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Quinones as the Redox Signal for the Arc Two-Component System of Bacteria

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Science  22 Jun 2001:
Vol. 292, Issue 5525, pp. 2314-2316
DOI: 10.1126/science.1059361

Abstract

The Arc two-component signal transduction system mediates adaptive responses of Escherichia coli to changing respiratory conditions of growth. Under anaerobic conditions, the ArcB sensor kinase autophosphorylates and then transphosphorylates ArcA, a global transcriptional regulator that controls the expression of numerous operons involved in respiratory or fermentative metabolism. We show that oxidized forms of quinone electron carriers act as direct negative signals that inhibit autophosphorylation of ArcB during aerobiosis. Thus, the Arc signal transduction system provides a link between the electron transport chain and gene expression.

Two-component signal transduction systems are widespread in prokaryotes and play extensive roles in adaptation to environmental changes (1, 2). The Arc two-component system of Escherichia coli, comprising the ArcB transmembrane sensor kinase and the cytosolic ArcA response regulator, modulates the expression of numerous regulons and operons (the Arc modulon) in response to changes in redox conditions of growth (3–5). In contrast to typical sensor kinases that have a substantial periplasmic domain between two transmembrane segments on the NH2-terminus for signal sensing, ArcB has a short periplasmic sequence of only 16 amino acids (6). ArcB is also unorthodox in having an elaborate cytosolic structure consisting of three catalytic domains: a primary transmitter with a conserved His residue (position 292), a receiver with a conserved Asp (position 576), and a secondary transmitter with a conserved His (position 717) (7, 8). ArcA is a classical response regulator that has an NH2-terminal receiver domain with a conserved Asp residue (position 54) and a COOH-terminal helix-turn-helix domain for DNA binding.

Under reducing conditions, ArcB autophosphorylates, then transphosphorylates ArcA through a His292 → Asp576 → His717 → Asp54phospho-relay, thereby increasing the affinity of ArcA for its DNA targets (9–11). Phosphorylated ArcA (ArcA-P) represses the expression of many genes involved in respiratory metabolism [e.g., enzymes of electron transport, the tricarboxylic acid cycle, and the glyoxalate shunt (3)] and activates other genes encoding proteins involved in fermentative metabolism [e.g., pyruvate formate lyase (12) and hydrogenase I (13)] by binding to a specific DNA sequence (14–16). Under oxidizing conditions, ArcB dephosphorylates ArcA-P through an Asp54 → His717 → Asp576 reverse phospho-relay (17).

Although much has been learned about the steps of signal transmission and decay as well as the identity of numerous target operons, the actual signal for ArcB remains to be identified. Oxygen has been excluded as a direct signal for inhibiting autophosphorylation, because during anaerobic respiratory growth the ArcA-P–dependent repression of a target operon can be lifted by a supplemented electron acceptor in accordance with its oxidizing power (midpoint potential) (3). Several fermentation metabolites (e.g., d-lactate, pyruvate, and acetate) have been shown to enhance the autophosphorylation rate of ArcB in vitro by up to three times the basal level by acting at a site in the cytosolic domain (18, 19). However, these compounds are likely to be allosteric activators. The true signal is expected to curtail autophosphorylation under oxidizing conditions; otherwise, many target operons would remain under permanent repression. Moreover, the cellular levels of metabolites such asd-lactate depend not only on the respiratory state of the cell but also on the oxygen-hydrogen ratio of the carbohydrate being fermented. In addition, if the inputs come solely from the cytosol, the transmembrane domain of ArcB would not have evolved (or have been maintained). A case in point is the non–membrane-associated kinase NtrB that senses the state of nitrogen supply from cytosolic elements (20). We therefore explored the possibility that the transmembrane domain of ArcB either participates directly in signal reception or serves as an anchor to keep the protein close to the source of the signal. The former was excluded because the amino acid sequence of the transmembrane domain can be altered without impairing signal transduction (6). It thus appears that membrane association promotes interaction between the cytoplasmic portion of the sensor protein and a redox signal.

The major quinones—ubiquinone-8 (Q8), menaquinone-8 (MK8), and demethylmenaquinone (DMK8)—are membrane-associated electron carriers that function as adapters between various electron-donating and electron-accepting enzyme complexes (21, 22) and were therefore assessed as possible direct signals for ArcB. When the soluble analogs ubiquinone-0 (Q0) or menadione (MK3) were incubated in the presence of [γ-32P]adenosine triphosphate (ATP) and purified ArcB78-778 [a truncated sensor kinase lacking the NH2-terminal transmembrane domain (10)], autophosphorylation of the protein (23) was inhibited (Fig. 1, A and B). Half-maximal inhibition of ArcB78-778 phosphorylation (24) by Q0 occurred at about 5 μM, whereas that by MK3 occurred at about 50 μM (25). Because quinones are reduced to quinols under fermentative conditions of growth, Q0 and MK3 were converted to their reduced form by treatment with excess dithionite (hydrosulfite) and then tested. Reduced Q0, reduced MK3, or dithionite alone did not affect the rate of ArcB78-778autophosphorylation (Fig. 1, A and B). The quinones act at a site in either the linker or the first transmitter domain, because Q0 also inhibited autophosphorylation of ArcB78-520 (10), which lacks the receiver domain and the secondary transmitter domain (25). To test whether inhibition of the kinase activity by Q0 and MK3is ArcB- specific, we examined two other sensor kinases: CpxA, a classical sensor kinase with a single transmitter domain (26), and BarA, which belongs to the ArcB subfamily of sensor kinases with three catalytic domains (27). Purified CpxA184-458 (28) and BarA198-918(29), both lacking the NH2-terminal transmembrane domain, were used. The presence of Q0 and MK3 in the oxidized form did not alter the autophosphorylation rate of the two sensor kinases (Fig. 1, C and D).

Figure 1

Effects of ubiquinone-0 and menadione on the rate of ArcB autophosphorylation. (A) Purified ArcB78-778 (2.5 μM) was incubated with [γ-32P]ATP in the presence or absence of ubiquinone-0 (250 μM) and/or dithionite (500 μM). Left panel: Autoradiograms of the gels. Right panel: Net increase of ArcB-P with time in the absence (▵) or presence of ubiquinone-0 (▪), dithionite (▴), or ubiquinone-0 and dithionite (□). (B) Purified ArcB78-778 was incubated with [γ-32P]ATP in the presence or absence of menadione (1 mM) and/or dithionite (1 mM). Left panel: Autoradiograms of the gels. Right panel: Net increase of ArcB-P with time in the absence (▵) or presence of menadione (⧫), dithionite (▴), or menadione and dithionite (◊). (C) Purified CpxA184-458 was incubated with [γ-32P]ATP at room temperature in buffer A in the presence or absence of ubiquinone-0 (250 μM) or menadione (1 mM). (D) Purified BarA198-918 was incubated with [γ-32P]ATP at room temperature in buffer A in the presence or absence of ubiquinone-0 (250 μM) or menadione (1 mM).

The in vitro results suggest that the physiological redox state is signaled to ArcB by the oxidized form of quinone electron carriers. This would explain previous observations that in a mutant lacking both cytochrome bo3 and cytochrome bd terminal oxidases, the aerobic expression of the ArcA-P–activatable Φ(cyd-lacZ) reporter was elevated, but that of the ArcA-P–repressible Φ(cyo-lacZ) reporter was lowered (30). In this mutant, the pool of Q8should be trapped in reduced form despite the oxidizing growth conditions. Because reduced quinones are unable to inhibit the kinase activity of ArcB, the level of ArcA-P should rise and accordingly alter the expression of the target operons.

To confirm the signaling role of Q8 in vivo, we compared the aerobic expressions of Φ(cydA′-lacZ) in aubiCA mutant that is blocked in the synthesis of the Q8 precursor 3-octaprenyl-4-hydroxybenzoate, a ubiCA arcB double mutant, and the isogenic wild-type parent (31). Blockage of Q8 synthesis increased the aerobic expression of Φ(cydA′-lacZ) by a factor of 3, but no increase was observed in the double mutant, indicating that increased aerobic expression of Φ(cydA′-lacZ) in the ubiCA mutant resulted from diminished inhibition of the ArcB kinase activity (Fig. 2). Together, the in vitro and in vivo data suggest that the oxidized forms of quinones are ArcB-specific signals that silence, rather than stimulate, ArcB kinase activity.

Figure 2

Effects of ubiquinone-8 deficiency on the aerobic expression of Φ(cydA′-lacZ) operon fusion. Strains ECL5001 (cyd+ λΦ[cydA′-lacZ]), ECL5039 (ΔubiCA::Kanr cyd+ λΦ[cydA′-lacZ]), and ECL5040 (ΔubiCA::KanrΔarcB::Tetr cyd+ λΦ[cydA′-lacZ]) were grown in 5 ml of minimal medium (30) containing 40 mMd-galactose as sole carbon and energy source in 250-ml baffled flasks at 37°C with shaking (300 rpm). β-Galactosidase activity was assayed and is expressed in Miller units. The data are averages of four experiments; standard deviations are indicated.

The quinones are ideally suited as redox signals for the Arc system, because the hydrophobicity provided by the isoprenoid chain not only ensures rapid lateral mobility within the membrane but may also facilitate limited vertical displacements, which could permit interactions of the reactive aromatic ring with the ArcB signal reception site. Signal reception by the cytosolic domain of a transmembrane protein is not unique for ArcB. The Aer transmembrane protein, the guiding element in bacterial aerotaxis, also lacks a periplasmic domain. This protein senses the cellular redox state by a flavin adenine dinucleotide molecule bound to the cytosolic domain (32, 33).

Anchoring of the sensor domain close to the cytoplasmic membrane ensures that all of the ArcB molecules are within reach of the signal. Silencing most of the ArcB molecules would not be possible if they were randomly distributed in the cytosol. Indeed, we found that liberation of ArcB from the membrane results in a constitutively active kinase in vivo (6). A part of the structural evolution of ArcB can thus be viewed as a tinkering process that enables a sensor kinase to interact with an electron transport element as signal, without the intervention of a periplasmic domain. The attainment of this interaction also confers an additional role for the quinones to serve as signals for gene expression.

  • * To whom correspondence should be addressed. E-mail: elin{at}hms.harvard.edu

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