Holtman C K, Pawlyk A C, Meadow N D, Pettigrew D W
Department of Biochemistry and Biophysics, Program in Microbial Genetics and Genomics, Texas A&M University, College Station, TX 77843-2128, USA.
J Bacteriol. 2001 Jun;183(11):3336-44. doi: 10.1128/JB.183.11.3336-3344.2001.
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, IIA(Glc) (formerly known as III(glc)), 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 IIA(Glc) 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.
葡萄糖磷酸转移酶系统的葡萄糖特异性磷酸载体IIA(Glc)（以前称为III(glc)）以及1,6-二磷酸果糖对大肠杆菌甘油激酶变构调节在体内的作用。这些变构效应物在葡萄糖对甘油利用和glpK基因表达的控制中所起的作用已被推测。基于同源重组的遗传学方法用于将具有已知特定突变的glpK等位基因置于三种不同遗传背景下glpK基因的染色体环境中。这些等位基因编码具有正常催化特性和变构调节特性特定改变的甘油激酶，这是通过对纯化酶的体外表征确定的。具有这些等位基因的大肠杆菌菌株表现出基于体外表征所预期的甘油激酶调节表型。使用具有不同glpR等位基因的菌株来评估甘油激酶变构调节与glpK基因表达的葡萄糖控制中的特异性阻遏之间的关系。这些研究结果表明，葡萄糖对甘油利用和甘油激酶表达的控制不受IIA(Glc)对甘油激酶抑制作用丧失的影响。相反，1,6-二磷酸果糖对甘油激酶的抑制是主要的变构控制机制，在其不存在时葡萄糖无法控制甘油的利用。葡萄糖对甘油利用的控制不需要特异性阻遏，并且葡萄糖控制的各种机制（分解代谢物阻遏、特异性阻遏和诱导物排除）在甘油利用中的相对作用与乳糖利用不同。
