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Journal of Bacteriology, June 2001, p. 3336-3344, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3336-3344.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

C. Kay Holtman,1 Aaron C. Pawlyk,1 Norman D. Meadow,2 and Donald W. Pettigrew1,*

Department of Biochemistry and Biophysics, Program in Microbial Genetics and Genomics, and Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128,1 and Department of Biology and McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 212182

Received 21 September 2000/Accepted 8 March 2001

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 glpK alleles 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. The E. 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 glpR alleles 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.

* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128. Phone: (979) 845-9621. Fax: (979) 845-9274. E-mail: dpettigrew{at}tamu.edu.

Journal of Bacteriology, June 2001, p. 3336-3344, Vol. 183, No. 11
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.11.3336-3344.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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