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A mutation in dnaK causes stabilization of the heat shock sigma factor σ32, accumulation of heat shock proteins and increase in toluene-resistance in Pseudomonas putida.

Heat shock gene expression is regulated by the cellular level and activity of the stress sigma factor σ(32) in Gram-negative bacteria. A toluene-resistant, temperature-sensitive derivative strain of Pseudomonas putida KT2442, designated KT2442-R2 (R2), accumulated several heat shock proteins (HSPs) under non-stress conditions. Genome sequencing of strain R2 revealed that its genome contains a number of point mutations, including a CGT to CCT change in dnaK resulting in an Arg445 to Pro substitution in DnaK. DNA microarray and real-time reverse transcription polymerase chain reaction analyses revealed that the mRNA levels of representative hsp genes (e.g. dnaK, htpG and groEL) were upregulated in R2 cells in the stationary phase. Wild-type and R2 cells showed similar heat shock responses at hsp mRNA and HSP levels; however, the σ(32) level in the mutant was not downregulated in the shut-off stage. Strain R2 harbouring plasmid-borne dnaK grew at 37°C, did not accumulate HSPs, and was more sensitive to toluene than strain R2. It is worth to note that that revertant of R2 able to grow at 37°C were isolated and exhibited a replacement of Pro445 by Ser or Leu in DnaK. Thus, the mutation in dnaK causes the temperature-sensitive phenotype, improper stabilization of σ(32) leading to HSP accumulation and increased toluene resistance in strain R2.