Intracellular concentration of cysteine in Escherichia coli and its relation to repression of the sulphate-activating enzymes.
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ABSTRACT:
SUBMITTER: Wheldrake JF
PROVIDER: S-EPMC1198366 | biostudies-other | 1967 Nov
REPOSITORIES: biostudies-other
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Project description:Escherichia coli does not routinely import manganese, but it will do so when iron is unavailable, so that manganese can substitute for iron as an enzyme cofactor. When intracellular manganese levels are low, the cell induces the MntH manganese importer plus MntS, a small protein of unknown function; when manganese levels are high, the cell induces the MntP manganese exporter and reduces expression of MntH and MntS. The role of MntS has not been clear. Previous work showed that forced MntS synthesis under manganese-rich conditions caused bacteriostasis. Here we find that when manganese is scarce, MntS helps manganese to activate a variety of enzymes. Its overproduction under manganese-rich conditions caused manganese to accumulate to very high levels inside the cell; simultaneously, iron levels dropped precipitously, apparently because manganese-bound Fur blocked the production of iron importers. Under these conditions, heme synthesis stopped, ultimately depleting cytochrome oxidase activity and causing the failure of aerobic metabolism. Protoporphyrin IX accumulated, indicating that the combination of excess manganese and iron deficiency had stalled ferrochelatase. The same chain of events occurred when mutants lacking MntP, the manganese exporter, were exposed to manganese. Genetic analysis suggested the possibility that MntS exerts this effect by inhibiting MntP. We discuss a model wherein during transitions between low- and high-manganese environments E. coli uses MntP to compensate for MntH overactivity, and MntS to compensate for MntP overactivity.
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Project description:In rat hepatocytes subjected to a fructose load, ATP content decreased from 3.8 to 2.6 micromol/g of cells. Under these conditions, the intracellular free Mg2+ ion concentration,as measured with mag-fura 2, increased from 0.25 to 0.43 micromol/g of cells and 0.35 micromol of Mg2+ ions were released per g of cells in the extracellular medium. Therefore the increase in the intracellular free Mg2+ ion concentration was less than expected from the decrease in ATP, indicating that approx. 80% of the Mg2+ ions released from MgATP2- were buffered inside the cells. When this buffer capacity was challenged with an extra Mg2+ ion load by blocking the fructose-induced Mg2+ efflux, again approx. 80% of the extra Mg2+ ion load was buffered. The remaining 20% appearing as free Mg2+ions in fructose-treated hepatocytes could act as second messenger for enzymes having a Km for Mg2+ in the millimolar range. Fructose activated glycogen synthase and glycogen phosphorylase, although both the time course and the dose-dependence of activation were different. This was reflected in a stimulation of glycogen synthesis with concentrations of fructose below 5 mM. Indeed, activation of glycogen synthase reached a maximum at 30 min of incubation and was observed with small (5 mM or less) concentrations of fructose, whereas the activation of glycogen phosphorylase was almost immediate (within 5 min) and maximal with large doses of fructose. The fructose-induced activation of glycogen phosphorylase, but not that of glycogen synthase, could be related to an increase in free Mg2+ ion concentration.
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Project description: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-beta-D-thiogalactopyranoside-induced overexpression of EIIA(Glc) 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. Two-dimensional 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.
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Project description:1. The intermediary metabolism of two strains of Escherichia coli has been examined. One strain (Q22) exhibits acute transient repression of beta-galactosidase synthesis when glucose is supplied to cells growing on glycerol; the other strain (W3110) does not. The two strains do not differ genetically in their lac operons. 2. Strain Q22 uses about twice as much glucose as strain W3110 per unit of cell mass produced. 3. Pentose phosphate-cycle activity in the presence of glucose is much stronger in strain Q22 than in strain W3110. 4. In strain Q22 the pool sizes of glucose 6-phosphate, 6-phosphogluconate, fructose 1,6-diphosphate and NADPH increase when glucose is added to cells growing on glycerol, and beta-galactosidase synthesis is severely inhibited. After about 1hr. the synthesis of beta-galactosidase is partly resumed, and the pool sizes of the four compounds fall. ATP, NADH and several other phosphorylated compounds show no concentration changes. 5. These concentration changes do not occur in strain W3110, in which beta-galactosidase synthesis is only rather weakly repressed by glucose. 6. It is suggested that repression of enzyme synthesis by glucose requires the rapid operation of the pentose phosphate cycle, and is mediated by one of the four substances whose concentration rises and later falls in strain Q22. A definite choice of effector from among these four possibilities cannot at present be made.
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