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Requirement of the Receiver and

Phosphotransfer Domains of ArcB for
Efficient Dephosphorylation of
Phosphorylated ArcA In Vivo
Gabriela R. Peña-Sandoval, Ohsuk Kwon and Dimitris
J. Bacteriol. 2005, 187(9):3267. DOI:

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which detect. ArcB undergoes ATP-depen- tions with both plant and animal hosts (1. at least in mophilus influenzae (2. In prokaryotes. Korea Research Institute of Bioscience and Biotechnology. 3. Under anoxic Downloaded from http://jb. All Rights Reserved. 187. both the cognate response regulator and the sen. Yusong-gu. E-mail: dimitris@ifc. Two-component signal transduction systems that depend idue at position 292. Upon cessation tabolism (23). 04510 Mexico City.1128/JB. This system comprises the vitro. no in vivo evidence was response regulator (15.JOURNAL OF BACTERIOLOGY. Furthermore. ArcB possesses three cytosolic catalytic domains: an teins. integrate these signals into a specific response. 25). Pen Departamento de Gene´tica Molecular. we address the question of whether the re- * Corresponding author. in turn. 717 (14.2005 Copyright © 2005. served Asp residue (Asp54) and a C-terminal helix-turn-helix The sensing and processing of these signals are carried out by domain for DNA binding. ArcB autophosphorylates and transphosphorylates ArcA. via an ArcAAsp54-P 3 ArcBHis717-P 3 ArcBAsp576-P 3 ArcB protein as the sensor kinase and the ArcA protein as the Pi reverse pathway (9). Universidad Nacional Auto ´noma de Me´xico. In a recent study. the kinase activity of of signaling. Phone: 52 55 volved in the in vivo dephosphorylation of ArcA-P. modulates the expression of numerous genes in response to the respiratory conditions of growth. was previously shown to proceed. sporulation. and orate. 29). and Hae. “sensory kinase” proteins and “response regulator” pro.3267– on February 25. Korea2 Received 29 October 2004/Accepted 21 January 2005 The Arc two-component system.2 and Dimitris Georgellis1* Gabriela R. Yersinia pestis. ArcA is a typical response regu. phosphorylated ArcA (ArcA-P) dephosphorylates and its transcriptional regulation is released. Fax: 52 55 5622 5611. in general as a transcriptional regulator. Under reducing conditions. regulatory activity. N-terminal transmitter domain (H1) with a conserved His res- tems. However.unam. 52 Oun-dong. First. The results demonstrate that the receiver and phos- photransfer domains of the tripartite sensor kinase ArcB are necessary and sufficient for efficient ArcA-P dephosphorylation in vivo. The ability to respond to a vast array of environmental lator comprising an N-terminal receiver domain with a con- signals is vital for the growth and survival of microorganisms. Daejeon 305-333.asm. a reaction needed to curtail its nella enterica serovar Typhimurium. 2013 by guest growth conditions. Instituto de Fisiologı´a Celular. by certain anaerobic metabolites such as D-lactate. represses the expression of residue and subsequently transphosphorylates a conserved Asp many operons involved in respiratory metabolism and activates residue in the cognate response regulator rendering it func. By contrast. dent autophosphorylation (10. a few operons encoding proteins involved in fermentative me- tional. 04510 Mexico City. May 2005. Under aerobic conditions. American Society for Microbiology. cell division. the sensor kinase undergoes Asp576 3 His717 3 Asp54 phosphorelay (10. and pathogenic interac. pairs. Under aerobic growth conditions. Requirement of the Receiver and Phosphotransfer Domains of ArcB for Efficient Dephosphorylation of Phosphorylated ArcA In Vivo ˜a-Sandoval. that belong to the large family of two-component sys. 13. Third. and a C-terminal trans- and acceptors have been reported to regulate diverse processes mitter domain (H2) with a conserved His residue at position that include energy metabolism. 16). 5622 a process that is enhanced The prototypical system of this kind comprises a membrane. there is no evident periplasmic domain (17. The dephosphorylation of ArcA-P has been shown to occur. 20). Mexico. chemotaxis.00⫹0 doi:10. No. Salmo. strated that the molecular mechanism of kinase silencing in- The Arc (anoxic redox control) two-component system is a volves the oxidation of two cytosol-located redox-active cys- complex signal transduction system that plays a key role in teine residues (Cys180 and Cys241) that participate in regulating energy metabolism at the level of transcription in intermolecular disulfide bond formation (24). acetate.187. and transphosphorylates ArcA via a His292 3 regulator. including the pathogens Vibrio cholerae. Second. In this study. Phosphor- an ATP-dependent autophosphorylation at a conserved His ylated ArcA (ArcA-P). and bound sensor kinase and a cytosol-located cognate response pyruvate (7. A shift to nonstimulating conditions leads to immediate 3267 . 9 0021-9193/05/$08. the cytosolic portion of the protein contains a putative these molecular circuits are typically organized by protein leucine zipper (9) and a PAS (Per-Amt-Sim) domain (31). Instituto de Fisiologı´a Celular. Mexico. provided to support the physiological significance of such a pathway. 17). at least in vitro. 12. Mailing address: Departamento de Ge- ne´tica Molecular.1 and Laboratory of Metabolic Engineering. it was demon- of the system. 3267–3272 Vol. ArcB is unusually elab- molecular circuits within the cell. 16). ArcB is inhibited by the quinone electron carriers that act as sor kinase undergo dephosphorylation that results in silencing direct negative signals (8). In this study. 18). amplify. symbiotic nitrogen fixation.9. comprising the ArcB sensor kinase and the ArcA response regulator. via an ArcAAsp54-P 3 ArcBHis717-P 3 ArcBAsp576-P 3 Pi reverse phosphorelay. the physiological significance of this pathway was assessed. p. bacteria (23). Upon signal reception. Universidad Nacio- ceiver (D1) and secondary transmitter (H2) of ArcB are in- nal Auto´noma de Me´xico.1 Ohsuk Kwon. a central receiver domain (D1) with a on histidine and aspartyl residues as phosphoryl group donors conserved Asp residue at position 576. which in turn represses or acti- vates its target operons. 30). dephosphorylation of ArcA-P.

As of the reporter. pQE30ArcB521-778. ArcA-P-dependent repression is released upon expression of D1-H2 under nonstimulating conditions. while the other pQE30ArcB (9). were cloned downstream from the ara promoter of plasmid pBAD30 (11) release of the Arc-dependent transcriptional control. stitutive ArcB kinase activity and also carries the ␭⌽(lldP⬘- repressible ␭⌽(lldP⬘-lacZ) reporter (20) was grown anaerobi.3. anaerobic growth. was shifted to aerobiosis.asm. was determined (depicted as ⫺10 min). and the time course of ␤-galactosidase activity was In this respect. and 20 mM L. Considering the fact that the in vitro half-life of the phospho-aspartyl bond of ArcA-P exceeds 60 min (9). H717Q (D1*- of some 40 operons. ArcB autophosphorylates cally in buffered Luria broth (LB) supplemented with 20 mM and transphosphorylates ArcA even during aerobic conditions L-lactate to induce expression of the reporter (5). Therefore. After an additional 10 min lactosidase assay was withdrawn (depicted as ⫺10 min). may contribute to the dephosphorylation of ArcB-P. closed circles. shift to aerobiosis. we argued that in vivo overexpression tions of growth.25. 1. the cultures were divided in two: one of the subcultures con. 2A). The generated plasmids were transformed into strain ylation. it has to be mentioned that the presence of monitored (Fig. Gen- erally. As seen in reporter was very low (Fig. 521-778. At an optical of growth. and pMX023 encod- anaerobic growth. shifting the anaerobic culture to aerobiosis caused a additional 10 min of growth. pected size at similar steady-state levels (Fig. a sample was withdrawn very low. Induction by arabinose revealed that all dephosphorylation of both ArcB-P and ArcA-P is probably resultant D1-H2 constructs overexpress a protein of the ex- necessary for release of the Arc-dependent transcriptional con. in which the wild-type arcB has been replaced system after a shift from anaerobic to aerobic conditions of with a tar-arcB hybrid (tab1) that encodes a protein with con- growth. containing for 1 h with 10-min intervals. the expression of the course of the ␤-galactosidase activity was followed. 2B) in all tested strains. one aliquot was withdrawn for measuring the ␤-galactosidase activity (depicted as ⫺10 min). In of growth. The EcoRI-HindIII 0 min. It was found that addition of arabinose in molecular oxygen leads to the oxidation of the quinone elec. production of an ArcA-P phosphatase and the expression level of the reporter. pMX021. should lower the amount of ArcA-P and augment expression tosidase units. phorylation of ArcA-P. peptides. ArcA-P dephosphorylates via an ArcAAsp54-P 3 ArcBHis717-P 3 ArcBAsp576-P 3 Pi reverse pathway (9).org/ on February 25. ArcB521-778. 1. rapid dephosphorylation of ArcA-P is also necessary for re- porter expression to resume after the shift to aerobiosis. transcriptional control in the absence of stimuli. During to generate pMX020. Since the receiver (D1) and secondary FIG. suggesting that the Arc the cultures to induce transcription of the plasmid-encoded system responds quickly to changes in respiratory conditions. the expression of the reporter was very low. After an Fig. 1). the ␤-galactosidase activity is always density at 600 nm (OD600) of ⬃0.3 mM arabinose was added in rapid increase in reporter expression. To gain some insight into the mechanism of dephosphor. H717Q (9).2. H717Q (this study). the ArcA response regulator is activated ing ArcB521-778 (D1-H2). and as a result. strain ECL5002 carrying the ArcA-P. linepropanesulfonic acid) (pH 7. lacZ) reporter fusion. 1. The already formed ArcB-P dephosphorylates. D576A. at least in vitro. H717Q upon phosphorylation by ArcB and regulates the transcription ArcB (D1-H2*). D576A. At time mutant D1-H2 (ArcB521-778) proteins. BACTERIOL. by the intrinsic lability of the phospho-aspartyl bond in D1 (10). However. pMX022. indica. 521-778. D576A (D1*-H2). 2B). the rate of dephosphorylation of response regulators appears to be controlled by the inherent lability of the mixed anhydride phospho-aspartyl bond and/or a phosphatase-like activity embodied in the cognate sensor kinase or another protein. SixA.3268 NOTES J. A shift to nonstimulating conditions leads to immediate release of the Arc-dependent transcriptional control. Also. trol. tron carriers that in turn inhibit ArcB phosphorylation (8) and thereby prevent further transphosphorylation of ArcA. the ribosomal binding site of the pQE30 vector and the arcB sequence that encodes the ArcB521-778 fragment.4). In this strain. the phosphohistidine phosphatase. respectively. rapid H2*). serving as control. The ArcA/B two- transmitter (H2) of ArcB are involved in the in vitro dephos- component system responds very rapidly to changes in redox condi. At an OD600 of 0. 20 mM D-xylose. fragments of plasmids pQE30ArcB521-778 (10). It has been previ- ously reported that. we examined the time lag for the response of the Arc ECL5047 (19). the growth medium of the strain carrying either pBAD30 or . we pos- Downloaded from http://jb. Open squares. D576A tinued growth under anaerobiosis.1 M MOPS (morpho. we constructed a series of plasmids expressing wild-type and lactate as inducer. which was previously reported to specifically dephosphorylate His717 of ArcB (28). When oxygen becomes available. expressed in ␤-galac. To this end. The transformants were grown aerobically in expected. most likely. The ␤-galactosidase activity was monitored and pQE30ArcB521-778. 2013 by guest tulated that an ArcA-P-specific phosphatase might be needed for its dephosphorylation during oxidizing conditions of growth. and ArcB521-778. the culture was shifted to aerobiosis and the time agreement with previous results (19). and a sample for the ␤-ga- tive of ArcA-P repression (Fig. Strain ECL5002 carrying the ␭⌽(lldP⬘-lacZ) reporter of these two domains might release the ArcA-P-dependent was grown anaerobically in LB containing 0. buffered LB to an OD600 of ⬃0. To test this.

3 the expression of either reporter. However. 20 mM D-xylose.4).0). was grown aerobically in LB containing 0. pMX021 (encodes D1*- H2). the plasmids grown aerobically in LB containing 1. the possibility of in- termolecular phosphoryl group transfer from H1 or H2 of ArcB to D1-H2* with concomitant hydrolysis of the Asp576-P cannot be excluded. the above results provide strong evidence that the presence of D1-H2 abolishes the ArcA-P-dependent transcriptional regulation under nonstimu- lating conditions. because the phosphoryl group will be transferred from ArcA-P to His717 of H2. with D1*-H2 showing a stronger effect than D1-H2*. at a considerably lower degree than the wild-type peptide.1 M MOPS (pH 7. a 1-ml sample was pelleted. which encodes a protein with constitutive ArcB the ␤-galactosidase activity was determined (Fig. It was kinase activity. expression of either D1*-H2 or D1-H2* re- activity was followed. It is of interest that in the cases where a sensor kinase facilitates dephosphorylation of its cognate response regulator. the ␭⌽(lldP⬘-lacZ) reporter fusion. and arcB hybrid (tab1). we tested whether expression of D1-H2 under of D1-H2 under nonstimulating conditions. ArcA-P-dependent repression is released upon expression phosphorelay. Expression of D1-H2 abrogates the ArcA-P transcriptional regulation under stimulating conditions. although pBAD30.VOL. In contrast. and the resolved proteins were visualized by Coomassie cydAB⫹ background. that is either the forward phosphorylation cascade or the reverse FIG. or pMX023 (encodes D1*-H2*) was dependent transcriptional control. washed with 1 ml of 10 mM Tris-HCl transformed into strains ECL5002 and ECL5003 carrying. closed squares. or pMX023. To this end. addition of arabinose in the growth medium of the strain carrying the D1-H2-express- ing plasmid (pMX020) resulted in an immediate increase of reporter expression. this does not exclude the possibility that the full- length wild-type ArcB maintains different folds dependent on . while nonstimulating conditions will have the opposite effect. does not seem to be a determinant favoring any of the two opposing reactions of phosphorylation and dephosphorylation. 2005 NOTES 3269 the D1*-H2*-expressing plasmid (pMX023) did not affect the expression of the reporter. (B) Strain ECL5047 (19) carrying a chromosomal tar. Samples of 10 ␮l were ArcA-P-activated ␭⌽(cydA⬘-lacZ) reporter in the lldPRD⫹ and subjected to electrophoresis in a sodium dodecyl sulfate-12% poly- acrylamide gel. tion of ␭⌽(cydA⬘-lacZ) and the repression of ␭⌽(lldP⬘-lacZ) lactate. by interfering with the phos- phorelay in ArcB and/or by sequestrating ArcA and thereby blocking its transphosphorylation. the ArcA-P-repressed ␭⌽(lldP⬘-lacZ) and the 2⫻ sodium dodecyl sulfate sample buffer. in agreement with the mM arabinose was added in each culture and the ␤-galactosidase previous result. To explore the possibility that distinct confor- mations might specify the reaction catalyzed by D1-H2.4. bically in the presence of arabinose to an OD600 of ⬃0. ECL5047 transformed with pMX020. At an OD600 of that express the wild-type or mutant D1-H2 proteins were ⬃0. pMX022. (A) Strain ECL5003 car. closed circles. closed diamonds. and pBAD30. The effect of D1*-H2 is to be expected. Nonetheless. closed triangles. it is not known whether the same conformation of the sensor allows both the phosphory- lation and dephosphorylation reactions to be catalyzed. 2013 by guest tion between ArcB and ArcA.2. found that while the presence of D1-H2 abolished the activa- pMX020. the presence of D1*-H2* did not affect the ␤-galactosidase activity (depicted as ⫺10 min). stimulating (anaerobic) conditions also abolishes the ArcA-P- rying pBAD30. 187. ECL5047 trans. The transformants were grown anaero- blue staining.or the D1-H2*-express- ing plasmid (pMX021 or pMX022) resulted in a moderate release of reporter repression. on February 25. and solubilized by incubation at 95°C for 5 min in 100 ␮l of spectively. At time 0 min. addition of inducer in the growth medium of the strains carrying either the D1*-H2. Also.3 mM arabinose. ECL5047 transformed with pMX021. ECL5047 transformed with pMX022. At an OD600 of 0. pMX020 (encodes D1-H2). one aliquot was withdrawn for measuring during anaerobiosis. pMX022 (encodes D1-H2*). pMX021. if the reverse phosphorelay is the actual in vivo pathway for ArcA-P dephosphorylation. a conformational change of the D1-H2 peptide per se formed with pMX023. Open squares. Finally. and 20 mM L. which reached a steady-state level 25 times higher than in the strains carrying either pBAD30 or pMX023. 3). re- (pH 8. If dis- tinct conformations of the sensor are needed to specify whether the kinase or the phosphatase activity will be allowed.4. 1. it can be assumed that under stimulating conditions activation of the kinase will be followed by concomitant inhibition of the phosphatase. An alternative should be that the mutant peptides attenuate the communica- Downloaded from http://jb. 2.asm. ECL5047 transformed with duced the ArcA-P-dependent transcriptional control. Thus.

carrying either the ␭⌽(lldP⬘-lacZ) or the ␭⌽(cydA⬘- D1-H2 acts directly on ArcA-P. at a final pH of 7. the cultures were harvested and the The ␭⌽(cydA⬘-lacZ)-bearing strains were cultured anaerobically in ␤-galactosidase activity was assayed. the cultures were harvested and the ␤-galactosidase bars. expression was 3.asm. most probably at the expense of acetyl phosphate. and the standard deviation values are on February 25. the ⌬arcB strains ECL5004 and ECL5012 . 4). 4). 1 ␮M ZnCl2. we took advantage of the facts that in the absence of their its cognate sensor kinase. Downloaded from http://jb. untransformed strains. hatched OD600 of 0. and experiments. cognate kinases many response regulators undergo in vivo au.1 M MOPS. At an untransformed ECL5004 (⌬arcB) and ECL5012 (⌬arcB). Expression of D1-H2 annuls the acetyl-phosphate-depen- dent phosphorylation of ArcA in vivo. For the growth of ␭⌽(lldP⬘-lacZ)-bearing strains. in the absence of this. The ␭⌽(lldP⬘-lacZ) and the mM pyruvate and 1. Solid bars. For the growth of ␭⌽(lldP⬘- ␭⌽(cydA⬘-lacZ) reporter strains ECL5002 and ECL5003 were trans. pMX022 (D1-H2*). and 0.1 M MOPS (pH 7. the above medium was supplemented with 20 mM L-lactate. mM L-lactate. carrying the wild-type ArcB. indicated. 20 mM (NH4)2SO4. 40 mM KCl. 21. It was found that the ␭⌽(cydA⬘-lacZ) should cause the same effect.3 mM MgSO4. interception of the cultures were harvested and the ␤-galactosidase activity was phosphoryl group transfer from ArcB to ArcA by D1-H2 determined (Fig. the lacZ) reporters. At an OD600 of 0. ArcA-P. activity was assayed and expressed in Miller units. the above medium was supplemented with 20 formed with either the wild-type or mutant D1-H2-expressing plasmid. Therefore. pMX021 (D1*-H2). the of ArcA-P dephosphorylation. bars. However.3 mM arabinose.4. Thus. and the standard deviation values are pMX023 (D1*-H2*). The data are the average of four buffered LB containing 0. strains transformed with plasmids pBAD30. At an OD600 of ⬃0.3270 NOTES J. we attempted to gen. pMX020 (D1-H2).4. ECL5012. pMX022 (D1-H2*).5-fold higher and the expression of ␭⌽(lldP⬘- erate ArcB-independent ArcA-P in vivo and examine whether lacZ) was 3-fold lower in the arcB-null background than in the D1-H2 is able to directly dephosphorylate ArcA-P. In doing wild-type arcB⫹ background (Fig. 4. or pMX023 (D1*-H2*). or average of four experiments. To this end. were grown aerobically in defined medium effect of D1-H2 on reporter expression is most likely the result supplemented with 20 mM pyruvate. The wild-type strains ECL5002 and the redox state of the cell that could play a decisive role in ECL5003 and their isogenic ⌬arcB strains ECL5004 and dictating the direction of the phosphoryl group transfer.3 mM arabinose. As mentioned earlier. The data are the pMX020 (D1-H2). The ␭⌽(lldP⬘-lacZ) and the ␭⌽(cydA⬘-lacZ) reporter strains ECL5002 and ECL5003 and their ⌬arcB isogenic strains ECL5012 and ECL5004 transformed with either the wild-type or mutant D1-H2-expressing plasmid were grown aero- bically in defined medium [1 mM KH2PO4. lacZ)-bearing strains. pMX021 (D1*-H2). Empty 1. 20 mM D-xylose. Expression of D1-H2 abrogates the ArcA-P transcriptional CaCl2. 6. ECL5002 and ECL5003. cally on pyruvate than on glycerol as the sole carbon and energy source (26). ECL5004 and ECL5012 transformed with plasmids pBAD30. solid bars.4.4). and mutant D1-H2 peptides on the dephosphorylation of phate is an order of magnitude higher in cells grown aerobi. 1 ␮M FeSO4. BACTERIOL. 34 mM NaCl. hatched bars. viding the means to examine the direct effect of the wild-type 32) and that the intracellular concentration of acetyl-phos. thus pro- tophosphorylation at the expense of acetyl-phosphate (4. ArcA is able to autophosphorylate. 2013 by guest FIG. 10 ␮M FIG. 3.6] supplemented with 20 regulation under stimulating conditions. 0.

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