Role of Cysteines in the Stability and DNA-Binding Activity of the Hypochlorite-Specific Transcription Factor HypT

Abstract

Reactive oxygen species are important components of the immune response. Hypochlorite (HOCl) is produced by neutrophils to kill invading microorganisms. The bactericidal activity of HOCl is due to proteome-wide unfolding and oxidation of proteins at cysteine and methionine residues. Escherichia coli cells are protected from HOCl-killing by the previously identified dodecameric transcription factor HypT (YjiE). Here, we aimed to unravel whether HOCl activates HypT directly or via a reaction product of HOCl with a cellular component. Bacterial viability assays and analysis of target gene regulation indicate that HypT is highly specific to activation by HOCl and that no reaction products of HOCl such as monochloramine, hydroxyl radicals, or methionine sulfoxide activate HypT in vivo. Surprisingly, purified HypT lost its DNA-binding activity upon incubation with HOCl or reaction products that oxidize HypT to form a disulfide-linked dimer, and regained DNA-binding activity upon reduction. Thus, we postulate that the cysteines in HypT contribute to control the DNA-binding activity of HypT in vitro. HypT contains five cysteine residues; a HypT mutant with all cysteines substituted by serine is aggregation-prone and forms tetramers in addition to the typical dodecamers. Using single and multiple cysteine-to-serine mutants, we identified Cys150 to be required for stability and Cys4 being important for oligomerization of HypT to dodecamers. Further, oxidation of Cys4 is responsible for the loss of DNA-binding of HypT upon oxidation in vitro. It appears that Cys4 oxidation upon conditions that are insufficient to stimulate the DNA-binding activity of HypT prevents unproductive interactions of HypT with DNA. Thus, Cys4 oxidation may be a check point in the activation process of HypT.

Tetramers Are the Activation-competent Species of the HOCl-specific Transcription Factor HypT

“…Given that the T m values of the HypT variants are comparable when obtained by thermal stability assay after dissociation and reassociation as described in this study and CD analysis as described previously (9,12), this indicates that the dissociation and reassociation procedure itself does not influence the thermal stability of HypT and mutants. Thus, the mixed reassociation between HypT mutants and wild-type HypT leads to an increase in thermal stability, showing that the effect is derived from wild-type HypT.…”

“…HypT is a LysR-type transcriptional regulator that specifically protects Escherichia coli cells from HOCl damage (9,12). LysR-type transcriptional regulators are ubiquitous regulators that typically form dimers and tetramers (e.g.…”

Metabolic Response of Escherichia coli upon Treatment with Hypochlorite at Sub-Lethal Concentrations

“…ROS differ in their reactivity with macromolecules and thus in their antimicrobial activity. Most ROS such as the superoxide anion and hydrogen peroxide (H 2 O 2 ) react rather selectively with macromolecules [ 6 ]. Hypochlorite (HOCl) is even more reactive and bactericidal and is thus used widely as a disinfectant in hospitals and households (commonly known as “bleach”) [ 7 ].…”

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli

“…Chlorine-specific resistance in E. coli involves three chlorine-sensitive transcription factors, hypT, rclR, and nemR, which are activated specifically by chlorine ion oxidation (10)(11)(12). The chlorine resistance of E. coli is also mediated through the RpoS-regulated general stress response (13), the oxidative stress regulons oxyR and soxR (14,15), and heat shock proteins (16).…”