Identification of a quinone-sensitive redox switch in the ArcB sensor kinase

Abstract

Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system. Under anaerobic conditions, the ArcB sensor kinase autophosphorylates and transphosphorylates ArcA, a global transcriptional regulator that controls the expression of numerous operons involved in respiratory or fermentative metabolism. Under aerobic conditions, the kinase activity of ArcB is inhibited by the quinone electron carriers that act as direct negative signals. Here, we show that the molecular mechanism of kinase silencing involves the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation, a reaction in which the quinones provide the source of oxidative power. Thus, a pivotal link in the Arc signal transduction pathway connecting the redox state of the quinone pool to the transcriptional apparatus is elucidated.

Fig. 1.

Effects of various modifying agents on the activity of ArcB. (A) Purified ArcB^78-778 (50 pmol) was incubated with [γ-³²P]ATP in the presence or absence of Q0 (0.250 mM), chloramine T (0.1 mM), H₂O₂ (2 mM), and DTT (5 mM), and the net phosphorylation of the protein was assayed by SDS/PAGE. (Left) Autoradiograms of the gels. (Right) Net increase of ArcB-P with time in the absence (□) or presence of Q0 (♦), chloramine T (•), H₂O₂ (▴), or DTT (▪). (B) Effect of DTT and glutathione on the aerobic expression of λΦ(cydA′-lacZ). Four parallel cultures of strain ECL5203 (6) and its isogenic ECL5204 (ΔarcB) (6) were grown aerobically in Luria-Bertani broth containing 0.1M MOPS (pH 7.4) and 20 mM d-xylose. At OD₆₀₀ of 0.3, one aliquot was withdrawn for measuring the β-galactosidase activity (depicted as -15 min). At time 0 min, DTT or glutathione was added to the cultures to final concentrations of 2.5 mM (♦), 5 mM (•), 10 mM (▴), or nothing (□), and the β-galactosidase activity was followed. × and + depict ECL5204 (ΔarcB) with no addition and 10 mM DTT, respectively. (Left) Addition of DTT. (Right) Addition of glutathione. The data represent the averages from four experiments (variations were <10% from the mean).

Fig. 2.

Effect of alkylation on the inhibition of ArcB by Q0 and chloramine T. Purified ArcB^78-778 (50 pmol) was preincubated with MMTS (1 mM) for 30 min, and the kinetics of [γ-³²P]ATP-dependent phosphorylation in the presence or absence of Q0 (0.250 mM) and chloramine T (0.1 mM) were assayed by SDS/PAGE. (Left) Autoradiograms of the gels. (Right) Time course of ArcB phosphorylation in the absence (□) or presence of Q0 (♦), chloramine T (•), MMTS (▵), MMTS and Q0 (⋄), or MMTS and chloramine T (○).

Fig. 3.

Requirement of the two cysteine residues for ArcB silencing by Q0. (A) A total of 50 pmol of purified ArcB^78-778 (ArcB), ArcB^{78-778, C180A} (ArcB^C1), ArcB^{78-778, C241A} (ArcB^C2), and ArcB^{78-778, C180A, C241A} (ArcB^CC) were incubated with [γ-³²P]ATP in the presence or absence of Q0 (0.250 mM), and the net phosphorylation of the protein was assayed by SDS/PAGE. (Upper) Autoradiograms of the gels. (Lower) Net increase of protein-P with time in the absence (open symbols) or presence of Q0 (filled symbols). The kinetics of ArcB phosphorylation are depicted in all panels as dotted lines. (B) Aerobic expression of λΦ(lldP′-lacZ). Strains ECL5002 (λΦ[lldP′-lacZ]) (6), IFC1001 (arcB^C180A Kan^r λΦ[lldP′-lacZ]), IFC1002 (arcB^C241A Kan^r λΦ[lldP′-lacZ]), and IFC1003 (arcB^{C180A, C241A} Kan^r λΦ[lldP′-lacZ]) were grown in Luria-Bertani broth containing 0.1 M MOPS (pH 7.4) and 20 mM d-xylose supplemented with 20 mM l-lactate as an inducer. β-Galactosidase activity was assayed and expressed in Miller units. The data are averages from four experiments (variations were <10% from the mean).

Fig. 4.

Redox state of ArcB. (A) Tagging ArcB with MAL-PEG. Purified ArcB peptides (50 pmol), untreated or pretreated with 0.5 mM Q0, were incubated with 1 mM MAL-PEG at 25°C. After 1 h, the reactions were terminated and differences in the mobility of the tagged and the untagged proteins were visualized by Western blot analysis with specific ArcB polyclonal antibodies. (B) Spectroscopic characteristics of NBD-modified ArcB. Purified ArcB peptides (10 nmol) were pretreated with either 0.5 mM Q0 or 10 mM DTT and incubated with NBD-Cl (1 mM). Final spectra (600 to 300 nm) were monitored after free NBD-Cl was removed by three cycles of concentration and redilution in a nanosep 10K. Solid line, pretreatment with DTT; dotted line, pretreatment with Q0. (C) Immunoblot analysis. Aerobically or anaerobically grown E. coli cells were harvested at an OD₆₀₀ ≈0.4-0.5 and treated with trichloroacetic acid to precipitate the proteins and stop further thiol/disulfide exchange. Precipitated proteins were dissolved in nonreducing SDS sample buffer and divided into two equal portions, one of which was treated with 100 mM DTT. Proteins were separated on nonreducing SDS/PAGE, followed by Western blot analysis with specific ArcB polyclonal antibodies. The 88-kDa band corresponds to the size of full-length ArcB monomer, and the 170-kDa band corresponds to an ArcB dimer.

Fig. 5.

The ArcB sensor kinase and a model for its redox regulation. (A) Schematic representation of ArcB. The linker region contains a putative leucine zipper (8) and a Per-Arnt-Sim (PAS) domain (9). Depicted, in the linker region, are also the cysteine residues 180 and 241. The primary transmitter domain (H1) contains the conserved His-292 and the catalytic determinants N, G1, and G2. The G1 and G2 sequences typify nucleotide-binding motifs. The receiver domain (D1) contains the conserved Asp-576, and the phosphotransfer domain (H2) contains the conserved His-717. (B) A simplified model for ArcB inactivation. Upon a shift from anaerobic to aerobic conditions of growth, the quinone pool shifts to its oxidized state. This allows the electron transfer from the Cys-180 of ArcB to quinones that leads to the formation of an intermolecular disulfide bond between the Cys-180 of two monomers, and results in a significant reduction of the kinase activity of ArcB. As the electrons rapidly flow toward O₂ via either cytochrome bd or bo oxidase, the quinone pool maintains its oxidized state and induces the formation of a second disulfide bond between the two Cys-241, resulting in the complete silencing of the ArcB kinase activity.