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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...

SummarymalT encodes the central activator of the maltose system in Escherichia coli, a gene that is typically under positive control of the cAMP/CAP catabolite repression system. When cells were grown in tryptone broth, the addition of glycerol reduced malT expression two-to threefold. Phosphorylation of glycerol to glycerol-3-phosphate (G3P) was necessary for this repression, but further metabolism to dihydroxyacetone phosphate was not. Mutants lacking adenylate cyclase and harbouring a crp* mutation (synthesizing a cAMP receptor protein that is independent of cAMP) no longer repressed a transcriptional malT±lacZ fusion but still repressed a translational malT±lacZ fusion. Similar results were obtained with a mutant lacking enzyme IIA Glc . For the translational fusion (in a cya crp* genetic background) to be repressed by glycerol, a drop to pH 5 of the growth medium was necessary. Thus, while transcriptional repression by glycerol requires enzyme IIA Glc , cAMP and CAP, pH-mediated translational repression is cAMP independent. Other sugars that are not transported by the phosphotransferase system, most notably D-xylose, showed the same effect as glycerol.

2-O-␣-Mannosyl-D-glycerate (MGs)has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strain MC4100. Uptake is mediated by the P-enolpyruvatedependent phosphotransferase system with the MGinducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent K m of MG uptake in induced cells was 10 M, and the V max was 0.65 nmol/min/ 10 9 cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with ␣-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system.