FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2084872777> ?p ?o ?g. }

• W2084872777 endingPage "9923" @default.
• W2084872777 startingPage "9917" @default.
• W2084872777 abstract "Under anaerobic growth conditions,Escherichia coli operates a two-component signal transduction system, termed Arc, that consists of ArcB protein, a transmembrane sensor kinase and ArcA protein, the cognate response regulator. In response to low oxygen levels, autophosphorylated ArcB phosphorylates ArcA, and the resulting phosphorylated ArcA (ArcA-P) functions as a transcriptional regulator of the genes necessary to maintain anaerobic growth. Under anaerobic conditions, cells maintain a slow growth rate, suggesting that the initiation of chromosomal replication is regulated to reduce the initiation frequency. DNase I footprinting experiments revealed that ArcA-P binds to the left region of the chromosomal origin, oriC. ArcA-P did not affect thein vitro replication of plasmid DNA containing the ColE1 origin nor the in vitro replication of viral DNAs; however, ArcA-P specifically inhibited in vitro E. coli chromosomal replication. This inhibition was caused by the prevention of open complex formation, a necessary step in the initiation of chromosomal replication. Our in vitro results suggest that the Arc two-component system participates in regulating chromosomal initiation under anaerobic growth conditions. Under anaerobic growth conditions,Escherichia coli operates a two-component signal transduction system, termed Arc, that consists of ArcB protein, a transmembrane sensor kinase and ArcA protein, the cognate response regulator. In response to low oxygen levels, autophosphorylated ArcB phosphorylates ArcA, and the resulting phosphorylated ArcA (ArcA-P) functions as a transcriptional regulator of the genes necessary to maintain anaerobic growth. Under anaerobic conditions, cells maintain a slow growth rate, suggesting that the initiation of chromosomal replication is regulated to reduce the initiation frequency. DNase I footprinting experiments revealed that ArcA-P binds to the left region of the chromosomal origin, oriC. ArcA-P did not affect thein vitro replication of plasmid DNA containing the ColE1 origin nor the in vitro replication of viral DNAs; however, ArcA-P specifically inhibited in vitro E. coli chromosomal replication. This inhibition was caused by the prevention of open complex formation, a necessary step in the initiation of chromosomal replication. Our in vitro results suggest that the Arc two-component system participates in regulating chromosomal initiation under anaerobic growth conditions. phosphorylated ArcB phosphorylated ArcA phosphorylation mixture containing carbamyl phosphate replicative form transphosphorylation reaction mixture base pair(s) adenosine 5′-(β,γ-methylenetriphosphate) Most organisms, including Escherichia coli, are able to adapt to variable growth conditions and environmental changes by regulating gene expression (1Parkinson J.S. Hoch J.A. Silhavy T.J. Two-component Signal Transduction.in: American Society for Microbiology, Washington, DC1995: 9-23Google Scholar, 2Ninfa, A. J., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 1, 1996, 1246, 1262, American Society for Microbiology, Washington DC.Google Scholar). Adaptation in bacterial cells is usually achieved through two-component signal transduction systems, which consist of a sensor kinase and a response regulator (2Ninfa, A. J., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 1, 1996, 1246, 1262, American Society for Microbiology, Washington DC.Google Scholar, 3Stock J.B. Stock A.M. Mottonen J.M. Nature. 1990; 344: 395-400Crossref PubMed Scopus (485) Google Scholar). The response regulator, phosphorylated by cognate sensor kinase in response to an external signal, serves as a transcriptional regulator optimizing gene expression under a given condition. In E. coli, the Arc (anoxic redox control) two-component signal transduction system operates in response to a shift from aerobiosis to anaerobiosis (4Lynch, A. S., Lin, E. C. C., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 1, 1996, 1526, 1538, American Society for Microbiology, Washington DC.Google Scholar). The Arc signal transduction system consists of the ArcB and ArcA proteins, a transmembrane sensor kinase and its cognate response regulator, respectively (5Iuchi S. Cameron D.C. Lin E.C. J. Bacteriol. 1989; 171: 868-873Crossref PubMed Google Scholar, 6Iuchi S. Cameron D.C. Lin E.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1888-1892Crossref PubMed Scopus (296) Google Scholar). In response to oxygen deficiency or redox change, ArcB autophosphorylates in an ATP-dependent manner and converts to phosphorylated ArcB (ArcB-P)1 via an intramolecular phospho-relay of His-292 → Asp-576 → His-717 (7Georgellis D. Lynch A.S. Lin E.C. J. Bacteriol. 1997; 179: 5429-5435Crossref PubMed Google Scholar). Subsequently, ArcB-P phosphorylates Asp-54 of ArcA, and the resulting ArcA-P (phosphorylated ArcA) functions as a transcriptional repressor for the sdh, gltA, lld,cyo, and sodA genes (4Lynch, A. S., Lin, E. C. C., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 1, 1996, 1526, 1538, American Society for Microbiology, Washington DC.Google Scholar, 6Iuchi S. Cameron D.C. Lin E.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1888-1892Crossref PubMed Scopus (296) Google Scholar, 8Iuchi S. Chepuri V. Fu H.A. Gennis R.B. Lin E.C. J. Bacteriol. 1990; 172: 6020-6025Crossref PubMed Google Scholar, 9Tardat B. Touati D. Mol. Microbiol. 1991; 5: 455-465Crossref PubMed Scopus (76) Google Scholar) and as an activator for cyd, pfl, and traY genes (4Lynch, A. S., Lin, E. C. C., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 1, 1996, 1526, 1538, American Society for Microbiology, Washington DC.Google Scholar, 8Iuchi S. Chepuri V. Fu H.A. Gennis R.B. Lin E.C. J. Bacteriol. 1990; 172: 6020-6025Crossref PubMed Google Scholar, 10Sawers G. Suppmann B. J. Bacteriol. 1992; 174: 3474-3478Crossref PubMed Google Scholar, 11Silverman P.M. Wickersham E. Harris R. J. Mol. Biol. 1991; 218: 119-128Crossref PubMed Scopus (41) Google Scholar) to sustain anaerobic growth. Anaerobic conditions that induce the Arc two-component signal transduction system lead to reduction in growth rate (12Iuchi S. Aristarkhov A. Dong J.M. Taylor J.S. Lin E.C. J. Bacteriol. 1994; 176: 1695-1701Crossref PubMed Google Scholar). Because the rate of chromosomal replication regulated mainly at the level of initiation is coupled to growth rate (13von Meyenburg, K., Hansen, F. G., Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M., Umbarger, H. E., 2, 1987, 1555, 1577, American Society for Microbiology, Washington DC.Google Scholar), the slow growth rate ofE. coli during times of oxygen deficiency suggests that the frequency of chromosomal initiation is reduced. It is therefore probable that regulation of initiation occurs at oriC, theE. coli origin of chromosomal replication (14Oka A. Sugimoto K. Takanami M. Hirota Y. Mol. Gen. Genet. 1980; 178: 9-20Crossref PubMed Scopus (113) Google Scholar). This unique sequence, which is the highly conserved origin of Gram-negative bacteria (15Zyskind J.W. Cleary J.M. Brusilow W.S. Harding N.E. Smith D.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1164-1168Crossref PubMed Scopus (117) Google Scholar), includes four DnaA boxes, AT-rich region containing three 13-mers, and an IHF binding site, all of which are required for proper chromosomal initiation of replication (16Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., New York1992: 521-533Google Scholar, 17Messer, W., Weigel, C., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 2, 1996, 1579, 1601, American Society for Microbiology, Washington DC.Google Scholar). Binding of the initiator protein, DnaA, to the DnaA boxes leads to unwinding of the AT-rich regions and allows for the entry of DnaB helicase, a required step for subsequent initiation processes (18Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 19Bramhill D. Kornberg A. Cell. 1988; 54: 915-918Abstract Full Text PDF PubMed Scopus (333) Google Scholar). Opening of the AT-rich region is facilitated by the binding of IHF protein to the IHF site, thereby bending oriC (20Polaczek P. New Biol. 1990; 2: 265-271PubMed Google Scholar, 21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar). Although IHF can be substituted by HU protein (21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar), in vivo footprinting experiments suggest that IHF may play a role in determining the timing of chromosomal initiation during the cell cycle (22Cassler M.R. Grimwade J.E. Leonard A.C. EMBO J. 1995; 14: 5833-5841Crossref PubMed Scopus (109) Google Scholar). Here we report that the binding of ArcA-P, phosphorylated by ArcB, tooriC results in the inhibition of chromosomal initiation. This result suggests that the Arc two-component signal transduction system plays a role in the regulation of chromosomal initiation atoriC in response to oxygen deficiency. Sources were as follows: [γ-32P]ATP (5000 Ci/mmol), [α-32P]dCTP (3000 Ci/mmol), and deoxynucleotides, Amersham Pharmacia Biotech; Long Ranger polyacrylamide, FMC BioProducts; DNase I, Life Technologies Inc.; nuclease P1, Roche Molecular Biochemicals; T4 polynucleotide kinase and Vent (exo−) DNA polymerase, New England BioLabs; calf intestinal alkaline phosphatase and restriction enzymes,Promega Corp. Unless indicated, reagents were purchased from Sigma Chemical Co. Monomeric DnaA protein from HMS174 (pKC596) (23Carr K.M. Kaguni J.M. Mol. Microbiol. 1996; 20: 1307-1318Crossref PubMed Scopus (59) Google Scholar) and HU protein (24Kaguni J.M. Kornberg A. Cell. 1984; 38: 183-190Abstract Full Text PDF PubMed Scopus (134) Google Scholar) were purified as previously described. E. coli strains MC1061 (25Johnston S. Lee J.H. Ray D.S. Gene. 1985; 34: 137-145Crossref PubMed Scopus (86) Google Scholar), W3110 (26Bachmann B.J. Bacteriol. Rev. 1972; 36: 525-557Crossref PubMed Google Scholar), and WM433(dnaA204) (27Beyersmann D. Messer W. Schlicht M. J. Bacteriol. 1974; 118: 783-789Crossref PubMed Google Scholar) were previously described. E. coli DH5α (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was used for isolation of plasmid DNA. The DNAs were as follows: pFToriC (21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar) for DNase I footprinting; pSBoriC (29Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23087-23091Abstract Full Text PDF PubMed Google Scholar) for nuclease P1 assay; pBluescript SK(+) (Stratagene Corp.) for cloning; M13RE85 RF (replicative form) DNA (30Baker T.A. Kornberg A. Cell. 1988; 55: 113-123Abstract Full Text PDF PubMed Scopus (172) Google Scholar), and single-stranded DNAs from phage G4 (31Stayton M.M. Kornberg A. J. Biol. Chem. 1983; 258: 13205-13212Abstract Full Text PDF PubMed Google Scholar), M13mp19 (Stratagene Corp.), φX174 (31Stayton M.M. Kornberg A. J. Biol. Chem. 1983; 258: 13205-13212Abstract Full Text PDF PubMed Google Scholar) for in vitrocomplementation assays. For the construction of pBADarcA and pBADarcB, coding regions of arcA containing the whole polypeptide and arcB containing from the amino acid residue 129 to the stop codon were amplified using polymerase chain reaction and inserted into the EcoRI/HindIII site of pBAD18 (32Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3959) Google Scholar) and the KpnI/XbaI site of pBAD24 (32Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3959) Google Scholar), respectively. E. colistrain MC1061 harboring either pBADarcA or pBADarcB was grown in LB media to an OD at 595 nm of 0.5 followed by the addition of l(+)-arabinose to a concentration of 0.2% to induce overproduction of the proteins. 2 h after induction, cells were harvested, resuspended in cell resuspension buffer (25 mm HEPES-KOH (pH 7.6), 1 mm EDTA, and 2 mm dithiothreitol) to an OD at 595 nm of 200 and frozen in liquid nitrogen. Cell lysis and ammonium sulfate precipitation were performed as previously described (33Hwang D.S. Kaguni J.M. J. Biol. Chem. 1988; 263: 10625-10632Abstract Full Text PDF PubMed Google Scholar), except that 0.37 g/ml ammonium sulfate instead of 0.28 g/ml was used. The pellet was resuspended in buffer A (25 mm HEPES-KOH (pH 7.8), 1 mm EDTA, 10% glycerol, and 2.86 mm2-mercaptoethanol), which was used throughout the purification procedure. Subsequently, the activities of ArcA and ArcB were determined by transphosphorylation reactions as described below. The resuspended ammonium sulfate-precipitated pellet containing ArcA protein was dialyzed to the conductivity equivalent of 50 mm KCl then subjected to FastQ column chromatography (60 ml of bed volume, Sigma) using 600 ml of a linear gradient ranging from 50 mm to 1 m KCl in buffer A. The fractions containing ArcA were pooled and dialyzed to the conductivity equivalent of 50 mm KCl then loaded onto a heparin-agarose column (12 ml of bed volume, Sigma). A 120-ml gradient ranging from 50 mm to 1 m KCl in buffer A was used for protein elution, with ArcA eluting at ∼150 mm KCl. The fractions containing ArcA were pooled, diluted with buffer A to the conductivity equivalent of 50 mm KCl, and loaded onto a FastS column (4 ml of bed volume, Sigma). The fractions containing ArcA were pooled, diluted to the conductivity equivalent of 50 mm KCl with buffer A, and subjected to Cibacron Blue column chromatography (2.5 ml of bed volume, Sigma). A 25-ml gradient ranging from 50 mmto 2 m KCl in buffer A was run over the column, yielding near homogeneous ArcA, which eluted at ∼0.5 m KCl. About 17 mg of ArcA protein was obtained from 15 liters culture of MC1061(pBADarcA). For ArcB protein purification, the ammonium sulfate precipitate obtained from 6 liters of MC1061(pBADarcB) culture as described above was resuspended in buffer A, dialyzed to a conductivity equivalent of 50 mm KCl, and subjected to FastQ column chromatography (90 ml of bed volume) using a 900-ml gradient ranging from 50 mm to 1 m KCl in buffer A. Fractions containing ArcB were pooled, then dialyzed to a conductivity equivalent of 50 mm KCl and loaded onto a Cibacron blue column (17 ml of bed volume). A 170-ml gradient ranging from 50 mm to 1m KCl in buffer A was run over the column. ArcB eluted in a broad range of fractions, which were pooled, and the protein was precipitated by the addition of 0.45 g/ml ammonium sulfate followed by centrifugation at 45,000 rpm for 30 min in a Ti70 rotor (Beckman). The pellet was resuspended with buffer A and subjected to Superose-12 gel filtration chromatography (Amersham Pharmacia Biotech, HR 10/30). ArcB eluted as a single peak. These fractions were pooled and loaded onto a MonoQ column (Amersham Pharmacia Biotech, HR 5/5). ArcB eluted from the MonoQ column at near homogeneity and was used in further experiments. A 6-liter culture of MC1061(pBADarcB) yielded about 9 mg of homogeneous ArcB. Transphosphorylation reactions (TP), including purified ArcA and ArcB, were performed as previously described (34Iuchi S. Lin E.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar) with minor modifications. 10 μl of the TP mixture contained 6 μg each of ArcA and ArcB, 0.1 mm ATP, 70 mm KCl, 10 mm MgCl2, 33 mm HEPES-KOH (pH 7.4), 0.1 mm EDTA, and 2 mm dithiothreitol. After incubation at 32 °C for 10 min, the reaction mixture (2 μl per each assay unless indicated) was immediately used for further experiments. To visualize the phosphorylated proteins, [γ-32P]ATP was added to the above mixture. The reaction was terminated by addition of gel-loading buffer (10% glycerol, 3% SDS, 3% β-mercaptoethanol, and 0.3% bromphenol blue). After incubation at 55 °C for 3 min, the mixture was subjected to 12% SDS-polyacrylamide gel electrophoresis. The gel was dried and visualized by autoradiography. Phosphorylation reaction of ArcA with carbamyl phosphate (CP) was performed as previously described (35Lynch A.S. Lin E.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar). 10 μl of the CP mixture containing the indicated amount of ArcA protein, 40 mmdilithium carbamyl phosphate,125 mm KCl, 10 mm MgCl2, and 100 mm Tris-HCl (pH 7.0) was incubated at 30 °C for 1 h and immediately used for experiments. DNase I protection assays were performed as previously described (36Lee Y.S. Kim H. Hwang D.S. Mol. Microbiol. 1996; 19: 389-396Crossref PubMed Scopus (29) Google Scholar) with minor modifications. A 435-bp XbaI/XhoI fragment from pFToriCwas labeled at either the XhoI or XbaI restriction site. 21.5 fmol of labeled fragments was mixed with the indicated proteins in 25 μl of standard reaction mixture containing 0.1 mm ATP, 50 mm potassium chloride, 10 mm magnesium acetate, 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, DNase I (5 ng in 1.5 μl of H2O) was added, incubated for 30 s, then stopped by the addition of 27 μl of 0.6 m sodium acetate, 0.4% SDS, 25 mmEDTA, and 0.1 mg/ml yeast tRNA. Proteins were removed by phenol/chloroform extraction, and DNA was precipitated by ethanol, followed by a 70% ethanol wash. DNA was subjected to electrophoresis through a 5% Long Ranger polyacrylamide sequencing gel containing 7m urea. The gel was dried and visualized by autoradiography. As previously described (37Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (237) Google Scholar, 38Fuller R.S. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5817-5821Crossref PubMed Scopus (189) Google Scholar), in vitro oriC plasmid replication with fraction II from WM433 (dnaA204) and purified DnaA protein was performed using 200 ng of M13RE85 RF DNA as a template. Single-stranded phage DNAs, such as G4, M13mp19, and φX174, and pBluescript containing the ColE1 replication origin, 200 ng each, were used for DnaA-independent replication. In these assays, all conditions were identical to oriC plasmid replication reactions, except that DnaA was omitted. Open complex formation at oriC was detected using single-strand specific P1 nuclease as previously described with modifications (21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar, 39Torheim N.K. Skarstad K. EMBO J. 1999; 18: 4882-4888Crossref PubMed Scopus (68) Google Scholar). 25 μl of opening reaction mixture containing the indicated amount of DnaA, 15 ng of HU, 200 ng of supercoiled pSBoriC, 4 mm ATP, 50 mm potassium glutamate, 2.5 mm magnesium acetate, 2.5 μg of bovine serum albumin, 40 mm HEPES-KOH (pH 7.6), and 17% glycerol, was incubated at 37 °C for 5 min. Then 3 units of P1 nuclease in 3 μl of 30 mm sodium acetate (pH 5.2) was added and incubated at 37 °C for 30 s. The cleavage reaction was quenched by the addition of 27 μl of stop solution (25 mm EDTA, 0.4m NaOH). After incubation at room temperature for 10 min, followed by addition of 6 μl of 3 m sodium acetate (pH 5.2), proteins were removed by phenol/chloroform extraction. With 2.5 μg of yeast tRNA as a carrier for precipitation, DNA samples were collected by ethanol precipitation followed by a 70% ethanol wash. The precipitated DNA was resuspended with 6 μl of H2O, and 2 μl was taken to be used as template for primer extension reactions. 6 μl of the primer extension mixture included 1.25 nmol of each dNTP, 0.25 pmol of 5′-end-labeled primer PA1 (21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar), and 0.42 unit of Vent (exo−) DNA polymerase in Vent (exo−) buffer with 5 mm MgSO4. The primer was 5′-end-labeled with [γ-32P]ATP and T4 polynucleotide kinase; unincorporated radioactivity was removed using a Bio-Gel 6 spin column (Bio-Rad). The mixture was subjected to primer extension reactions in a thermocycler, for 20 cycles (95 °C for 1 min except for 4 min in the first cycle, 55 °C for 1 min, and 72 °C for 1 min except for 6 min in the last cycle). The reaction was stopped by the addition of 4 μl of sequencing gel loading buffer, then subjected to electrophoresis through a 5% Long Ranger polyacrylamide sequencing gel containing 7 m urea. The gel was dried and visualized by autoradiography. To investigate the interaction of ArcA and ArcB with oriC DNA, ArcA and ArcB proteins were overproduced and purified to near homogeneity using column chromatography (Fig. 1 A). Due to poor expression of the ArcB protein, we instead expressed and purified a truncated ArcB lacking the N terminus from amino acid residues from 1 to 128 as described previously (34Iuchi S. Lin E.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar). The ArcA and ArcB proteins used in this study were distinctive from the proteins containing the hexa-histidine (7Georgellis D. Lynch A.S. Lin E.C. J. Bacteriol. 1997; 179: 5429-5435Crossref PubMed Google Scholar, 35Lynch A.S. Lin E.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar, 40Colloms S.D. Alen C. Sherratt D.J. Mol. Microbiol. 1998; 28: 521-530Crossref PubMed Scopus (45) Google Scholar, 41Strohmaier H. Noiges R. Kotschan S. Sawers G. Hogenauer G. Zechner E.L. Koraimann G. J. Mol. Biol. 1998; 277: 309-316Crossref PubMed Scopus (48) Google Scholar) or renatured from SDS-polyacrylamide gels (34Iuchi S. Lin E.C. J. Bacteriol. 1992; 174: 5617-5623Crossref PubMed Google Scholar) as used in previous studies. The purified ArcA and ArcB proteins were active in transphosphorylation reactions. In the presence of ATP, ArcB underwent autophosphorylation and subsequently transferred the phosphoryl group from itself to ArcA, forming ArcA-P, whereas ArcA alone was not phosphorylated (Fig.1 B). Binding of the transphosphorylation mixture (TP mixture), which contains ArcA, ArcB, and ATP, to the oriC region of theE. coli chromosome was detected using a DNase I protection assay (Fig. 2). Increasing the amount of TP mixture added to the footprinting reaction revealed that an ∼150-bp region located at the left end of oriC, including the three 13-mer AT-rich regions, DnaA box R1, and IHF binding site, was protected from DNase I cleavage (Fig. 2). The omission of ArcA, ArcB, or both from the TP mixture abolished the protection pattern observed at oriC, indicating that the oriCbinding activity requires both ArcA and ArcB proteins. Neither ADP nor the nonhydrolyzable ATP-analogue AMPPNP was able to substitute ATP for the oriC protection activity of the TP mixture, implying the requirement of ATP hydrolysis for binding (Fig.3 A). Efficient oriCprotection activity required more than 50 μm ATP. Without ArcB and ATP, it has been shown that the phosphoryl group donors carbamyl phosphate and acetyl phosphate can phosphorylate ArcA (35Lynch A.S. Lin E.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar). Therefore, we incubated ArcA with carbamyl phosphate in the absence of ArcB and ATP, and found an identical DNase I cleavage protection pattern (Fig. 3 B). These results indicate that ArcA-P, produced by phosphorylation of ArcA protein either by ArcB and ATP or by carbamyl phosphate, binds to the left end of oriC. The AT-rich regions and DnaA box R1 found at oriC are indispensable for both in vitro andin vivo initiation of chromosomal replication (18Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 29Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23087-23091Abstract Full Text PDF PubMed Google Scholar, 42Asai T. Takanami M. Imai M. EMBO J. 1990; 9: 4065-4072Crossref PubMed Scopus (59) Google Scholar). Therefore, the effect of ArcA-P, which was found to bind those regions of oriC, on chromosomal initiation was examined using anin vitro oriC plasmid replication assay (37Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (237) Google Scholar, 38Fuller R.S. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5817-5821Crossref PubMed Scopus (189) Google Scholar). This assay resembles in vivo chromosomal initiation in many aspects, including dependence upon the oriC sequence, requirement of replicative proteins, and bidirectional replication fromoriC (37Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (237) Google Scholar, 43Kaguni J.M. Fuller R.S. Kornberg A. Nature. 1982; 296: 623-627Crossref PubMed Scopus (39) Google Scholar). Addition of purified DnaA protein to the oriC plasmid replication assay sustained the replication of oriC plasmid M13RE85 RF DNA, in which the oriC region has been inserted into M13mp8 RF DNA (Fig. 4 A). The presence of the TP mixture in the assay, however, inhibited DnaA-dependent oriC plasmid replication. Omission of ArcA, ArcB, or both from the TP mixture eliminated the inhibitory activity of the TP mixture (Fig. 4 B), indicating that the inhibitory activity is dependent on both ArcA and ArcB. However, incubation of ArcA protein with carbamyl phosphate instead of ArcB and ATP also inhibited oriC plasmid replication, whereas carbamyl phosphate or ArcA alone was not inhibitory (Fig.4 C). These results imply that ArcA-P, formed either by ArcB and ATP or by carbamyl phosphate, inhibits the oriC plasmid replication, and ArcB is not a requirement. To determine whether ArcA-P specifically inhibits initiation atoriC, the effect of ArcA-P on other origins was studied. Single-stranded viral DNAs φX174, M13, and G4 replicate from single- to double-stranded RF DNA using unique initiation processes, with each viral origin using varying proteins (16Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., New York1992: 521-533Google Scholar). In the absence of DnaA, the soluble proteins of fraction II, used for oriC plasmid replication, are sufficient to replicate single-stranded viral DNAs to RF DNAs (37Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (237) Google Scholar). Plasmid pBluescript, which contains the ColE1 origin, can also be replicated by fraction II in the absence of DnaA, but with less efficiency than the DnaA-dependent replication oforiC plasmid DNAs (37Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (237) Google Scholar). Addition of the TP mixture to these reactions did not affect replication of any of the tested single-stranded viral DNAs or pBluescript DNA (Fig. 4 D), indicating that ArcA-P specifically inhibits the initiation of replication at oriC. Because DNase I footprinting revealed the binding region of ArcA-P at a 150-bp region of the left end of oriC containing both DnaA box R1 and the IHF binding site, DNase I footprinting was further performed to determine whether ArcA-P inhibits initiation of replication at oriC by blocking the interaction of DnaA or IHF with oriC (Fig. 5). Addition of increasing amounts of DnaA to oriC (Fig.5 A, lanes 2–4) resulted in the protection of DnaA boxes R1 to R4 and IHF bound to the IHF binding site (Fig.5 B, lanes 2–4) both as previously described (21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar). DnaA and IHF added prior to or after ArcA-P did not allow the binding of ArcA-P to DnaA box R1 nor the IHF site, respectively; however, neither protein affected the binding of ArcA-P to the AT-rich region (Fig. 5, A and B, lanes 5–11). These results indicate that the binding of DnaA to DnaA box R1 and IHF to the IHF site is preferred over the binding of ArcA-P to those sites. However, DnaA and IHF do not affect ArcA-P binding to the AT-rich region. To further study the binding of ArcA-P to the AT-rich region oforiC, competition experiments using IciA were performed. IciA protein specifically binds to the three 13-mers in the AT-rich region and inhibits the initiation stage of in vitro oriCreplication (52Hwang D.S. Kornberg A. Cell. 1990; 63: 325-331Abstract Full Text PDF PubMed Scopus (58) Google Scholar). Interestingly, addition of IciA to a preformed ArcA-P·oriC complex displaced ArcA-P to generate a footprint similar that of IciA only. Conversely, ArcA-P displaced IciA bound to oriC (Fig. 5 B, lanes 14–16). At the onset of initiation, DnaA protein unwinds the AT-rich regions of oriC with the aid of HU or IHF, forming the open complex, a step that is prerequisite for the subsequent stages of initiation (18Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 19Bramhill D. Kornberg A. Cell. 1988; 54: 915-918Abstract Full Text PDF PubMed Scopus (333) Google Scholar). DnaA-dependent strand opening of the AT-rich region can be observed using single-stranded-specific P1 nuclease and primer extension assays (Fig.6), as previously described (21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar, 39Torheim N.K. Skarstad K. EMBO J. 1999; 18: 4882-4888Crossref PubMed Scopus (68) Google Scholar). Addition of the TP mixture prior to DnaA protein in the assays resulted in inhibition of open complex formation. However, the addition of the TP mixture after open complex formation did not significantly inhibit open complex formation. These results imply that ArcA-P functions prior to the DnaA-dependent strand opening of the AT-rich region. The amounts of TP mixture required for oriC binding (Fig.2), inhibition of oriC replication (Fig. 4 B), and inhibition of open complex formation (Fig. 6) were comparable to each other. These results suggest that binding of ArcA-P to oriCinhibits oriC initiation by blocking open complex formation. ArcA protein phosphorylated by ArcB and ATP or by carbamyl phosphate binds the left end of oriC. This bound region includes the AT-rich 13-mers, DnaA box R1, and the IHF binding site, regions all highly conserved in chromosomal replication origins of Gram-negative bacteria (15Zyskind J.W. Cleary J.M. Brusilow W.S. Harding N.E. Smith D.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1164-1168Crossref PubMed Scopus (117) Google Scholar) and all of which are essential for initiation of E. coli chromosomal replication (18Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 29Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23087-23091Abstract Full Text PDF PubMed Google Scholar, 42Asai T. Takanami M. Imai M. EMBO J. 1990; 9: 4065-4072Crossref PubMed Scopus (59) Google Scholar). In chromosomal initiation events, the AT-rich region of oriCis unwound upon binding of DnaA proteins to the DnaA boxes (18Bramhill D. Kornberg A. Cell. 1988; 52: 743-755Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 19Bramhill D. Kornberg A. Cell. 1988; 54: 915-918Abstract Full Text PDF PubMed Scopus (333) Google Scholar). Interaction of DnaA protein with the AT-rich region, leading to strand opening, is facilitated by the bending of oriC by IHF protein bound to the IHF site (20Polaczek P. New Biol. 1990; 2: 265-271PubMed Google Scholar, 21Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar). Binding of ArcA-P blocks DnaA-dependent strand opening of the AT-rich regions (Fig.6), a step required for subsequent stages of chromosomal initiation to occur. Binding of ArcA-P also results in the inhibition of in vitro oriC plasmid replication (Fig. 4). Because ArcA-P does not appear to affect the binding of DnaA and IHF to their loci (Fig. 5), such inhibition may be caused by ArcA-P binding to the AT-rich regions of oriC, thereby inhibiting a proper interaction between DnaA protein and the AT-rich region, an interaction which is thought to be required for the opening of the AT-rich region by DnaA (29Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23087-23091Abstract Full Text PDF PubMed Google Scholar,44Yung B.Y. Kornberg A. J. Biol. Chem. 1989; 264: 6146-6150Abstract Full Text PDF PubMed Google Scholar). Aside from our reported binding of ArcA-P to oriC, ArcA-P binds to a number of promoter regions (35Lynch A.S. Lin E.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar, 44Yung B.Y. Kornberg A. J. Biol. Chem. 1989; 264: 6146-6150Abstract Full Text PDF PubMed Google Scholar, 45Drapal N. Sawers G. Mol. Microbiol. 1995; 16: 597-607Crossref PubMed Scopus (55) Google Scholar, 46Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar), including thepfl promoter that controls expression of pyruvate formate lyase (46Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar). We observed that ArcA-P possessed similar affinities to theoriC and pfl promoters using gel-shift assays (data not shown). However, we could not match the suggested ArcA-P binding consensus sequence (49Verma M. Moffat K.G. Egan J.B. Mol. Gen. Genet. 1989; 216: 446-454Crossref PubMed Scopus (17) Google Scholar) with the oriC sequences, the only commonality we found was a richness of A and T. Although unphosphorylated ArcA binds to pfl and sodApromoter regions in a several- to 10-fold excess over ArcA-P, with the same pattern of ArcA-P binding (46Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar, 47Pellicer M.T. Fernandez C. Badia J. Aguilar J. Lin E.C. Baldom L. J. Biol. Chem. 1999; 274: 1745-1752Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), we could not detect any binding of unphosphorylated ArcA to oriC, even when using a 15- to 20-fold excess (up to 15 μm) as compared with ArcA-P in the DNase I footprinting assay (data not shown). ArcA-P, even with a molecular mass of 27 kDa, protected a wide region, ∼150 bp, of oriC from DNase I cleavage (Figs. 2 and 3). This broad binding pattern was also observed at other ArcA-P binding sites (35Lynch A.S. Lin E.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar, 46Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar, 48McGuire A.M. De Wulf P. Church G.M. Lin E.C. Mol. Microbiol. 1999; 32: 219-221Crossref PubMed Scopus (22) Google Scholar). The increased amount of ArcA-P enhanced the intensity of protected regions, rather than resulting in the broadening of the bound region, which would be evident if sequential bindings of ArcA-P were taking place (Fig. 2). These binding patterns suggest that multimerized or oligomerized ArcA-P, formed prior to DNA binding, as suggested from other binding sites (35Lynch A.S. Lin E.C. J. Bacteriol. 1996; 178: 6238-6249Crossref PubMed Google Scholar, 46Tardat B. Touati D. Mol. Microbiol. 1993; 9: 53-63Crossref PubMed Scopus (103) Google Scholar, 47Pellicer M.T. Fernandez C. Badia J. Aguilar J. Lin E.C. Baldom L. J. Biol. Chem. 1999; 274: 1745-1752Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), may bind to the left half oforiC. The DNase I-hypersensitive sites with ∼10-bp intervals (Figs. 2 and 3) suggest that multimerized ArcA-P wraps the left end of oriC. Chromosomal replication is regulated mainly at the level of initiation; the rate of chromosomal replication is coupled to growth rate (13von Meyenburg, K., Hansen, F. G., Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M., Umbarger, H. E., 2, 1987, 1555, 1577, American Society for Microbiology, Washington DC.Google Scholar). Different physiological conditions determine unique growth rates, reflected by the frequency of chromosomal initiation. Slow growth conditions require a reduction in the frequency of chromosomal initiations to meet the retarded growth. However, it has been scarcely documented how organisms set the initiation frequency in response to various physiological conditions or environmental stresses. In E. coli, it was reported that UV irradiation inhibits the initiation of chromosomal replication from oriC (50Iuchi S. Furlong D. Lin E.C. J. Bacteriol. 1989; 171: 2889-2893Crossref PubMed Google Scholar), however, there are no further studies showing the regulatory factors involved or underlying mechanisms. The pleiotrophic effects of arcA mutations (4Lynch, A. S., Lin, E. C. C., Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., Neidhardt, F. C., Curtiss, R., III, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E., 1, 1996, 1526, 1538, American Society for Microbiology, Washington DC.Google Scholar, 5Iuchi S. Cameron D.C. Lin E.C. J. Bacteriol. 1989; 171: 868-873Crossref PubMed Google Scholar, 6Iuchi S. Cameron D.C. Lin E.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1888-1892Crossref PubMed Scopus (296) Google Scholar, 41Strohmaier H. Noiges R. Kotschan S. Sawers G. Hogenauer G. Zechner E.L. Koraimann G. J. Mol. Biol. 1998; 277: 309-316Crossref PubMed Scopus (48) Google Scholar, 51Nystrom T. Larsson C. Gustafsson L. EMBO J. 1996; 15: 3219-3228Crossref PubMed Scopus (98) Google Scholar,52Hwang D.S. Kornberg A. Cell. 1990; 63: 325-331Abstract Full Text PDF PubMed Scopus (58) Google Scholar) present challenges in performing in vivo experiments and make interpretation of the results difficult. Even under aerobic condition, arcA mutants possess reduced chromosome numbers compared with the wild type (data not shown). Although there is no clear in vivo data available, our in vitroresults suggest that Arc, a two-component signal transduction system operated under anaerobic conditions in E. coli, plays a role in regulation of chromosomal initiation. Under oxygen depletion stress, the response regulator ArcA, phosphorylated by sensor kinase ArcB, may bind to oriC and reduce chromosomal initiation. We thank Dr. Edmund C. C. Lin and Dr. Ohsuk Kwon for helpful discussions and Gillian Newman for careful editing of this manuscript." @default.
• W2084872777 created "2016-06-24" @default.
• W2084872777 creator A5018058696 @default.
• W2084872777 creator A5024856047 @default.
• W2084872777 creator A5040681944 @default.
• W2084872777 creator A5042013176 @default.
• W2084872777 date "2001-03-01" @default.
• W2084872777 modified "2023-09-30" @default.
• W2084872777 title "The Arc Two-component Signal Transduction System Inhibitsin Vitro Escherichia coli Chromosomal Initiation" @default.
• W2084872777 cites W1484866107 @default.
• W2084872777 cites W1491301761 @default.
• W2084872777 cites W1495304854 @default.
• W2084872777 cites W1513191274 @default.
• W2084872777 cites W1519460907 @default.
• W2084872777 cites W1527739172 @default.
• W2084872777 cites W1530522271 @default.
• W2084872777 cites W1588043867 @default.
• W2084872777 cites W1608860139 @default.
• W2084872777 cites W1651805553 @default.
• W2084872777 cites W1698914072 @default.
• W2084872777 cites W1741228629 @default.
• W2084872777 cites W189144760 @default.
• W2084872777 cites W1912451872 @default.
• W2084872777 cites W1962352625 @default.
• W2084872777 cites W1969206674 @default.
• W2084872777 cites W1975665069 @default.
• W2084872777 cites W1977926334 @default.
• W2084872777 cites W1999051466 @default.
• W2084872777 cites W2002108207 @default.
• W2084872777 cites W2007377116 @default.
• W2084872777 cites W2019257874 @default.
• W2084872777 cites W2020551760 @default.
• W2084872777 cites W2024235703 @default.
• W2084872777 cites W2051830305 @default.
• W2084872777 cites W2060703599 @default.
• W2084872777 cites W2061228928 @default.
• W2084872777 cites W2063045156 @default.
• W2084872777 cites W2063443838 @default.
• W2084872777 cites W2069176646 @default.
• W2084872777 cites W2078327540 @default.
• W2084872777 cites W2081945314 @default.
• W2084872777 cites W2084874442 @default.
• W2084872777 cites W2088891507 @default.
• W2084872777 cites W2091303980 @default.
• W2084872777 cites W2106833221 @default.
• W2084872777 cites W2140809061 @default.
• W2084872777 cites W2142120140 @default.
• W2084872777 cites W2147568984 @default.
• W2084872777 cites W2152393685 @default.
• W2084872777 cites W2154678954 @default.
• W2084872777 cites W2160084442 @default.
• W2084872777 cites W2169828861 @default.
• W2084872777 cites W80165809 @default.
• W2084872777 doi "https://doi.org/10.1074/jbc.m008629200" @default.
• W2084872777 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11133990" @default.
• W2084872777 hasPublicationYear "2001" @default.
• W2084872777 type Work @default.
• W2084872777 sameAs 2084872777 @default.
• W2084872777 citedByCount "38" @default.
• W2084872777 countsByYear W20848727772012 @default.
• W2084872777 countsByYear W20848727772013 @default.
• W2084872777 countsByYear W20848727772014 @default.
• W2084872777 countsByYear W20848727772015 @default.
• W2084872777 countsByYear W20848727772016 @default.
• W2084872777 countsByYear W20848727772017 @default.
• W2084872777 countsByYear W20848727772018 @default.
• W2084872777 countsByYear W20848727772019 @default.
• W2084872777 countsByYear W20848727772020 @default.
• W2084872777 countsByYear W20848727772021 @default.
• W2084872777 crossrefType "journal-article" @default.
• W2084872777 hasAuthorship W2084872777A5018058696 @default.
• W2084872777 hasAuthorship W2084872777A5024856047 @default.
• W2084872777 hasAuthorship W2084872777A5040681944 @default.
• W2084872777 hasAuthorship W2084872777A5042013176 @default.
• W2084872777 hasBestOaLocation W20848727771 @default.
• W2084872777 hasConcept C104317684 @default.
• W2084872777 hasConcept C121332964 @default.
• W2084872777 hasConcept C15152581 @default.
• W2084872777 hasConcept C153911025 @default.
• W2084872777 hasConcept C168167062 @default.
• W2084872777 hasConcept C185592680 @default.
• W2084872777 hasConcept C202751555 @default.
• W2084872777 hasConcept C2524010 @default.
• W2084872777 hasConcept C33923547 @default.
• W2084872777 hasConcept C54355233 @default.
• W2084872777 hasConcept C547475151 @default.
• W2084872777 hasConcept C55493867 @default.
• W2084872777 hasConcept C62478195 @default.
• W2084872777 hasConcept C83415579 @default.
• W2084872777 hasConcept C86803240 @default.
• W2084872777 hasConcept C95444343 @default.
• W2084872777 hasConcept C97355855 @default.
• W2084872777 hasConceptScore W2084872777C104317684 @default.
• W2084872777 hasConceptScore W2084872777C121332964 @default.
• W2084872777 hasConceptScore W2084872777C15152581 @default.
• W2084872777 hasConceptScore W2084872777C153911025 @default.
• W2084872777 hasConceptScore W2084872777C168167062 @default.
• W2084872777 hasConceptScore W2084872777C185592680 @default.

• next