The DnaK/DnaJ Chaperone System Enables RNA Polymerase-DksA Complex Formation in Salmonella Experiencing Oxidative Stress

Our previous biochemical approaches showed that the oxidoreductase activity of the DnaJ protein facilitates the interaction of oxidized DksA with RNA polymerase. Investigations herein demonstrate that under biologically relevant conditions the DnaJ- and DksA-codependent activation of the stringent response in Salmonella undergoing oxidative stress involves the DnaK chaperone. Oxidation of DksA cysteine residues stimulates redox-based and holdase interactions with zincbinding and C-terminal domains of DnaJ. Genetic and biochemical evidence indicates that His(33) in the liPD motif in the J domain of DnaJ facilitates interactions of unfolded DksA with DnaK. A mutation in His(33) in the J domain prevents the presentation of unfolded DksA to DnaK without limiting the oxidoreductase activity mapped to DnaJ's zinc-2 site. Thr(199) in the ATPase catalytic site of DnaK is required for the formation of the DksA/RNA polymerase complex. The DnaK/DnaJ/DksA complex enables the formation of an enzymatically active RNA polymerase holoenzyme that stimulates transcription of branched-chain amino acid and histidine metabolic genes in Salmonella exposed to reactive oxygen species. The DnaK/DnaJ chaperone protects Salmonella against the cytotoxicity associated with reactive oxygen species generated by the phagocyte NADPH oxidase in the innate host response. The antioxidant defenses associated with DnaK/DnaJ can in part be ascribed to the elicitation of the DksA-dependent stringent response and the protection this chaperone system provides against protein carbonylation in Salmonella undergoing oxidative stress. IMPORTANCE DksA was discovered 30years ago in a screen for suppressors that reversed the thermosensitivity of Escherichia colt mutant strains deficient in DnaK/DnaJ, raising the possibility that this chaperone system may control DksA function. Since its serendipitous discovery, DksA has emerged as a key activator of the transcriptional program called the stringent response in Gram-negative bacteria experiencing diverse adverse conditions, including nutritional starvation or oxidative stress. DksA activates the stringent response through the allosteric control this regulatory protein exerts on the kinetics of RNA polymerase promoter open complexes. Recent investigations have shown that DksA overexpression protects dnaKl mutant bacteria against heat shock indirectly via the ancestral chaperone polyphosphate, casting doubt on a possible complexation of DnaK, DnaJ, and DksA. Nonetheless, research presented herein demonstrates that the cochaperones DnaK and DnaJ enable DksA/RNA polymerase complex formation in response to oxidative stress.

Automated summary

The study demonstrates that under oxidative stress conditions, the DnaJ and DksA co-dependently activate the stringent response in Salmonella with the assistance of DnaK, facilitating interactions between unfolded DksA and DnaK in a redox-based manner. This complex of DnaK/DnaJ/DksA enables the formation of an active RNA polymerase holoenzyme, which enhances the expression of specific genes related to amino acid and histidine metabolism in Salmonella exposed to reactive oxygen species.