The Molecular Chaperone DnaJ is Involved in the Peroxide-Induced Oxidative Stress Response in Escherichia coli

Abstract

The chaperone protein DnaJ and sigma (σ) factor σ32 (often called RpoH) play important roles in the heat-shock response in Escherichia coli. Previous studies have demonstrated increased heat tolerance accompanied by increased RpoH concentrations in E. coli treated with a sub-minimal inhibitory concentrations (MIC) of kanamycin. DnaJ, along with other chaperone proteins, forms a complex with RpoH to facilitate its degradation in the absence of stressful stimuli. In this study, we explored the relative importance both proteins in the oxidative stress response in the presence and absence of a sub-MIC of kanamycin. Phagocytes of the human immune system often use reactive oxygen species upon contact with foreign bacteria to facilitate killing and prevent potential infection. Tolerance of E. coli strain C600, dna J mutant strain MF634, and Δ rpo H strain K165 to peroxide-induced oxidative stress was assessed by (i) monitoring cell growth in broth cultures containing H O and by (ii) measuring 2 2 zones of H O growth inhibition in a solid phase diffusion disc assay In broth cultures, growth of the wild-type 2 2. – strain was less negatively impacted by H O compared to the dna J and Δ rpo H mutants. In the disc diffusion assay, 2 2 zones of inhibition for WT were significantly smaller than those of the dna J mutant. The presence of a sub-MIC of kanamycin did not produce smaller zones of inhibition for either WT or mutant strains in the disc diffusion assay. Western blot detection analysis showed that in the absence of a sub-MIC of kanamycin, RpoH concentrations were – higher in the dna J mutant compared to the WT strain. Results of this study indicate that DnaJ is an important mediator of the oxidative stress and that a lack of a functional copy of dna J is sufficient to cause impairment of the oxidative stress response in E. coli.