William W. Kay

Genetic control of the metabolism of propionate by Escherichia coli K12

Abstract 1. 1. Many strains of Escherichia coli K12 are unable to utilize propionate as a sole source of carbon. Spontaneous mutants were isolated from both wild-type and mutant strains with enzyme defects in pathways of carbon flow related to the tricarboxylic acid cycle. 2. 2. From the analysis of such mutants and of propionate-metabolizing mutants of the parent strain it was concluded that propionate is first transported by the acetate transport system then metabolized via conversion to acetate by oxidation through pyruvate and also via the hydroxyglutarate pathway. 3. 3. These propionate-utilizing mutants were found to be derepressed for both the enzymes of the hydroxyglutarate pathway and also the glyoxylate shunt. 4. 4. Acetate was shown to repress the oxidation of propionate to pyruvate.

Transport of C4-dicarboxylic acids in Neurospora crassa

1. 1. The transport of tricarboxylic acid cycle dicarboxylic acids by Neurospora crassa was induced 80-fold by growth on acetate whereas tricarboxylic acid enzymes were induced only 2-6-fold and glyoxylate cycle enzymes were induced 20-fold. 2. 2. From the kinetics of uptake and competitive inhibition it was concluded that a single system was responsible for the transport of succinate, fumarate, l-malate and possibly α-ketoglutarate as well as a number of structural analogues. The system appears to be less specific than the known bacterial systems. 3. 3. Succinate transport was strongly inhibited by various known inhibitors of energy metabolism — primarily by proton conductors. Transport was also severely inhibited by various sulfhydryl reagents. 4. 4. Succinate, fumarate and l-malate uptake were identically pH dependent and evidence is presented which implicates the monoionic species as the molecular form of the dicarboxylic acid transported.

A Dicarboxyclic acid transport system inBacillus subtilis

The transport of compounds related to the tricarboxylic acid cycle (TCA) in microorganisms has only recently been investigated in detail. In Escherichia coli and Salmonella typhimurium [ 1, 21 L-aspartate, fumarate, Lmalate and succinate share a common transport system and mutants devoid of this system have been described. Citrate transport in S. typhimurium (W.W. Kay, unpublished results) is mediated by a separate system as in Aerobacter aerogenes [3] and B. subtilis [4] however, the specificity of this system in B. subtilis has not been reported although growth on C4-dicarboxylic acid; repressed citrate transport. This report describes an investigation which determines the existence and properties of a C4-dicarboxylic acid active transport system in B. subtilis. The organism was selected for several obvious advantages: it is easily grown on simple media, it is genetically defined, and it is a gram-.positive microorganism a group of microorganisms in which transport systems have received little attention. The latter characteristic also permits the ready isolation of membrane preparations free of cell wall material [5,6], an important advantage for detailed investigations into the biochemistry of transport systems.

Acidic amino acid transport in Neurospora crassa mycelia

Abstract 1. 1. l -Aspartate transport activity was optimally induced following 6 h growth on acetate minimal media. 2. 2. The observed aspartate (and glutamate) transport activity was attributed to the general amino acid transport system since l -phenylalanine, but not l -cysteic acid, inhibited transport activity at all pH values tested. 3. 3. Changes in K m values for l -aspartate and l -glutamate transport following growth on acetate minimal media indicated that apparent “affinities” of the “acidic amino acid transport systems” dramatically increased during the stages of active growth.

Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The Km values for the phosphate or sulfate requirement were approx. 3 mM. Biphasic kinetics were characteristic of both the succinate (Km values 0.1 and 1 mM), and inorganic phosphate (Km values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media. A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective. Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport. The transport of C4-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.

Transport of 5-hydroxytryptamine (Serotonin) by canine blood platelets

Abstract 1. 1. 5-Hydroxytryptamine (5-HT) was transported into canine blood platelets by a system characterized by distinct temperature (Q10 2.3) and pH optima (37°C, 7.0 respectively) and saturation kinetics (Km 1.6 × 10−7 M). 2. 2. Competitive inhibition studies with 14 structural analogues indicated a high degree of stereospecificity. 3. 3. N-acetyl-5-hydroxytryptamine (NAHT) was not actively transported by the same system but was an effective competitive inhibitor of 5-HT uptake. 4. 4. Canine platelets, when immobilized on membrane filters, did not release labelled 5-HT unless washed with adenosinediphosphate (ADP) or thrombin, however filter immobilized platelets were still able to undergo shape changes. Under these conditions accumulated 5-HT only slowly exchanged with exogenous 5-HT or NAHT. 5. 5. Of several metabolic inhibitors tested only the respiratory poisons potassium ferricyanide and sodium amytal were exceptionally strong inhibitors of 5-HT transport and caused the release of 5-HT from pre-loaded platelets. N-ethylmaleimide (NEM) was similarly inhibitory.

Sodium-dependent transport of 5-hydroxytryptamine (serotonin) by canine blood platelets

Abstract The transport of 5-hydroxytryptamine (5-HT) was shown to be strongly dependent on the presence of Na+ in the incubation medium whereas divalent cations were without effect. The Km for the Na+ requirement was 16.8 m m . The addition of Na+ to Na+-depleted platelets restored maximum 5-HT transport within 3 min. The affinity of the 5-HT carrier for its substrate was directly proportional to the concentration of Na+; however, below 25 m m Na+ unique reversible morphological changes in platelet shape occurred as revealed by scanning electron microscopy which resulted in a drastically reduced affinity for 5-HT. K+, choline (Ch+), or Li+ could be used as counterbalancing cations to maintain osmolarity, and the affinity for 5-HT was also dependent on the concentrations of these ions. Ouabain as well as various ionophores at low concentrations inhibited 5-HT uptake. The inhibition was the result of the destruction of the Na + K + gradient across the cytoplasmic membrane. Ionophores, however, did not cause the depletion of either intracellular ATP or 5-HT.

Suppression of a dicarboxylic acid transport mutant phenotype in Escherichia coli K12

Abstract 1. 1. Propionate-utilizing mutants of Escherichia coli K12 supress the dicarboxylic transport mutant phenotype; that is, these C 4 -acid transport mutants regain the capacity to grow on a dicarboxylic acid as a carbon source. 2. 2. Mutants harboring both the propionate and transport mutations are unable to transport C 4 -dicarboxylic acids at low concentrations (1o −5 M), however, they apparently metabolize them at higher concentrations. 3. 3. The propionate mutation acts as a physiological suppressor of a transport defect by causing the simultaneous increase in activity of a number of tricarboxylic acid cycle enzymes concerned with dicarboxylic acid metabolism.