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

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.

[1]  R C Stewart,et al.  The short form of CheA couples chemoreception to CheA phosphorylation , 1994, Journal of bacteriology.

[2]  G. L. Hazelbauer,et al.  Mutations specifically affecting ligand interaction of the Trg chemosensory transducer , 1986, Journal of bacteriology.

[3]  J. Ferry,et al.  Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase. , 1989, The Journal of biological chemistry.

[4]  J. Ferry,et al.  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. , 1987, The Journal of biological chemistry.

[5]  B. Wanner Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? , 1992, Journal of bacteriology.

[6]  D. Hanahan Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[7]  R. Thauer,et al.  Methane formation from acetyl phosphate in cell extracts of Methanosarcina barkeri Dependence of the reaction on coenzyme A , 1988 .

[8]  H. Berg,et al.  Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase , 1993, Journal of bacteriology.

[9]  J. Messing [2] New M13 vectors for cloning , 1983 .

[10]  T. Henkin,et al.  Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA , 1994, Journal of bacteriology.

[11]  M. Welch,et al.  Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch. , 1992, Biochemistry.

[12]  W. Holms,et al.  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. , 1986, Current topics in cellular regulation.

[13]  G W Luli,et al.  Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations , 1990, Applied and environmental microbiology.

[14]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[15]  F. Lipmann,et al.  ACETYLATION OF SULFANILAMIDE BY LIVER HOMOGENATES AND EXTRACTS , 1945 .

[16]  H. Kornberg,et al.  The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. , 1977, Journal of general microbiology.

[17]  T. Chou,et al.  Separation of acetyl transfer enzymes in pigeon liver extract. , 1952, The Journal of biological chemistry.

[18]  P. Berg Acyl adenylates; an enzymatic mechanism of acetate activation. , 1956, The Journal of biological chemistry.

[19]  E. Southern Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[20]  S. Garges A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. By Jeffrey H. Miller. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1992. , 1993 .

[21]  T. Henkin,et al.  Regulation of the Bacillus subtilis acetate kinase gene by CcpA , 1993, Journal of bacteriology.

[22]  H. Fritz,et al.  Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli , 1984, Cell.

[23]  J. Ferry,et al.  Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis. , 1988, The Journal of biological chemistry.

[24]  L. Enquist,et al.  Experiments With Gene Fusions , 1984 .

[25]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[26]  L. G. Davis,et al.  Basic methods in molecular biology , 1986 .

[27]  W. Holms,et al.  Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures. , 1989, Journal of general microbiology.

[28]  A. Wolfe,et al.  Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids , 1994, Journal of bacteriology.

[29]  T. Nyström 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 , 1994, Molecular microbiology.

[30]  G. Wu,et al.  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. , 1992, Journal of general microbiology.

[31]  A. Ninfa,et al.  Is acetyl phosphate a global signal in Escherichia coli? , 1993, Journal of bacteriology.

[32]  S. Roseman,et al.  Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. , 1986, The Journal of biological chemistry.

[33]  A. Zehnder,et al.  Carbon monoxide dehydrogenase and acetate thiokinase in Methanothrix soehngenii , 1984 .

[34]  A. Guranowski,et al.  Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase , 1994, Journal of bacteriology.

[35]  A. Wolfe,et al.  Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli , 1994, Molecular microbiology.

[36]  K. Isono,et al.  The physical map of the whole E. coli chromosome: Application of a new strategy for rapid analysis and sorting of a large genomic library , 1987, Cell.

[37]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[38]  B. Eikmanns,et al.  Methanogenesis from Acetate by Methanosarcina barkeri: Catalysis of Acetate Formation from Methyl Iodide, CO2 , and H2 by the Enzyme System Involved , 1987 .

[39]  T. Henkin,et al.  Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis , 1993, Molecular microbiology.

[40]  W R Strohl,et al.  Acetate metabolism by Escherichia coli in high-cell-density fermentation , 1994, Applied and environmental microbiology.

[41]  R. Simons,et al.  Improved single and multicopy lac-based cloning vectors for protein and operon fusions. , 1987, Gene.

[42]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[43]  J. Hoch,et al.  Phosphotransacetylase from Bacillus subtilis: purification and physiological studies. , 1973, Biochimica et biophysica acta.

[44]  J. Messing New M13 vectors for cloning. , 1983, Methods in enzymology.

[45]  M. Grunberg‐Manago,et al.  Enzymatic phosphorylation of acetate. , 1954, The Journal of biological chemistry.

[46]  H. G. Trüper Tricarboxylic acid cycle and related enzymes in Hydrogenomonas strain H16G+ grown on various carbon sources. , 1965, Biochimica et biophysica acta.

[47]  F. Blattner,et al.  Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes. , 1993, Nucleic acids research.

[48]  H. Berg,et al.  Acetyladenylate plays a role in controlling the direction of flagellar rotation. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[49]  E. Chen,et al.  Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. , 1985, DNA.

[50]  E. Freese,et al.  Control of metabolite secretion in Bacillus subtilis. , 1973, Journal of general microbiology.

[51]  A. Hoffmann,et al.  Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. , 1991, Nucleic acids research.

[52]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Jeffrey H. Miller Experiments in molecular genetics , 1972 .

[54]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.