Ubiquinone and menaquinone electron carriers represent the yin and yang in the redox regulation of the ArcB sensor kinase

Abstract

The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to respiratory growth conditions. Under aerobic growth conditions, the ubiquinone electron carriers were proposed to silence the kinase activity of ArcB by oxidizing two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we confirm the role of the ubiquinone electron carriers as the silencing signal of ArcB in vivo, we show that the redox potential of ArcB is about -41 mV, and we demonstrate that the menaquinols are required for proper ArcB activation upon a shift from aerobic to anaerobic growth conditions. Thus, an essential link in the Arc signal transduction pathway connecting the redox state of the quinone pool to the transcriptional apparatus is elucidated.

Fig 1

Requirement of ubiquinones for ArcB silencing in vivo. Cultures of strain ECL5001 (wt) (A), its isogenic ECL5039 strain (ΔubiCA) (B), and ECL5039 harboring plasmid pMX537 (pubi⁺) (C), all of which carry the ArcB activatable cydA′-lacZ reporter, were grown anaerobically in Luria-Bertani broth containing 0.1 M MOPS (pH 7.4) and 20 mM d-xylose. At an OD₆₀₀ of 0.2, one aliquot was withdrawn for measuring the β-galactosidase activity (depicted as 0 min) and the rest of the culture was divided in two. One part was kept under anaerobic conditions (filled diamonds) as a control, whereas the other was shifted to aerobiosis (empty diamonds), and the time course of the β-galactosidase activity was followed. (D) Strain IFC5007 (wt) and its isogenic IFC5008 strain (ΔubiCA), both carrying the ArcB repressible sdh-lacZ reporter, were grown anaerobically in the above-described medium. At an OD₆₀₀ of 0.2, the culture was divided in two. One part was kept under anaerobic conditions (empty bars), whereas the other was shifted to aerobiosis (solid bars), and after 2 h of incubation, the β-galactosidase activity was assayed and expressed in Miller units. (E) Strain ECL5001 (wt) and its isogenic ECL5039 strain (ΔubiCA), both carrying the ArcB activatable cydA′-lacZ reporter, were grown anaerobically (empty bars) or aerobically (solid bars) in defined mineral medium with glucose as the sole carbon source. The cells were harvested at mid-exponential growth phase, and the β-galactosidase activity was assayed and expressed in Miller units. The data represent the averages from four independent experiments, and the standard deviation values are indicated.

Fig 2

Determination of the redox potential of ArcB. (A) Purified ArcB^78-778 (top) or full-length ArcB-enriched inverted membrane vesicles (bottom) were incubated with [γ⁻³²P]ATP in the presence of 1 mM: lane 1, 1,4-benzoquinone (E′° = +274 mV); lane 2, 1,2-naphtoquinone (E′° = +134 mV); lane 3, 1,4-naphtoquinone (E′° = +69 mV); lane 4, juglone (E′° = +30 mV); lane 5, menadione (E′° = ±0 mV); lane 6, plumbagin (E′° = −29 mV); lane 7, lawsone (E′° = −137 mV); lane 8, anthraquinone-2-sulfonate (E′° = −225mV); and lane 9, nothing. After 2.5 min, the reactions were terminated by addition of an equal volume of 4× SDS sample buffer and the mixtures were immediately subjected to SDS-PAGE. Representative autoradiograms of the dried gels are presented. (B) A concentration of ∼1 μM full-length ArcB embedded in inverted membrane vesicles was incubated with buffers containing various cysteine and cystine concentrations for 3 h at 25°C. Subsequently, the protein was incubated with [γ⁻³²P]ATP for 2.5 min, the reactions were terminated by addition of an equal volume of 4× SDS sample buffer, and the mixtures were immediately subjected to SDS-PAGE. Gel stained with Coomassie blue (top), an autoradiogram of phosphorylated proteins on the dried gel (middle), and a plot of ArcB net phosphorylation versus [Cys]²/[Cystine] (bottom) are shown. Data represent the averages from seven independent experiments, and the standard deviation values are indicated. The plot was interpreted by the equations given in Materials and Methods, and the K_eq and E′° values for ArcB were determined to be 4.2 × 10⁻⁶ M and −41 mV, respectively.

Fig 3

Requirement of menaquinones for ArcB activation in vivo. Cultures of strain ECL5003 (wt) (A), its isogenic strain IFC5006 (ΔmenFDHB) (B), IFC5006 complemented with plasmid pMX538 (pmen⁺) (C), and IFC5006 in the presence of 1,4-dihydroxy-2-naphtoic acid (DHNA) (D), all bearing the ArcB activatable cydA′-lacZ reporter, were grown aerobically in LB broth containing 0.1 M MOPS (pH 7.4) and 20 mM d-xylose. At an OD₆₀₀ of 0.2, one aliquot was withdrawn for measuring the β-galactosidase activity (depicted as 0 min) and the rest of the culture was divided in two. One part was kept under aerobic conditions (empty diamonds) as a control, whereas the other was shifted to anaerobiosis (filled diamonds), and the time course of the β-galactosidase activity was followed. Data represent the averages from four experiments, and the standard deviation values are indicated.

Fig 4

A simplified model for ArcB regulation. During aerobic growth conditions or upon a shift from anaerobic to aerobic growth, ubiquinones (UQ/UQH₂; E′° = +100 mV) constitute the major quinones in the respiratory chain of E. coli. As the electrons flow toward O₂ via the cytochromes (Cyt), the ubiquinone pool maintains its oxidized state, allowing the electron transfer from the cysteine residues of ArcB (E′° = −41 mV) to UQ. This results in disulfide bond formation and immediate silencing of the ArcB kinase activity. During microaerobic growth conditions or upon a shift from aerobic to anaerobic growth, the ubiquinone pool is gradually replaced by menaquinones (MQ/MQH₂; E′° = −74 mV). The low redox potential of MQ/MQH₂ permits the electron transfer from menaquinol (MQH₂) to the cysteine residues of ArcB, resulting in disulfide bond breakage and activation of the ArcB kinase activity. The pool of menaquinol is restored by the electron flow from the NADH dehydrogenases (NDH).