• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. May 1995; 177(10): 2878–2886.
PMCID: PMC176962

Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli.

Abstract

Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993). We constructed a mutant allele, delta acs::Km, with the central 0.72-kb BclI-BclI portion of acs deleted, and recombined it into the chromosome. Whereas wild-type cells grew well on acetate across a wide range of concentrations (2.5 to 50 mM), those deleted for acs grew poorly on low concentrations (< or = 10 mM), those deleted for ackA and pta (which encode Ack and Pta, respectively) grew poorly on high concentrations (> or = 25 mM), and those deleted for acs, ackA, and pta did not grow on acetate at any concentration tested. Expression of acs from a multicopy plasmid restored growth to cells deleted for all three genes. Relative to wild-type cells, those deleted for acs did not activate acetate as well, those deleted for ackA and pta displayed even less activity, and those deleted for all three genes did not activate acetate at any concentration tested. Induction of acs resulted in expression of a 72-kDa protein, as predicted by the reported sequence. This protein immunoreacted with antiserum raised against purified Acs isolated from an unrelated species, Methanothrix soehngenii. The purified E. coli Acs then was used to raise anti-E. coli Acs antiserum, which immunoreacted with a 72-kDa protein expressed by wild-type cells but not by those deleted for acs. When purified in the presence, but not in the absence, of coenzyme A, the E. coli enzyme activated acetate across a wide range of concentrations in a coenzyme A-dependent manner. On the basis of these and other observations, we conclude that this open reading frame encodes the acetate-activating enzyme, Acs.

Full Text

The Full Text of this article is available as a PDF (698K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aceti DJ, Ferry JG. Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis. J Biol Chem. 1988 Oct 25;263(30):15444–15448. [PubMed]
  • Barak R, Welch M, Yanovsky A, Oosawa K, Eisenbach M. Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch. Biochemistry. 1992 Oct 20;31(41):10099–10107. [PubMed]
  • BERG P. Acyl adenylates; an enzymatic mechanism of acetate activation. J Biol Chem. 1956 Oct;222(2):991–1013. [PubMed]
  • Bergmeyer HU, Moellering H. Enzymatische Bestimmung von Acetat. Biochem Z. 1966 Mar 28;344(2):167–189. [PubMed]
  • Blattner FR, Burland V, Plunkett G, 3rd, Sofia HJ, Daniels DL. Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes. Nucleic Acids Res. 1993 Nov 25;21(23):5408–5417. [PMC free article] [PubMed]
  • Brown TD, Jones-Mortimer MC, Kornberg HL. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol. 1977 Oct;102(2):327–336. [PubMed]
  • Chen EY, Seeburg PH. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. [PubMed]
  • CHOU TC, LIPMANN F. Separation of acetyl transfer enzymes in pigeon liver extract. J Biol Chem. 1952 May;196(1):89–103. [PubMed]
  • Dailey FE, Berg HC. Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase. J Bacteriol. 1993 May;175(10):3236–3239. [PMC free article] [PubMed]
  • el-Mansi EM, Holms WH. Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures. J Gen Microbiol. 1989 Nov;135(11):2875–2883. [PubMed]
  • Fox DK, Roseman S. Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. J Biol Chem. 1986 Oct 15;261(29):13487–13497. [PubMed]
  • Grundy FJ, Turinsky AJ, Henkin TM. Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA. J Bacteriol. 1994 Aug;176(15):4527–4533. [PMC free article] [PubMed]
  • Grundy FJ, Waters DA, Allen SH, Henkin TM. Regulation of the Bacillus subtilis acetate kinase gene by CcpA. J Bacteriol. 1993 Nov;175(22):7348–7355. [PMC free article] [PubMed]
  • Grundy FJ, Waters DA, Takova TY, Henkin TM. Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis. Mol Microbiol. 1993 Oct;10(2):259–271. [PubMed]
  • Guranowski A, Günther Sillero MA, Sillero A. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase. J Bacteriol. 1994 May;176(10):2986–2990. [PMC free article] [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Hoffmann A, Roeder RG. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. [PMC free article] [PubMed]
  • Holms WH. The central metabolic pathways of Escherichia coli: relationship between flux and control at a branch point, efficiency of conversion to biomass, and excretion of acetate. Curr Top Cell Regul. 1986;28:69–105. [PubMed]
  • Kleman GL, Strohl WR. Acetate metabolism by Escherichia coli in high-cell-density fermentation. Appl Environ Microbiol. 1994 Nov;60(11):3952–3958. [PMC free article] [PubMed]
  • Kohara Y, Akiyama K, Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. [PubMed]
  • Kramer B, Kramer W, Fritz HJ. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Luli GW, Strohl WR. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol. 1990 Apr;56(4):1004–1011. [PMC free article] [PubMed]
  • Lundie LL, Jr, Ferry JG. Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase. J Biol Chem. 1989 Nov 5;264(31):18392–18396. [PubMed]
  • McCleary WR, Stock JB, Ninfa AJ. Is acetyl phosphate a global signal in Escherichia coli? J Bacteriol. 1993 May;175(10):2793–2798. [PMC free article] [PubMed]
  • Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. [PubMed]
  • Nyström T. The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival. Mol Microbiol. 1994 Jun;12(5):833–843. [PubMed]
  • Park C, Hazelbauer GL. Mutations specifically affecting ligand interaction of the Trg chemosensory transducer. J Bacteriol. 1986 Jul;167(1):101–109. [PMC free article] [PubMed]
  • Prüss BM, Nelms JM, Park C, Wolfe AJ. Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids. J Bacteriol. 1994 Apr;176(8):2143–2150. [PMC free article] [PubMed]
  • Prüss BM, Wolfe AJ. Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli. Mol Microbiol. 1994 Jun;12(6):973–984. [PubMed]
  • Rado TA, Hoch JA. Phosphotransacetylase from Bacillus subtilis: purification and physiological studies. Biochim Biophys Acta. 1973 Sep 15;321(1):114–125. [PubMed]
  • ROSE IA, GRUNBERG-MANAGO M, KOREY SR, OCHOA S. Enzymatic phosphorylation of acetate. J Biol Chem. 1954 Dec;211(2):737–756. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Simons RW, Houman F, Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. [PubMed]
  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Speck EL, Freese E. Control of metabolite secretion in Bacillus subtilis. J Gen Microbiol. 1973 Oct;78(2):261–275. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Terlesky KC, Barber MJ, Aceti DJ, Ferry JG. EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate. J Biol Chem. 1987 Nov 15;262(32):15392–15395. [PubMed]
  • Trüper HG. Tricarboxylic acid cycle and related enzymes in Hydrogenomonas strain H16G+ grown on various carbon sources. Biochim Biophys Acta. 1965 Dec 16;111(2):565–568. [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Wanner BL. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J Bacteriol. 1992 Apr;174(7):2053–2058. [PMC free article] [PubMed]
  • Wolfe AJ, Conley MP, Berg HC. Acetyladenylate plays a role in controlling the direction of flagellar rotation. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6711–6715. [PMC free article] [PubMed]
  • Wolfe AJ, McNamara BP, Stewart RC. The short form of CheA couples chemoreception to CheA phosphorylation. J Bacteriol. 1994 Aug;176(15):4483–4491. [PMC free article] [PubMed]
  • Wu G, Williams HD, Zamanian M, Gibson F, Poole RK. Isolation and characterization of Escherichia coli mutants affected in aerobic respiration: the cloning and nucleotide sequence of ubiG. Identification of an S-adenosylmethionine-binding motif in protein, RNA, and small-molecule methyltransferases. J Gen Microbiol. 1992 Oct;138(10):2101–2112. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...