Redox-based signaling is fundamental to the capacity of bacteria to sense, and respond to, nitrosative and oxidative stress encountered in natural and host environments. The conserved RNA polymerase regulatory protein DksA is a thiol-based sensor of reactive nitrogen and oxygen species. DksA-dependent transcriptional control promotes antinitrosative and antioxidative defenses that contribute to Salmonella pathogenesis. The specific adaptive changes mediated by DksA in response to reactive species, however, have not been elucidated. Herein, we characterize DksA-dependent changes in gene expression in Salmonella enterica experiencing nitrosative stress. Genome-wide expression analysis of wild-type and ΔdksA Salmonella exposed to the nitric oxide ((•)NO) donor DETA NONOate demonstrated (•)NO- and DksA-dependent regulatory control of 427 target genes. Transcriptional changes centered primarily on genes encoding aspects of cellular metabolism. Several antioxidants and oxidoreductases important in redox buffering, (•)NO detoxification, and damage repair were also observed to be up-regulated in an (•)NO- and DksA-dependent manner. Compared to wild-type bacteria, (•)NO-treated ΔdksA Salmonella exhibited a de-repression of genes encoding components of iron homeostasis and failed to activate sulfur assimilation and cysteine biosynthetic operons. As cysteine is integral to efficient antinitrosative and antioxidative defense and repair programs, we further examined the redox-responsive transcriptional control of cysteine biosynthesis by DksA. These investigations revealed that the activation of genes comprising cysteine biosynthesis also occurs in response to hydrogen peroxide, is dependent upon the redox-sensing zinc finger motif of DksA, and requires the transcriptional regulator CysB. Our observations demonstrate that DksA mediates global adaptation to nitrosative stress in Salmonella and provide unique insight into a novel regulatory mechanism by which cysteine biosynthesis is controlled in response to reactive oxygen and nitrogen species.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response.

We show that thiols in the 4-cysteine zinc-finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide- or nitric oxide (NO)-mediated modifications of thiols in the DksA 4-cysteine zinc-finger motif release the metal cofactor and drive reversible changes in the α-helicity of the protein. Wild-type and relA spoT mutant Salmonella, but not isogenic dksA-deficient bacteria, experience the downregulation of r-protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA-dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc-bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine-tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys(114) , and neighbouring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress.