The 4‐cysteine zinc‐finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress

Abstract

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 Cys114, and neighboring 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.

Persister formation in Escherichia coli can be inhibited by treatment with nitric oxide

“…Several of those effects that could affect persister formation include inhibition of catalase activity (Brown, 1995), alteration of DksA activity (Henard, et al, 2014), and formation of peroxynitrite through reaction of NO with superoxide (Brunelli, et al, 1995). Since oxidative stress can increase persister levels (Vega, et al, 2012; Wu, et al, 2012) and NO impairs catalase activity, we treated an E. coli strain devoid of catalases (Δ katE Δ katG ) with DPTA at 4 h of growth, and measured type I persistence at 24 h. We observed that NO reduces type I persister levels of Δ katE Δ katG to a similar extent as wild type (Supplementary Figure S5A), which indicated that inhibition of catalase activity by NO does not explain the phenomenon described here.…”

“…NO is a critical antimicrobial used by the human immune system (Hibbs, et al, 1988), and exogenous administration of NO to infection sites using a number of different delivery mechanisms has produced promising results (Friedman, et al, 2011; Jones, et al, 2010; Robinson, et al, 2014; Sulemankhil, et al, 2012; Sun, et al, 2012) This molecule with diverse functions easily diffuses through the bacterial membranes and reacts with iron-sulfur clusters, superoxide, and oxygen, whereas its oxidized forms damage thiols, DNA, and residues on proteins (Robinson, et al, 2014). This reactivity provides NO with an ability to inhibit or disrupt a diverse set of enzymes and regulatory proteins, which include catalases, respiratory cytochromes, and bacterial reductases (Brown, 1995; Brunelli, et al, 1995; Henard, et al, 2014; Mason, et al, 2009). Such pleotropic effects lead to a complex relationship between NO and antibiotic tolerance.…”

Antioxidant Defense by Thioredoxin Can Occur Independently of Canonical Thiol-Disulfide Oxidoreductase Enzymatic Activity

“…Binding of recombinant TrxA-6His or TrxA C32A C35A-6His to GST-SsrB was analyzed as described previously (Henard et al, 2014). Briefly, 1 nmol of GST-SsrB proteins were incubated with 200 µl of Glutathione-Sepharose 4B beads (BioWorld, Dubin, OH) at 4°C for 2 h, washed with 20 bed volume of 50 mM Tris-HCl, pH 7.5, and then incubated with 2 nmole of TrxA-6His or TrxA C32A C35A-6His proteins at 4°C for 2 h with agitation.…”

“…Full-length TrxA and a C-terminal SsrB fragment encompassing residues 137–212 expressed as GST fusions from pGEX6P1 (GE Healthcare Biosciences, Fairfield, CT) were purified as described previously for DksA (Henard et al, 2014). Where indicated, 25 µM recombinant SsrB was treated with 250 µM H 2 O 2 for 1 h at 37°C.…”

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

“…In some bacterial pathogens, DksA proteins are critical for bacterial virulence (20)(21)(22)(23)(24). It was recently proposed that Salmonella DksA may act as a sensor for reactive oxygen and nitrogen species due to its redox-active thiols (25). Of the two DksA-like proteins present in Rhodobacter sphaeroides, only one was shown to regulate the stringent response and to be necessary for photosynthetic growth and utilization of exogenous amino acids (19).…”