Reverse Genetics of Escherichia coliGlycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo

ABSTRACT Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (formerly known as IIIglc), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpKalleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. TheE. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpRalleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIAGlc inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.

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

[2]  D. W. Pettigrew,et al.  Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily. , 1998, Archives of biochemistry and biophysics.

[3]  S. Roseman,et al.  The bacterial phosphoenolpyruvate: glycose phosphotransferase system. , 1990, Annual review of biochemistry.

[4]  S. Silver,et al.  Phosphate in Microorganisms: Cellular and Molecular Biology , 1994 .

[5]  T. Larson,et al.  Interaction at a distance between multiple operators controls the adjacent, divergently transcribed glpTQ-glpACB operons of Escherichia coli K-12. , 1992, The Journal of biological chemistry.

[6]  T. Larson,et al.  Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants , 1994, Journal of bacteriology.

[7]  E. Lin,et al.  Glycerol-Specific Revertants of a Phosphoenolpyruvate Phosphotransferase Mutant: Suppression by the Desensitization of Glycerol Kinase to Feedback Inhibition , 1971, Journal of bacteriology.

[8]  M. Saier.  Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. , 1989, Microbiological reviews.

[9]  D. W. Pettigrew,et al.  Unexpected Presence of Defective glpRAlleles in Various Strains of Escherichia coli , 2001, Journal of bacteriology.

[10]  Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system , 1978, Journal of bacteriology.

[11]  M. Saier,et al.  Allosteric regulation of glycerol kinase by enzyme IIIglc of the phosphotransferase system in Escherichia coli and Salmonella typhimurium , 1985, Journal of bacteriology.

[12]  P. Postma,et al.  Autoregulation of lactose uptake through the LacYpermease by enzyme IIAGlc of the PTS in Escherichia coli K‐12 , 1999, Molecular microbiology.

[13]  H. Kornberg,et al.  Genetic control of inducer exclusion by Escherichia coli , 1974, FEBS letters.

[14]  J. Philo.  An improved function for fitting sedimentation velocity data for low-molecular-weight solutes. , 1997, Biophysical journal.

[15]  G. Stewart,et al.  pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. , 1986, Plasmid.

[16]  E. Lin,et al.  Glycerol Kinase, the Pacemaker for the Dissimilation of Glycerol in Escherichia coli , 1970, Journal of bacteriology.

[17]  T. Holzman,et al.  Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. , 1984, Biochemistry.

[18]  G R Jacobson,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. , 1993, Microbiological reviews.

[19]  J. R. Fresco,et al.  Feedback Inhibition of Glycerol Kinase, a Catabolic Enzyme in Escherichia coli , 1966, Science.

[20]  H. Takahashi,et al.  Inducer exclusion in Escherichia coli by non‐PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc , 1998, Molecular microbiology.

[21]  D. W. Pettigrew,et al.  Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme. , 1988, The Journal of biological chemistry.

[22]  T. Larson,et al.  Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K‐12 , 1997, Molecular microbiology.

[23]  T. Doering,et al.  Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes. , 1987, The Journal of biological chemistry.

[24]  E. C. Lin,et al.  Glycerol dissimilation and its regulation in bacteria. , 1976, Annual review of microbiology.

[25]  S J Remington,et al.  Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase , 1993, Science.

[26]  S. Roseman,et al.  Signal transduction by the bacterial phosphotransferase system. Diauxie and the crr gene (J. Monod revisited). , 1990, The Journal of biological chemistry.

[27]  P. Postma,et al.  Interaction between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and glycerol kinase of Salmonella typhimurium , 1984, Journal of bacteriology.

[28]  S. Remington,et al.  Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc. , 1994, Biochemistry.

[29]  T. Larson,et al.  Multiple promoters are responsible for transcription of the glpEGR operon of Escherichia coli K-12. , 1998, Biochimica et biophysica acta.

[30]  T. Larson,et al.  Purification and Characterization ofglpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coli , 2000, Journal of bacteriology.

[31]  N. Wittekindt,et al.  Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12. , 1992, The Journal of biological chemistry.

[32]  A. Grossman,et al.  A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. , 1989, Microbiological reviews.

[33]  S. Roseman,et al.  A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo , 1996, Journal of bacteriology.

[34]  T. Inada,et al.  Mechanism responsible for glucose–lactose diauxie in Escherichia coli: challenge to the cAMP model , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[35]  S J Remington,et al.  Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation. , 1998, Structure.

[36]  S. Roseman,et al.  Cation-promoted association of Escherichia coli phosphocarrier protein IIAGlc with regulatory target protein glycerol kinase: substitutions of a Zinc(II) ligand and implications for inducer exclusion. , 1998, Biochemistry.

[37]  Saul Roseman,et al.  Signal transduction by the bacterial phosphotransferase system , 1990 .

[38]  P. Postma,et al.  Inducer exclusion by glucose 6‐phosphate in Escherichia coli , 1998, Molecular microbiology.

[39]  S. Remington,et al.  Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate. , 1998, Biochemistry.

[40]  W. Hillen,et al.  Carbon catabolite repression in bacteria. , 1999, Current opinion in microbiology.