Glycerol-3-Phosphate-Induced Catabolite Repression inEscherichia coli

Abstract

The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA Glc , as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA Glc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA Glc . Isopropyl-␤-D-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA Glc . A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA Glc is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Twodimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.Catabolite repression refers to the reduction in transcription of sensitive operons that is caused by certain carbon sources in the medium, most prominently by glucose (glucose effect). In Escherichia coli, phosphoenolpyruvate-dependent phosphotransferase system (PTS)-mediated uptake of glucose is crucial for this effect. The model largely accepted for E. coli focuses on the level of cyclic AMP (cAMP) synthesized by the membranebound adenylate cyclase (29,30,38). EIIA Glc , an intermediate in the phosphorylation cascade of the PTS for glucose, in its phosphorylated form is thought to stimulate adenylate cyclase. The basal level of adenylate cyclase activity would be present in the absence of EIIA Glc or in the presence of unphosphorylated EIIA Glc . As a consequence, CAP, the catabolite activator protein (or cAMP receptor protein [CRP]) that is needed for the transcription of sensitive operons (22) is linked in its activity to the PTS (31). The glucose PTS is also responsible for inducer exclusion, i.e., inhibition of the different transport systems by unphosphorylated EIIA Glc (31). Even though participation of the PTS in catabolite repression and inducer exclusion in E. coli has been documented very well, the effects of non-PTS sugars are less clear. Thus, gluc...

Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

“…On the other hand, the glycerol uptake rate was enhanced in the glpK mutant most probably because of reduced inhibitory effect of fructose 1,6-bisphosphate on GlpK, as shown previously for this mutant 10 . This results in increased transfer of phosphate from phosphorylated EIIA Glc to produce glycerol-3-phosphate 28 . The resulting decrease in phosphorylated EIIA Glc will in turn reduce adenylate cyclase activity and cAMP production.…”

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale

“…It has been previously reported that a similarly truncated mutant of adenylate cyclase had lower activity and was not repressed by glycerol-3-phosphate 22 . We hypothesize that wild-type strain MG1655 grows slowly on glycerol because some critical genes are misexpressed as a result of inappropriate levels of catabolite repression and that the attenuating mutations in cyaA and pdxK/crr relieve this misexpression.…”

Computer-aided rational design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli

“…coli prefers glucose rather than other sugars for its growth and glucose represses the catabolism of other sugars (Hogema et al, 1997). The glucose PTS is related to catabolite repression, chemotaxis regulation, glycogen phosphorylase, and multiple regulatory interactions (Lux et al, 1995;Postma et al, 1996;Seok et al, 1997;Saier, 1998;Eppler et al, 2002). Adenylate cyclase is activated when glucose in the medium is depleted.…”