10, 20, and 30 min). The solubilized proteins were analyzed on 10% SDS-polyacrylamide gels and used for protein staining (prepared from cells in a culture of 100 μL, A) and immunoblotting (prepared from cells in a culture of 50 μL, B). (A) Heat shock response (HSR) in terms of protein levels. Coomassie Brilliant Blue staining of gels showed that the temperature shift induced representative heat shock proteins (ClpB, DnaK, HtpG, and GroEL), which were identified by mass spectrometry. The positions of relevant proteins are marked in the right margin. (B) HSR in terms of σ32 levels. The cellular level of σ32 during the temperature up-shift was examined by western blotting with an antiserum against Serratia marcescens σ32. (C) Effect of gene disruptions on HSR in terms of protein levels. The representative heat shock proteins (Hsps) were induced at 42°C, but not significantly at 45°C. Temperature up-shifts downregulated levels of elongation-factor (EF) G and ribosomal protein S1. The positions of Hsps are shown in the middle margin, and those of EF-G (EF-G, ○) and ribosomal protein S1 (RpsA, •) are shown in the left margin.

Mentions: We examined whether insertional inactivation mutations of clpB and the J-domain protein genes could cause any noticeable changes on the pattern of total cell proteins. Deletion of clpB, cbpA, and djlA apparently did not alter the pattern of cellular proteins in overnight-grown P. putida cells, and that of dnaJ increased the amounts of DnaK and GroEL slightly in the mutant, but their levels were much less than those in R2 (data not shown). We next monitored the pattern of total cell proteins in the wild-type strain upon various degrees of up-shift of the ambient temperature to examine the effect of the inactivation mutations on the HSR (Fig. 5A). When logarithmically growing cell cultures were transferred from 30°C to 33°C, the increase of Hsps was not significant. At 35°C, slight increases of DnaK, GroEL, and HtpG were detectable, and protein bands for ClpB emerged. Larger up-shifts of temperature induced these proteins further, up to 42°C; however, at 45°C, the amounts of DnaK, GroEL, and HtpG increased for the first 10 min only, whereas that of ClpB seemed to increase continually (Fig. 5A). The increase of Hsps was not obvious at 50°C. The HSR in terms of protein synthesis in P. putida mutant strains KTΔclpB, KTΔdnaJ, KTΔcbpA, and KTΔdjlA were also examined at 42°C and 45°C (Fig. 5C). All strains exhibited essentially the same response pattern as the wild-type strain at each temperature. We noticed that two major protein species of 84 and 66 kDa were significantly decreased at both temperatures, especially in KTΔclpB. Time-of-flight mass spectrometry analyses revealed that they were elongation factor-(EF)-G and ribosomal protein S1, respectively.


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 dependence of the heat shock response. Cells of Pseudomonas putida strains were grown in LB broth to the logarithmic growth phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture temperature was shifted to 42°C and samples were taken at every 10 min three times (10, 20, and 30 min). The solubilized proteins were analyzed on 10% SDS-polyacrylamide gels and used for protein staining (prepared from cells in a culture of 100 μL, A) and immunoblotting (prepared from cells in a culture of 50 μL, B). (A) Heat shock response (HSR) in terms of protein levels. Coomassie Brilliant Blue staining of gels showed that the temperature shift induced representative heat shock proteins (ClpB, DnaK, HtpG, and GroEL), which were identified by mass spectrometry. The positions of relevant proteins are marked in the right margin. (B) HSR in terms of σ32 levels. The cellular level of σ32 during the temperature up-shift was examined by western blotting with an antiserum against Serratia marcescens σ32. (C) Effect of gene disruptions on HSR in terms of protein levels. The representative heat shock proteins (Hsps) were induced at 42°C, but not significantly at 45°C. Temperature up-shifts downregulated levels of elongation-factor (EF) G and ribosomal protein S1. The positions of Hsps are shown in the middle margin, and those of EF-G (EF-G, ○) and ribosomal protein S1 (RpsA, •) are shown in the left margin.
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Related In: Results  -  Collection

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fig05: Temperature dependence of the heat shock response. Cells of Pseudomonas putida strains were grown in LB broth to the logarithmic growth phase (OD600 ˜ 1.0) at 30°C. After a sample was taken at time 0, the culture temperature was shifted to 42°C and samples were taken at every 10 min three times (10, 20, and 30 min). The solubilized proteins were analyzed on 10% SDS-polyacrylamide gels and used for protein staining (prepared from cells in a culture of 100 μL, A) and immunoblotting (prepared from cells in a culture of 50 μL, B). (A) Heat shock response (HSR) in terms of protein levels. Coomassie Brilliant Blue staining of gels showed that the temperature shift induced representative heat shock proteins (ClpB, DnaK, HtpG, and GroEL), which were identified by mass spectrometry. The positions of relevant proteins are marked in the right margin. (B) HSR in terms of σ32 levels. The cellular level of σ32 during the temperature up-shift was examined by western blotting with an antiserum against Serratia marcescens σ32. (C) Effect of gene disruptions on HSR in terms of protein levels. The representative heat shock proteins (Hsps) were induced at 42°C, but not significantly at 45°C. Temperature up-shifts downregulated levels of elongation-factor (EF) G and ribosomal protein S1. The positions of Hsps are shown in the middle margin, and those of EF-G (EF-G, ○) and ribosomal protein S1 (RpsA, •) are shown in the left margin.
Mentions: We examined whether insertional inactivation mutations of clpB and the J-domain protein genes could cause any noticeable changes on the pattern of total cell proteins. Deletion of clpB, cbpA, and djlA apparently did not alter the pattern of cellular proteins in overnight-grown P. putida cells, and that of dnaJ increased the amounts of DnaK and GroEL slightly in the mutant, but their levels were much less than those in R2 (data not shown). We next monitored the pattern of total cell proteins in the wild-type strain upon various degrees of up-shift of the ambient temperature to examine the effect of the inactivation mutations on the HSR (Fig. 5A). When logarithmically growing cell cultures were transferred from 30°C to 33°C, the increase of Hsps was not significant. At 35°C, slight increases of DnaK, GroEL, and HtpG were detectable, and protein bands for ClpB emerged. Larger up-shifts of temperature induced these proteins further, up to 42°C; however, at 45°C, the amounts of DnaK, GroEL, and HtpG increased for the first 10 min only, whereas that of ClpB seemed to increase continually (Fig. 5A). The increase of Hsps was not obvious at 50°C. The HSR in terms of protein synthesis in P. putida mutant strains KTΔclpB, KTΔdnaJ, KTΔcbpA, and KTΔdjlA were also examined at 42°C and 45°C (Fig. 5C). All strains exhibited essentially the same response pattern as the wild-type strain at each temperature. We noticed that two major protein species of 84 and 66 kDa were significantly decreased at both temperatures, especially in KTΔclpB. Time-of-flight mass spectrometry analyses revealed that they were elongation factor-(EF)-G and ribosomal protein S1, respectively.

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