Glucose Catabolite Repression in Escherichia coli K12 Mutants Defective in Methyl-alpha-d-Glucoside Transport

Abstract

1. Two spontaneous Escherichia coli K12 mutants resistant to glucose catabolite repression were isolated using minimal agar plates with methyl a-D-glucoside. Mutants grow well on glucose and mannitol.2. Glucose does not inhibit the inducible enzyme synthesis in isolated mutants: mutant cell (in contrast to parent cells) produce high levels of j-galactosidase and L-tryptophanase under the conditions of glucose catabolite repression.3. The isolated mutants are negative in methyl-a-D-glucoside transport; glucose uptake is not severely damaged. But the mutants (named tgl, transport of glucose) retained the ability to phosphorylate methyl a-D-glucoside in vitro at the expense of phosphoenolpyruvate.4. The tgf mutation is cotransduced with purB and pyrC markers, i.e. locates near 24 min of the E. coli chromosome map.5. It is thought that E. coli cells possess two glucose transport systems. The first one is represented by the glucose-specific enzyme I1 of the phosphoenolpyruvate-dependent phosphotransferase system. The second glucose transport system (coded for tgf gene) functions as permease and possesses high affinity to methyl a-D-ghcoside. The integrity of glucose permease determine the sensitivity of the cell to glucose catabolite repression.The transfer of glucose into Escherichia coli K 12 is mediated by the phosphoenofpyruvate : carbohydrate phosphotransferase system [l]. The phosphotransferase reaction coupled with the glucose transport involves the necessary activity of some proteins. Enzyme I catalyses the transfer of the phosphate group from phosphoenolpyruvate to a small histidine containing protein (Hpr) [2,3]. The phosphorylated form of Hpr (phospho -Hpr) is the phosphate donor in the second step of the phosphotransferase reaction at which the membrane-linked enzyme 11, specific for glucose, carries out phosphorylation of the carbohydrate [2,4]. At present there is some evidence that Genetic Nou~w~claturr. tgl = lack of thc glucose permease; gptA = lack of the phospho -Hpr : glucose phosphotransferase activity; um,y = lack of the methyl-a-D-glucoside uptake system. Other symbols are standard, see [9].Enzyme.s. P-Galactosidase (EC 3.2.1.23); L-tryptophanase (EC 4.2.1.20).in E. coli a cytoplasmic factor I11 may participate in the last step of the phosphotransferase reaction [4].Recently it has been reported that there exist two phospho -Hpr : glucose phosphotransferase systems in crude extracts of E. coli cells. The two systems differ in their substrate specificity [ S ] . In the preliminary communication it was mentioned that system A phosphorylates both glucose and methyl a-D-glucoside (nonmetabolizable glucose analogue) ; in the wild type of E. coli 40 % of the glucose phosphotransferase activity is due to system A. The remaining 60 % of the activity is maintained by system €3 which phosphorylates only glucose and has little affinity for methyl a-D-glucoside. Curtis and Epstein consider that system B represents the membrane-linked glucose-specific enzyme I1 but not the glucokinase or some other soluble...

Defective enzyme II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system leading to uncoupling of transport and phosphorylation in Salmonella typhimurium

“…In those cases, phosphorylation and transport of glucose, catalyzed by II-BGlc, are completely absent, even at high external concentrations. The pts-418 mutation is also different from the tgl mutation described by Gershanovitch and coworkers in E. coli (1,5). tgl mutants are still able to phosphorylate methyl a-glucoside, provided it can reach its phosphorylation site inside the cell.…”

“…Enzyme II-B('c could be composed of more than one subunit. ptsG might code for a protein that is involved in methyl a-glucoside phosphorylation, whereas a second gene might code for a protein which is responsible for transport per se (1,5,6). If those two genes are closely linked, introduction of the ptsG: :TnlO mutation would at the same time eliminate also the pts-418 mutation, which is responsible for the transport of glucose in AptsHI galP strains.…”

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation

“…Mutational events could possibly lead to altered enzymes II with dissociated phosphorylation and transport activities. For instance, Bourd et al and Erlagaeva et al (2,4) and Kornberg and Jones-Mortimer (8) have described mutations in E. coli, designated tgl, which impair methyl a-glucoside transport but allow normal phosphorylation of this sugar in sonic extracts or in cells made permeable by toluene treatment. On the other hand, we have described mutants of S. typhimurium in which the enzyme II specific for mannose and glucose may catalyze the facilitated diffusion of galactose in the absence of phosphorylation (15).…”

Fructose Transport in Bacillus subtilis

“…However, the authors did not , obtain more structural information about the role of these proteins in the transport process. Bourd et al [30] reported in Escherichia coli the isolation of a mutant called tgl, defective in the transport of the a-methylglucoside, the mutation leaving the phosphotransferase activity undamaged. This mutant was isolated as resistant to x-methylglucoside-mediated catabolite repression.…”