Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida.
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.
Affiliation: Graduate School of Life Sciences, Toyo University, Gunma.Show MeSH
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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.
Affiliation: Graduate School of Life Sciences, Toyo University, Gunma.