Multimerization of Phosphorylated and Non-phosphorylated ArcA Is Necessary for the Response Regulator Function of the Arc Two-component Signal Transduction System

Abstract

To adapt to anaerobic conditions,Escherichia coli operates the Arc two-component signal transduction system, consisting of a sensor kinase, ArcB, and a response regulator, ArcA. ArcA is converted to the active form, phosphorylated ArcA (ArcA-P), by ArcB-mediated phosphorylation. The active ArcA-P binds to the promoter regions of target genes, thereby regulating their transcriptional activities. The phosphoryl group of ArcA-P is unstable with a half-life of 30 min. However, we were able to inhibit the dephosphorylation for more than 12 h by the addition of EDTA; this allowed us to characterize ArcA-P. Gel-filtration and glycerol sedimentation experiments demonstrated that ArcA exists as a homo-dimer. ArcA phosphorylated by either ArcB or carbamyl phosphate multimerizes to form a tetramer of dimers; this multimer binds to the ArcA DNA binding site. Isoelectric focusing gel electrophoresis and nitrocellulose-filter binding analyses indicated that the ArcA multimer is composed of both ArcA-P and ArcA in a ratio, 1:1. The ArcA(D54E) mutant protein was unable to be phosphorylated by ArcB. This defect resulted in the inability of ArcA(D54E) to form a multimer or to bind to the ArcA DNA binding site. These results indicate that phosphorylation of ArcA induces multimerization prior to DNA binding, and the multimerization is a prerequisite for binding. Our results suggest a novel model that phosphorylation of ArcA by ArcB regulates multimerization of ArcA, which in turn functions as a response regulator. To adapt to anaerobic conditions,Escherichia coli operates the Arc two-component signal transduction system, consisting of a sensor kinase, ArcB, and a response regulator, ArcA. ArcA is converted to the active form, phosphorylated ArcA (ArcA-P), by ArcB-mediated phosphorylation. The active ArcA-P binds to the promoter regions of target genes, thereby regulating their transcriptional activities. The phosphoryl group of ArcA-P is unstable with a half-life of 30 min. However, we were able to inhibit the dephosphorylation for more than 12 h by the addition of EDTA; this allowed us to characterize ArcA-P. Gel-filtration and glycerol sedimentation experiments demonstrated that ArcA exists as a homo-dimer. ArcA phosphorylated by either ArcB or carbamyl phosphate multimerizes to form a tetramer of dimers; this multimer binds to the ArcA DNA binding site. Isoelectric focusing gel electrophoresis and nitrocellulose-filter binding analyses indicated that the ArcA multimer is composed of both ArcA-P and ArcA in a ratio, 1:1. The ArcA(D54E) mutant protein was unable to be phosphorylated by ArcB. This defect resulted in the inability of ArcA(D54E) to form a multimer or to bind to the ArcA DNA binding site. These results indicate that phosphorylation of ArcA induces multimerization prior to DNA binding, and the multimerization is a prerequisite for binding. Our results suggest a novel model that phosphorylation of ArcA by ArcB regulates multimerization of ArcA, which in turn functions as a response regulator. phosphorylated ArcB phosphorylated ArcA phosphorylation reaction with carbamyl phosphate isoelectric focusing polymerase chain reaction transphosphorylation reaction base pair(s) Bacteria are able to adapt to a wide variety of environmental conditions through regulation of gene expression (1Parkinson J.S. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington, DC1995: 9-23Google Scholar, 2Ninfa A.J. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, DC1996: 1246-1262Google Scholar). This gene regulation occurs by the activation of two-component signal transduction systems, a number of which have been identified. One such system found in Escherichia coli, the Arc two-component signal transduction system, is activated in response to anaerobic growth conditions (3Lynch A.S. Lin E.C.C. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, DC1996: 1526-1538Google Scholar). The Arc system is comprised of a transmembrane sensor kinase, ArcB, and a response regulator, ArcA (4Iuchi S. Cameron D.C. Lin E.C. J. Bacteriol. 1989; 171: 868-873Crossref PubMed Google Scholar, 5Iuchi S. Cameron D.C. Lin E.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1888-1892Crossref PubMed Scopus (294) Google Scholar). Upon a shift from aerobic to anaerobic conditions, ArcB undergoes autophosphorylation, presumably due to a change of redox signals in the membrane (6Iuchi S. Lin E.C.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar, 7Georgellis D. Kwon O. Lin E.C. C J. Biol. Chem. 1999; 274: 35950-35954Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It is known that the phosphoryl group on phosphorylated ArcB (ArcB-P)1 undergoes intramolecular phospho-relay from His-292 to Asp-576 to His-717 and then is finally transferred to ArcA (8Georgellis D. Lynch A.S. Lin E.C.C. J. Bacteriol. 1997; 179: 5429-5435Crossref PubMed Google Scholar, 9Kwon O. Georigellis D. Lin E.C.C. J. Bacteriol. 2000; 182: 3858-3862Crossref PubMed Scopus (81) Google Scholar). Although the exact phosphorylation site of ArcA is unknown, comparison of thearcA gene sequence with other response regulators such ascheY suggests a putative phosphorylation site at Asp-54 (6Iuchi S. Lin E.C.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar,8Georgellis D. Lynch A.S. Lin E.C.C. J. Bacteriol. 1997; 179: 5429-5435Crossref PubMed Google Scholar). Phosphorylated ArcA (ArcA-P) positively and negatively regulates the transcription of a number of operons involved either directly or indirectly in cellular metabolism under both aerobic and anaerobic conditions (5Iuchi S. Cameron D.C. Lin E.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1888-1892Crossref PubMed Scopus (294) Google Scholar). In some of these genes, for example pfl(10Drapal N. Sawers G. Mol. Microbiol. 1995; 16: 597-607Crossref PubMed Scopus (55) Google Scholar), cyd (11Lynch A.S. Lin E.C.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar), and sodA (12Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar), it has been demonstrated that ArcA-P functions as a transcriptional regulator by binding their promoter region. Recently, it was reported that ArcA-P binds to oriC, the origin of chromosomal replication, thereby specifically inhibiting in vitro E. coli chromosomal replication (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). It has been reported that, in DNase I protection assays, ArcA-P protects a wide region of DNA, more than 60 bp (up to 150 bp in theoriCregion), from the DNase I cleavage (10Drapal N. Sawers G. Mol. Microbiol. 1995; 16: 597-607Crossref PubMed Scopus (55) Google Scholar, 11Lynch A.S. Lin E.C.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar, 12Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar). In this report, we examined the interaction of ArcA protein with the wide regions of DNA as defined by the DNase I protection assays, and we show that phosphorylation of an ArcA dimer induces formation of an ArcA multimer, consisting of a tetramer of dimers that contains both ArcA and ArcA-P in a 1:1 ratio. In addition, we demonstrate that this multimerization occurs prior to DNA binding. Sources were as follows: [γ-32P]ATP (5000 Ci/mmol), Amersham Pharmacia Biotech; Long Ranger polyacrylamide, FMC BioProducts; DNase I, Life Technologies, Inc.; Pfu DNA polymerase and DpnI, Stratagene; T4 polynucleotide kinase, New England BioLabs; calf intestinal alkaline phosphatase and restriction enzymes, Promega Corp. Other reagents, unless otherwise indicated, were purchased from Sigma Chemical Co. ArcA and ArcB proteins were purified as previously described (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To generate mutant ArcA(D54E) protein, polymerase chain reaction (PCR)-based site-directed mutagenesis was conducted as recommended by Stratagene. Mutagenic primers (5′-GGTGATCATGGAAATCAATCTGCC-3′ and 5′-GGCAGATTGATTTCCATGATCACC-3′) were designed to incorporate a Glu substitution at Asp-54; PCR was performed using pBADarcA (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) as template DNA. Parental DNA templates were removed from the PCR products using DpnI restriction enzyme digestion, then the mixture containing nicked plasmids harboring the desired mutation was transformed into DH5α (14Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). Plasmids were isolated, and the proper amino acid substitution was confirmed by DNA sequencing. The resultant mutated DNA, pBADarcAD54E, was transformed into MC4100ΔarcA (gift from Edmund C. C. Lin) for purification to avoid contamination of wild type protein during purification procedures. ArcA(D54E) protein was purified in the same way as wild type ArcA purification as previously described (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), except that the FastS column step was omitted. Approximately 6.5 mg of ArcA(D54E) protein was purified from 18 liters of MC4100ΔarcA(pBADarcAD54E) culture. Transphosphorylation reactions (TP) containing ArcB and ArcA or ArcA(D54E) were performed as previously described (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) with minor modifications. 10 µl of TP mixture contained 6 µg of ArcA, 0.6 µg of ArcB, 0.1 mmATP, 70 mm KCl, 10 mm MgCl2, 33 mm HEPES-KOH (pH 7.4), 0.1 mm EDTA, 2 mm dithiothreitol, and 10% glycerol. After incubation at 25 °C for 10 min, 0.5 m EDTA was immediately added to the reaction mixture for a final concentration of 50 mm(except for DNase I protection assays). To visualize the phosphorylated proteins, [γ-32P]ATP was included in the TP mixture. The reaction was terminated by the addition of an equal volume of gel-loading buffer (20% glycerol, 8% SDS, 10% β-mercaptoethanol, 0.1 m EDTA, and 0.003% bromphenol blue). After incubation at 55 °C for 3 min, the mixture was subjected to 13% SDS-polyacrylamide gel electrophoresis. The gel was dried, and bands were visualized by autoradiography. The phosphorylation reaction of ArcA with carbamyl phosphate (CP) was performed as previously described (11Lynch A.S. Lin E.C.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar). The reaction was terminated by the addition of 0.5 m EDTA to 50 mm. To construct pBSpfl used for DNA binding assays, the pfl promoter regulatory region of −458 to +23 (10Drapal N. Sawers G. Mol. Microbiol. 1995; 16: 597-607Crossref PubMed Scopus (55) Google Scholar) was amplified by PCR using the primers, 5′-CGGGGATCCACGCGTTTGCTGCACATCAG-3′ and 5′-CGGGAATTCAGCTTTCACACTAACTCTCTC-3′ (BamHI and EcoRI sites are in boldface), usingE. coli genomic DNA as a template. The PCR product was digested with both BamHI and EcoRI and cloned into the same sites of a pBluescript II SK(+) (Stratagene). Gel-shift assays using the 324-bp BamHI/DraI fragment isolated from pBSpfl were performed essentially as previously described (11Lynch A.S. Lin E.C.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar). 21.5 fmol of 5′-end-labeled DNA was mixed with 20 µl of TP mixtures containing 50 mm potassium chloride, 2.5 µg of bovine serum albumin, 40 mm HEPES-KOH (pH 7.6), and 10% glycerol. After incubation at 32 °C for 10 min, each sample was loaded onto a 4.5% polyacrylamide gel containing 10% glycerol. For the DNase I protection assay, the 532-bpXbaI/XhoI fragment from pBSpfl was 5′-end-labeled at the XbaI restriction site, and the assay was conducted as previously described (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), unless 10 ng of DNase I (in 1.5 µl of H2O) was used. The IEF gel contained 5% polyacrylamide, 9 m urea, 2% Triton X-100, 2% ampholine (pH 4.0–6.0), and 0.4% ampholine (pH 3.0–10.0). 10 mmphosphoric acid and 20 mm sodium hydroxide were used as anolyte and catholyte, respectively. Each of the protein samples was mixed with an equal volume of IEF gel loading buffer (9 murea, 2% Triton X-100, 2% ampholine (pH 4.0–6.0), 0.4% ampholine (pH 3.0–10.0), 1% β-mercaptoethanol, and 0.02% bromphenol blue). After removing insoluble materials through centrifugation for 10 min in a microcentrifuge, samples were loaded to the anode. The gel was run at 150 V for 30 min, followed by 200 V for 2.5 h. TP reaction mixtures containing [γ-32P]ATP were filtered through nitrocellulose filters (Millipore, HA), equilibrated with buffer A (25 mmHEPES-KOH (pH 7.8), 1 mm EDTA, 2.8 mmβ-mercaptoethanol, 50 mm KCl, and 10% glycerol), followed by an additional washing with 5 ml of this buffer. The filters were then dried, and radioactivities were determined by was using the system Pharmacia A 12 column or 6 column was equilibrated with buffer A. 30 µl of each indicated reaction mixture was to the column and at a of 40 of proteins in the was by at and 30 µl of each was an ml was Each of these was using IEF gel electrophoresis and For glycerol µl of TP or mixture was loaded onto ml of a glycerol in buffer followed by a centrifugation for 25 h at in an at µl each of was from the ArcA and ArcB proteins were purified as previously described (13Lee Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google both purified proteins were active in transphosphorylation reactions In addition, which was by the of ArcB in the of was able to ArcA as previously (6Iuchi S. Lin E.C.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar, Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). the ArcB protein as both a and a it was to the ArcA to ArcB to ArcA-P transphosphorylation we determined that a of ArcA to ArcB the concentration of ArcA-P. Although the of protein to ArcA has been it is which ArcA amino is phosphorylated by the ArcA amino acid sequence was with other two-component response a putative phosphorylation site at amino acid an acid J.S. E.C. 1992; PubMed Scopus Google Scholar). we to the of Asp-54 in ArcA phosphorylation by To we the ArcA mutant protein in which Asp-54 of ArcA was with using site-directed The ArcA(D54E) mutant protein was purified to using column which was able to wild type ArcA was unable to the purified ArcA(D54E) we to the of wild type ArcA and mutant protein to bind the pfl anaerobic conditions, binding of ArcA-P to the pfl promoter induces the expression of phosphoryl such as carbamyl phosphate and phosphate two-component response regulators in the of sensor and (1Parkinson J.S. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington, DC1995: 9-23Google Scholar), ArcA was with either carbamyl phosphate or ArcB. phosphorylated by either carbamyl phosphate or ArcB, binding to the as determined by assays The of ArcA to bind the pfl promoter was ArcA was able to bind the binding was from that of ArcA-P. In addition, with either carbamyl phosphate or ArcB, a binding to the pfl promoter as ArcA the binding of ArcA and ArcA(D54E) to the pfl promoter in assays, their were examined using DNase I The ArcA-P by incubation with either carbamyl phosphate or ArcB to binding to to the site of in the pfl promoter as previously (10Drapal N. Sawers G. Mol. Microbiol. 1995; 16: 597-607Crossref PubMed Scopus (55) Google Scholar). Although a of ArcA as with the ArcA in assay was used in the protein a protection from DNase I In addition, with either carbamyl phosphate or ArcB, was unable to the the binding of ArcA or ArcA(D54E) in the assays be to binding to DNA. This binding in the of These results indicate that the phosphorylation of ArcA is for DNA binding, and Asp-54 of ArcA is for proper DNA binding. It has been reported that the phosphoryl group of response regulators such as Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: PubMed Scopus Google Scholar) and J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar) are found that ArcA-P phosphoryl group with a half-life of 30 min In addition, the phosphoryl group of of EDTA immediately the transphosphorylation reaction the dephosphorylation of both ArcA-P and In the of EDTA, the phosphoryl of both phosphorylated proteins for more than 12 h. The of EDTA is presumably due to of the which be a in the dephosphorylation EDTA was added to a concentration of 50 mm for experiments to the phosphoryl Isoelectric focusing gel electrophoresis proteins on in isoelectric phosphorylation the of a protein, a phosphorylated form of a protein be from form using IEF gel electrophoresis. ArcA, a of and ArcA(D54E) as a protein phosphorylated by ArcB, an additional the of which was using serum indicated that the protein bands in the IEF gel to ArcA or the was in the of ArcA(D54E) with ArcB. To this was due to an of ArcB to ArcA, ArcA and ArcA(D54E) were with ArcB and then subjected to IEF gel electrophoresis followed by or The was in the ArcA and was into These results indicate that the shift of ArcA with ArcB and [γ-32P]ATP was due to the phosphorylation of ArcA and the to ArcA-P. To the of carbamyl phosphate phosphorylation on ArcA and both proteins were with carbamyl phosphate IEF other protein which were the ArcB in the of the was to that of the ArcA-P by ArcB in the ArcA(D54E) with carbamyl However, the was in the ArcA(D54E) with carbamyl These results indicate that carbamyl phosphate amino of ArcA other than through phosphorylation or of the of in the IEF gel the other bands by carbamyl phosphate be from the ArcA-P C. Mol. Microbiol. PubMed Scopus Google Scholar). Although the gene a (6Iuchi S. Lin E.C.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar), ArcA-P have that ArcA-P protects wide regions 60 of DNA from DNase I cleavage (10Drapal N. Sawers G. Mol. Microbiol. 1995; 16: 597-607Crossref PubMed Scopus (55) Google Scholar, 11Lynch A.S. Lin E.C.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar, Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). we wide regions be by the of ArcA. ArcA-P and ArcA were subjected to gel ArcA as a of protein, the transphosphorylation mixture an that contained of the protein IEF gel electrophoresis of the that the contained both ArcA-P and ArcA, and that the contained ArcA In assays, of promoter binding was in the the contained of the protein, these results indicate that the phosphorylation of ArcA induces multimerization of ArcA prior to DNA binding. ArcB and at the The ArcA with carbamyl phosphate In the IEF the to ArcA-P was in the the In the other bands by carbamyl phosphate were in both of promoter binding was from the The that the by carbamyl phosphate has an volume with the by ArcB the of a formation ArcA-P and ArcB. In addition, the of ArcA(D54E) was a with the same volume of the of ArcA was was than that of ArcA This of ArcA(D54E) was by the of ArcB or the carbamyl The of ArcA, which were by incubation with either ArcB or carbamyl contained ArcA-P 5 and were in the of ArcA(D54E) and presumably the protein be phosphorylated by ArcB 1 and The of the with that of ArcB and the of the in the ArcA(D54E) with carbamyl phosphate indicated that the in ArcA(D54E) with ArcB was as due to of ArcB of ArcA-P was confirmed using glycerol sedimentation experiments The glycerol sedimentation of ArcA a by ArcB the with the ArcA These results confirmed that ArcA-P as in experiments and The sedimentation and of the and were as and respectively. These a of the ArcA protein to be 50 that ArcA is a of The sedimentation as and as of the and containing ArcA-P a of a multimer of These results suggest that the multimer is a tetramer of ArcA-P dimers or an composed of ArcA-P The comprised containing both ArcA and ArcA-P proteins and the experiments were performed in the of EDTA, which dephosphorylation of ArcA-P the of ArcA due to dephosphorylation of ArcA-P in these is we to the of ArcA and ArcA-P in these To the of in TP mixtures containing [γ-32P]ATP was using nitrocellulose binding assays The binding assay indicated that of the ArcA was converted to the ArcA-P. Although the binding assay both and in the TP the radioactivities were with the radioactivities of the ArcA-P in the IEF gel that of the ArcA was converted to ArcA-P. the ArcB was from the of TP mixture contained ArcB in addition to ArcA-P and in the of the protein in the to that in the was These results indicate that of the multimer is composed of ArcA-P. This to be in with the ArcA-P in we that the multimer is composed of both ArcA and ArcA-P with a of of ArcA-P in TP binding experiments with TP mixtures containing [γ-32P]ATP were performed as described under and assays were and in a binding experiments with TP mixtures containing [γ-32P]ATP were performed as described under and assays were and the response regulators of two-component signal transduction systems, the of which is the response regulator has been by and other J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, 1989; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The and of this protein form an acid and the is phosphorylated J.S. E.C. 1992; PubMed Scopus Google Scholar). to amino acid sequence with and other response regulators of two-component systems, it was that and Asp-54 of ArcA protein form an acid and the Asp-54 is the site for ArcB phosphorylation (8Georgellis D. Lynch A.S. Lin E.C.C. J. Bacteriol. 1997; 179: 5429-5435Crossref PubMed Google Scholar). However, the of these amino we demonstrate that an ArcA mutation at Y. S. J. S. J. B. and D. S. as as a mutation at Asp-54 resulted in the inability of these mutant proteins to be phosphorylated by ArcB. In addition, the of ArcA(D54E) as with that of wild type ArcA in gel suggests that ArcA(D54E) a from ArcA. Although the phosphorylation site was determined these results indicate that the and Asp-54 are to a proper for ArcA. phosphate and phosphate the response regulators of two-component (1Parkinson J.S. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington, DC1995: 9-23Google Scholar). However, for the phosphorylation has been In addition to ArcA-P by ArcB incubation of ArcA-P with carbamyl phosphate at additional ArcA proteins were in IEF gel electrophoresis and The ArcA proteins were incubation of the ArcA(D54E) mutant protein with carbamyl these ArcA proteins be by to ArcA-P The of these proteins in both the and in that the the multimerization of ArcA-P with ArcA. of proteins induces a protein In two-component systems, a sensor an acid of response regulator, in activation of the regulator PubMed Scopus Google Scholar). of the protein induces and the resultant dimer binds to DNA binding sites U. J. 1995; PubMed Scopus Google Scholar, Mol. Microbiol. 1996; PubMed Scopus Google Scholar). In addition, the phosphorylated dimers and as by experiments J. Biol. Chem. Full Text PDF PubMed Google Scholar). phosphorylation of induces interaction dimers that results in the formation of The proteins and bind to target DNA S. 1993; PubMed Scopus Google Scholar). a protein binds or to DNA, the region in experiments by the of the the of ArcA-P the region the of the region and region of Y.S. Han J.S. Jeon Y. Hwang D.S. J. Biol. Chem. 2001; 276: 9917-9923Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). These that multimerization of ArcA occurs prior to DNA binding, and the ArcA multimer binds to DNA. Our results indicate that the phosphorylation of the ArcA dimer induces the formation of a tetramer of dimers an that is composed of both ArcA-P and ArcA. The of ArcA-P to ArcA in the multimer to be These suggest ArcA multimer is composed of of which is or dimers of of which are form a tetramer with ArcA The formation of an ArcA multimer with ArcA-P and ArcA be in the that ArcA proteins to phosphorylation in response to an anaerobic This the activation of ArcA, thereby to a of E. coli to anaerobic for of this