Acetate Metabolism in a pta Mutant ofEscherichia coli W3110: Importance of Maintaining Acetyl Coenzyme A Flux for Growth and Survival

ABSTRACT In order to study the physiological role of acetate metabolism inEscherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate,d-lactate, and l-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by thepta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The ptamutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-β-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

[1]  J. Bailey,et al.  Effect of alteration of the acetic acid synthesis pathway on the fermentation pattern of escherichia coli , 1991, Biotechnology and bioengineering.

[2]  Chankyu Park,et al.  Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR , 1995, Journal of bacteriology.

[3]  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.

[4]  K. Izui,et al.  Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. II. Kinetic studies with a reaction system containing physiological concentrations of ligands. , 1981, Journal of biochemistry.

[5]  D. Clark,et al.  Anaerobic fermentation balance of Escherichia coli as observed by in vivo nuclear magnetic resonance spectroscopy , 1989, Journal of bacteriology.

[6]  T. Ferenci,et al.  Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress , 1993, Journal of bacteriology.

[7]  R. Rosenzweig,et al.  Microbial evolution in a simple unstructured environment: genetic differentiation in Escherichia coli. , 1994, Genetics.

[8]  B D Davis,et al.  Regulation of alpha-ketoglutarate dehydrogenase formation in Escherichia coli. , 1965, The Journal of biological chemistry.

[9]  H. Kaback,et al.  Mechanisms of active transport in isolated bacterial membrane vesicles. 18. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone. , 1974, Archives of biochemistry and biophysics.

[10]  D. Clark,et al.  Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase , 1989, Journal of bacteriology.

[11]  A. Ninfa,et al.  Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli , 1992, Journal of bacteriology.

[12]  J. Liao,et al.  Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle , 1992, Journal of bacteriology.

[13]  A. Ninfa,et al.  Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli , 1998, Molecular microbiology.

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

[15]  A. Steinbüchel,et al.  Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter , 1991, Journal of bacteriology.

[16]  T. Ferenci,et al.  Adaptation to life at micromolar nutrient levels: the regulation of Escherichia coli glucose transport by endoinduction and cAMP. , 1996, FEMS microbiology reviews.

[17]  J. Bailey,et al.  Comparative studies of Escherichia coli strains using different glucose uptake systems: Metabolism and energetics. , 1997, Biotechnology and bioengineering.

[18]  F. Neidhardt,et al.  Physiology of the bacterial cell : a molecular approach , 1990 .

[19]  D. Clark,et al.  The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. , 1997, Microbiology.

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

[21]  H. Holms,et al.  Flux analysis and control of the central metabolic pathways in Escherichia coli. , 1996, FEMS microbiology reviews.

[22]  A. Wolfe,et al.  Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli , 1995, Journal of bacteriology.

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

[24]  H. Kaback,et al.  Mechanisms of active transport in isolated bacterial membrane vesicles. XV. Purification and properties of the membrane-bound D-lactate dehydrogenase from Escherichia coli. , 1973, The Journal of biological chemistry.

[25]  Armin Fiechter,et al.  Mass culture of Escherichia coli: Medium development for low and high density cultivation of Escherichia coli B/r in minimal and complex media , 1985 .

[26]  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.

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

[28]  K. Izui,et al.  Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. Estimation of the activities in the cells grown on various compounds. , 1980, Journal of biochemistry.

[29]  David P. Clark,et al.  The IdhA Gene Encoding the Fermentative Lactate Dehydrogenase of Escherichia Coli , 1997 .

[30]  B. Prüß,et al.  Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli , 1998, Archives of Microbiology.

[31]  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.

[32]  W. Bentley,et al.  An integrated metabolic modeling approach to describe the energy efficiency of Escherichia coli fermentations under oxygen‐limited conditions: Cellular energetics, carbon flux, and acetate production , 1993, Biotechnology and Bioengineering.

[33]  J. Stock,et al.  Acetyl phosphate and the activation of two-component response regulators. , 1994, The Journal of biological chemistry.

[34]  S. Park,et al.  Aerobic regulation of the sucABCD genes of Escherichia coli, which encode alpha-ketoglutarate dehydrogenase and succinyl coenzyme A synthetase: roles of ArcA, Fnr, and the upstream sdhCDAB promoter , 1997, Journal of bacteriology.

[35]  J. Guest,et al.  Structure, Expression, and Protein Engineering of the Pyruvate Dehydrogenase Complex of Escherichia coli a , 1989, Annals of the New York Academy of Sciences.

[36]  J. Liao,et al.  Pathway analysis, engineering, and physiological considerations for redirecting central metabolism. , 1996, Biotechnology and bioengineering.

[37]  F. Neidhardt,et al.  2-Oxoacid dehydrogenase complexes of Escherichia coli: cellular amounts and patterns of synthesis , 1983, Journal of bacteriology.

[38]  T. Ferenci,et al.  The importance of the binding-protein-dependent Mgl system to the transport of glucose in Escherichia coli growing on low sugar concentrations. , 1993, Research in microbiology.

[39]  K. Andersen,et al.  Are growth rates of Escherichia coli in batch cultures limited by respiration? , 1980, Journal of bacteriology.

[40]  S. Ichihara,et al.  Construction of Pta-Ack pathway deletion mutants of Escherichia coli and characteristic growth profiles of the mutants in a rich medium. , 1994, Bioscience, biotechnology, and biochemistry.

[41]  J. Cronan,et al.  Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS(katF) gene , 1994, Molecular microbiology.