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S C Quay

Publications and source records attributed to S C Quay.

46 records · Page 3Linked to original sources
PubMed1977

Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.

The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.

Biological Transport, Active↗
PubMed1977

Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur.

Amino Acids↗
PubMed1976

Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12.

Two mutant loci resulting in derepression of, respectively, the L-leucine-specific transport system (lstR) and both the leucine-specific and the general branched-chain amino acid transport LIV-I systems (livR) were mapped by conjugation and transduction. Both livR and lstR were found to be closely linked to aroA at min 20 on the Escherichia coli genetic map. The merodiploid livR+/livR displayed wild-type regulation of L-leucine transport, indicating that the livR product is a diffusible, negative controlling element for high-affinity leucine transport systems. Isogenic strains carrying lstR, livR, and wild-type transport alleles were compared for leucine uptake kinetic parameters and leucine-binding protein levels. The higher levels of leucine transport in the mutant strains under repressing conditions were generally due to increased high-affinity systems, which were accompanied by striking increases in the level of leucine-binding proteins.

Biological Transport, Active↗
PubMed1976

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.

Bacterial Proteins↗
PubMed1975

Role of leucyl-tRNA synthetase in regulation of branched-chain amino-acid transport.

The regulation of the transport of leucine, isoleucine, and valine in Escherichia coli B/r was studied in a mutant with a complete deletion of the leucine biosynthetic operon and a temperature-sensitive leucyl-tRNA synthetase [L-leucine:tRNALeu ligase (AMP-forming), EC 6.1.1.4]. Under conditions of excess leucine and a functional leucyl-tRNA synthetase transport activity was repressed. Shifting the culture to a temperature at which the activation of leucine to an appropriate tRNA species became growth-rate-limiting led to a large increase in the high-affinity transport of leucine, isoleucine, and valine (system LIV-I) while the uptake of histidine and proline was unchanged. A similar increase was observed for branched-chain amino-acid binding protein activity. The temperature change did not alter the transport activity for any of these substrates or the level of the binding proteins in an isogenic strain with a normal leucyl-tRNA synthetase. The increase in transport activity observed in the mutant was prevented by inhibitors of protein and RNA synthesis and probably represents an increase in the differential rate of synthesis of the protein(s) required for transport. These experiments demonstrate that the repression of branched-chain amino-acid transport involves the interaction of leucine with its aminoacyl-tRNA synthetase and its cognate leucyl-tRNA species.

Amino Acids↗
PubMed1975

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.

Biological Transport, Active↗
PubMed1975

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.

Acetolactate Synthase↗
PubMed1974

Glucose uptake and phosphorylation in Pseudomonas fluorescens.

Pseudomonas fluorescens ATCC 13525 and a particulate glucose oxidase (d-glucose:oxygen oxidoreductase, EC 1.1.3.4) mutant of this organism, gox-7, were examined to determine if glucose oxidation via particulate glucose oxidase is a required first step for glucose uptake. Initial [(14)C]glucose-uptake rates in parent and gox-7 cells were qualitatively similar. Initial [(14)C]glucose-uptake product analysis revealed that glucose was accumulated via active transport and was rapidly metabolized to glucose-6-phosphate and gluconate-6-phosphate in both parent and gox-7 cells. Cell extracts contained soluble adenosine 5'-triphosphate specific kinase activity for phosphorylation of glucose. Glucose uptake was induced by glucose and not gluconate, thus, establishing independent regulation of glucose transport and glucose catabolism in p. fluorescens. The results prove that glucose oxidase was not an obligatory reaction for glucose carbon permeation in P. fluorescens. A general unifying scheme for glucose utilization in the aerobic fluorescent pseudomonads is suggested for the purpose of clarifying glucose uptake in these bacteria.

Aerobiosis↗
PubMed1972

Gluconate regulation of glucose catabolism in Pseudomonas fluorescens.

Induction of Entner-Doudoroff pathway enzymes in Pseudomonas fluorescens was investigated to study the role of gluconate as a possible inducer. Glucose oxidase-deficient mutants were isolated and characterized. One of these mutants, gox-7, was deficient in particulate glucose oxidase; another mutant, gox-17, was deficient in particulate glucose and gluconate oxidase activities. Gluconate, but not glucose, induced synthesis of gluconokinase and 6-phosphogluconate dehydratase in both mutants. High constitutive levels of 2-keto-3-deoxy-6-phosphogluconate aldolase were found when both mutants were grown on glucose. Growth of parent and both mutant strains on glycerol also resulted in high levels of Entner-Doudoroff pathway enzymes. It was concluded that glucose cannot serve as an inducer molecule for derepression of Entner-Doudoroff pathway enzymes in P. fluorescens. Evidence presented provides good support for gluconate being the true inducer of this pathway in P. fluorescens. A relationship is presented for explaining distribution of the Entner-Doudoroff pathway in certain groups of bacteria.

Alcohol Oxidoreductases↗