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B U Feucht

Publications and source records attributed to B U Feucht.

9 recordsLinked to original sources
PubMed1990

Physiological consequences of the complete loss of phosphoryl-transfer proteins HPr and FPr of the phosphoenolpyruvate:sugar phosphotransferase system and analysis of fructose (fru) operon expression in Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in the proteins of the fructose operon [fruB(MH)KA], the fructose repressor (fruR), the energy-coupling enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (ptsH and ptsI), and the proteins of cyclic AMP action (cya and crp) were analyzed for their effects on cellular physiological processes and expression of the fructose operon. The fru operon consists of three structural genes: fruB(MH), which encodes the enzyme IIIFru-modulator-FPr tridomain fusion protein of the PTS; fruK, which encodes fructose-1-phosphate kinase; and fruA, which encodes enzyme IIFru of the PTS. Among the mutants analyzed were Tn10 insertion mutants and lacZ transcriptional fusion mutants. It was found that whereas a fruR::Tn10 insertion mutant, several fruB(MH)::Mu dJ and fruK::Mu dJ fusion mutants, and several ptsHI deletion mutants expressed the fru operon and beta-galactosidase at high constitutive levels, ptsH point mutants and fruA::Mu dJ fusion mutants retained inducibility. Inclusion of the wild-type fru operon in trans did not restore fructose-inducible beta-galactosidase expression in the fru::Mu dJ fusion mutants. cya and crp mutants exhibited reduced basal activities of all fru regulon enzymes, but inducibility was not impaired. Surprisingly, fruB::Mu dJ crp or cya double mutants showed over 10-fold inducibility of the depressed beta-galactosidase activity upon addition of fructose, even though this activity in the fruB::Mu dJ fusion mutants that contained the wild-type cya and crp alleles was only slightly inducible. By contrast, beta-galactosidase activity in a fruK::Mu dJ fusion mutant, which was similarly depressed by introduction of a crp or cya mutation, remained constitutive. Other experiments indicated that sugar uptake via the PTS can utilize either FPr-P or HPr-P as the phosphoryl donor, but that FPr is preferred for fructose uptake whereas HPr is preferred for uptake of the other sugars. Double mutants lacking both proteins were negative for the utilization of all sugar substrates of the PTS, were negative for the utilization of several gluconeogenic carbon sources, exhibited greatly reduced adenylate cyclase activity, and were largely nonmotile. These phenotypic properties are more extreme than those observed for tight ptsH and ptsI mutants, including mutants deleted for these genes. A biochemical explanation for this fact is proposed.

Bacterial Proteins↗
PubMed1987

Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli.

The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.

Alanine↗
PubMed1978

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.

Biological Transport↗
PubMed1976

Regulation of carbohydrate permeases and adenylate cyclase in Escherichia coli. Studies with mutant strains in which enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system is thermolabile.

Carbohydrate uptake and cyclic adenosine 3':5'-monophosphate (cyclic AMP) synthesis were studied employing mutant strains of Escherichia coli in which Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was heat-labile. Partial loss of Enzyme I activity, which resulted from incubation of cells at the nonpermissive temperature, depressed the rate and extent of methyl alpha-glucoside uptake. Temperature inactivation of Enzyme I also rendered cyclic AMP synthesis and the uptake of several carbohydrates (glycerol, maltose, melibiose, and lactose) hypersensitive to inhibition by methyl alpha-glucoside. Protein synthesis did not appear to be required for these effects. The parental strains and "revertant" strains in which Enzyme I was less sensitive to temperature did not exhibit heat-enhanced regulation. Inhibition was abolished by the crr mutation. The results suggest that Enzyme I functions as a catalytic component of the regulatory system. Simple positive selection procedures are described for the isolation of bacterial mutants which are deficient for either Enzyme I or the heat-stable protein of the phosphotransferase system.

Adenylyl Cyclases↗
PubMed1976

Regulation of carbohydrate uptake and adenylate cyclase activity mediated by the enzymes II of the phosphoenolpyruvate: sugar phosphotransferase system in Escherichia coli.

The uptake of various carbohydrates and the synthesis of adenosine 3':5'-monophosphate (cyclic AMP) are subject to inhibition by sugar substrates of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli. The induced synthesis of the sugar-specific components of the phosphotransferase system was studied and correlated with the induction of regulatory interactions controlling glycerol uptake and net cyclic AMP synthesis. Activities of the Enzyme II complexes specific for glucose, fructose, and mannitol were measured both in vivo and in vitro. These activities were induced 8- to 40-fold by growth in the presence of the appropriate substrate-inducers. Cross inducer specificities were noted. Maximal inhibition of glycerol uptake and cyclic AMP synthesis by a sugar substrate of the phosphotransferase system required induction of the Enzyme II complex specific for that sugar and was abolished by mutations which destroyed Enzyme II activity. The inducer specificities of the regulatory systems and of the Enzymes II were found to be the same. A mutation which depressed the cellular activity of Enzyme I of the phosphotransferase system did not relieve sensitivity to inhibition. The results suggest that adenylate cyclase and several carbohydrate permeases are subject to coordinate regulation by a mechanism which depends on the catalytic activities of the protein components of the phosphotransferase system.

Adenylyl Cyclases↗
PubMed1975

Regulation of intracellular adenosine cyclic 3':5'-monophosphate levels in Escherichia coli and Salmonella typhimurium. Evidence for energy-dependent excretion of the cyclic nucleotide.

Sugars and other energy sources were found to lower intracellular concentrations of adenosine 3':5'-monophosphate (cyclic AMP) in strains of Escherichia coli and Salmonella typhimurium which were deficient for cyclic AMP phosphodiesterase. This effect required the presence of the specific transport system responsible for entry of that sugar into the cell and depended on the intracellular catabolic enzymes. Metabolizable sugars were more effective than nonmetabolizable sugars in reducing cellular cyclic AMP levels, and this reduction was blocked partially by uncouplers of oxidative phosphorylation. Electron donors such as lactate and ascorbate plus phenazine methosulfate reduced internal cyclic AMP levels in bacterial membrane vesicles which had been preloaded with the cyclic nucleotide. Uncouplers of oxidative phosphorylation, but not arsenate, blocked the energy-stimulated loss of intravesicular cyclic AMP. Employing intact cells, sugars were shown to have two primary effects on cyclic AMP metabolism: (a) they inhibited net synthesis of the cyclic nucleotide while promoting its degradation, and (b) they stimulated efflux of cyclic AMP into the extracellular fluid. While the former effect was elicited by metabolizable and nonmetabolizable sugars alike, stimulation of cyclic nucleotide excretion was only observed with metabolizable sugars. The results suggest that the extrusion of cyclic AMP from the bacterial cell is energy-dependent and is driven by an energized membrane state.

Adenosine Triphosphate↗
PubMed1975

Coordinate regulation of adenylate cyclase and carbohydrate permeases by the phosphoenolpyruvate:sugar phosphotransferase system in Salmonella typhimurium.

Adenylate cyclase (EC 4.6.1.1) and several carbohydrate permeases are inhibited by D-glucose and other substrates of the phosphoenolpyruvate:sugar phosphotransferase system. These activities are coordinately altered by sugar substrates of the phosphotransferase system in a variety of bacterial strains which contain differing cellular levels of the protein components of the phosphotransferase system: Enzyme I, a small heat-stable protein, and Enzyme II. It is suggested that the activities of adenylate cyclase and the permease proteins are subject to allosteric regulation and that the allosteric effector is a regulatory protein which can be phosphorylated by the phosphotransferase system.

Adenylyl Cyclases↗
PubMed1975

A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium.

Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome.

Biological Transport↗