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 K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m 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.     

Isolation and Properties of Mutant Strains of Escherichia coli Defective in Succinate Transport System

To investigate the stereo-specificity and the genetic control of a succinate transport system, mutants of Escherichia coli defective in the transport of succinate were isolated. The mutants showed no detectable growth on fumarate and malate, as well as on succinate. All of the revertant strains from one of the transport defective mutants, T5, could grow either on succinate, fumarate or malate. The T5 cells accumulated only a trace amount of ¹⁴C-succinate or ¹⁴C-fumarate. These results indicated that at least succinate, fumarate, and malate were transported by the system involving the same component. From the competition experiments, it was suggested that oxalacetate was also transported by the same system. A partial participation of this system for the transport of aspartate was suggested.     

Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K_S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [¹⁴C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK_a2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate²⁻ accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

Succinate transport in Rhizobium leguminosarum.

The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.     

Succinate transport inRhizobium meliloti: Characteristics and impact on symbiosis

The transport of succcinate was studied inRhizobium meliloti M5N1. Succinate accumulation was a saturable function of the succinate concentration, and the apparent Km and Vmax values were respectively 2.9 M and 79 nmoles/min·mg protein. Strong inhibition of succinate transport was observed in the presence of 10 mM DNA and 4 mM azide, whereas arsenate and fluorure had no effect. Fumarate competitively inhibited succinate transport; the Ki value was between 3 and 6 M.A succinate transport mutant ofR. meliloti M5N1 was selected after nitrosoguanidine mutagenesis. It failed to grow on succinate, malate, or fumarate, but grew on arabinose, glutamate, pyruvate, and other carbohydrates. The mutant strain formed white (presumably leghemoglobin deficient) and ineffective nodules, since the acetylene reduction assay showed no nitrogenase activity.     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

A Transport System for C4-Dicarboxylic Acids in Isolated Membrane Preparations from Escherichia coli

To investigate the mechanism of succinate transport system in Escherichia coli, the isolated membranes were prepared from E. coli W2252 and T5, a mutant defective in succinate uptake derived from W2252. Uptakes of ¹⁴C-substrates by W2252 and T5 membranes and the dilution of accumulated radioactivity by unlabeled C₄-dicarboxylic acids, indicated that C₄-dicarboxylic acids in the tricarboxylic acid cycle are transported by the same system in E. coli which requires a suitable energy source such as NADH, D-lactate or reduced phenazine methosulfate. The uptakes of succinate by W2252 membranes were inhibited by an anaerobic incubation or some of the inhibitors of electron transport chain. Difference spectra of reduced versus oxidized membranes from W2252 and T5 indicated the reduction of flavoproteins and cytochromes by dithionite, NADH or D-lactate. From these results it was concluded that the uptake of the C₄-dicarboxylic acids in isolated membranes is coupled to an electron transport chain involving a specific dehydrogenase system.     

Transport of succinate by Pseudomonas putida

Induced succinate uptake and transport (defined as transport of a compound followed by its metabolism and transport in the absence of subsequent metabolism) by Pseudomonas putida are active processes resulting in intracellular succinate concentrations 10-fold that of the initial extracellular concentration. Uptake was studied with the wild-type strain P. putida P2 and transport with a mutant deficient in succinate dehydrogenase activity. Addition of succinate, fumarate, or malate to the growth medium induces both processes above a basal level. Induction is dependent on protein synthesis and subject to catabolite repression. When extracts of induced and noninduced wild-type cells were assayed for succinate dehydrogenase, fumarase, and malate dehydrogenase only malate dehydrogenase increased in specific activity. Transport is inhibited by iodoacetamide, KCN, NaN₃, and 2,4-dinitrophenol and shows pH and temperature optima of 6.2 and 30 °C. Kinetic parameters are: basal uptake (cells grown on glutamate) K_m 11.6 μm, v 0.32 nmoles per min per mg dry cell mass; induced uptake (cells grown on succinate plus NH₄Cl) K_m 12.5 μm, v 5.78 nmoles per min per mg dry cell mass; induced transport (cells grown on nutrient broth plus succinate) K_m 10 μm, V 0.98 nmoles per min per mg dry cell mass. It was not possible to determine the kinetic parameters of basal transport. Malate and fumarate were the only compounds exhibiting competitive inhibition of uptake and transport suggesting common transport system for all three compounds. The K_i values for competitive inhibition and the K_m for succinate indicate the order of affinity for both uptake and transport are succinate > malate > fumarate. Data from kinetic parameters of uptake and transport and studies on succinate metabolism provide evidence consistent with concurrent increases in transport and metabolism to account for induced succinate uptake by P. putida.     

Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.     

Expression of Galactose Permease and Pyruvate Carboxylase in Escherichia coli ptsG Mutant Increases the Growth Rate and Succinate Yield under Anaerobic Conditions

In Escherichia coli, disruption of ptsG, which encodes the glucose-specific permease of the phosphotransferase transport system (PTS) protein EIICB^Glc, is crucial for high succinate production. This mutation can, however, cause very slow growth and low glucose consumption rates. The ptsG mutant (TUQ2), from wild type E. coli W1485, and E. coli galP (encoding galactose permease) and glk (encoding glucose kinase) gene expression plasmids were constructed. TUQ2 increased the generation time to approximately 4 h and gave a higher final cell density of 0.5 g/l by expression of galP. However, glk expression had no effect on the mutant. After expression of pyruvate carboxylase (PYC) and galactose permease, the ptsG mutant showed higher succinate yield (1.2 mol/mol glucose) and the specific rate of glucose consumption from 0.33 to 0.6 g/1 h. Received 31 August 2005; Revisions requested 27 September 2005; Revisions received 1 November 2005; Accepted 2 November 2005 An erratum to this article is available at .     

Interrelationship between Dilauryl Succinate Feeding and Nutritional Encephalomalacia in Starting Chicks

Various levels of dilauryl succinate with or without additional d-α-tocopheryl acetate and of diethyl succinate were fed to chicks for 4 weeks to examine the interrelationship between the esters and nutritional encephalomalacia.

Chicks fed dilauryl succinate at the level higher than 3% died with lesions in the cerebellum. The lesions were prevented by the supplementation of 25 mg or more of d-α-tocopheryl acetate per kg of diet. Median lethal dietary level for males of meat-type and egg-type chicks at 3 weeks of age was 6.3 and 6.0%, respectively. That for females of meat-type at 3 weeks of age was 9.0%, suggesting that males were more sensitive than females. Diethyl succinate did not induce encephalomalacia.     

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength.

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Synthesis of Cyclotene and Its Related Compounds by the Acyloin Condensation

A mutant which required glutamate for growth as the sole nitrogen source was derived from alkalophilic Bacillus No. 8–1 by UV irradiation. The relationship was examined between cell growth and glutamate transport into cells.

Cell growth and glutamate transport into cells were dependent on extracellular pH in the presence of Na⁺, and both were maximum between pH 9 and 10. The quantitative relation between specific growth rate and glutamate uptake rate indicated that the amount of glutamate required for growth at pH 7 and 9 was consistent with that of glutamate transported at pH 7 and 9, respectively. But the amount of glutamate transported at pH 7 was not sufficient to support growth at pH 9. The glutamate transport system of this mutant strain evidently had an effect on growth.     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

Oxydation of substrates in organic acids utilization negative mutants and the wild typeRhizobium meliloti strain S'14

Two mutants defective in succinate utilization were isolated by NTG mutagenesis of the effective wild typeRhizobium meliloti strain S₁₄. The mutants used carbon sources in a fashion similar to strain S₁₄, but they were not able to grow on succinate, fumarate or malate. The mutants nodulated alfalfa plants but did not exhibit any nitrogenase activity. The mutants oxidized glucose and fructose, but were not able to oxidize organic acids. Cultured free-living bacteria of strain S₁₄ appeared to have an inducible C₄-dicarboxylic acid uptake system and a constitutive glucose uptake system. When S₁₄ cells were grown on glucose in the presence of 5mM or more succinate or malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression like phenomenom. Contribution no. 301, Station de Recherches, Agriculture Canada.     

Succinate accumulation in man during exercise

It has been demonstrated in several diving vertebrates that succinate, a component of the Krebs cycle, accumulates in blood during breath-hold dives. The production of succinate is thought to result from amino acid catabolism. Our purpose was to determine whether succinate accumulation occurs in man during muscular activity requiring anaerobic energy contribution. Experiments using an endurance athlete included apneic work on an underwater ergometer and treadmill running to exhaustion. During 1 min breath-hold [(V)\dot]\dot V O₂max, venous succinate increased from 42 [(V)\dot]\dot V O₂max and increased succinate from a similar resting value to 93 M×10^–6. Increases in alanine, lactate, and pyruvate were observed for both types of exercise. The findings confirm that succinate accumulation also occurs in man. It was suggested that amino acid catabolism may provide a source of anaerobic energy production in addition to glycolysis. However, the importance of the proposed energy pathway remains to be quantified.     

Hexose metabolism in pancreatic islets: Succinate dehydrogenase activity in islet homogenates

Succinate dehydrogenase activity was measured in rat pancreatic islet homogenates incubated in the presence of [1,4-¹⁴C]succinate, the reaction velocity being judged through the generation of ¹⁴CO₂ in the auxiliary reactions catalysed by pig heart fumarase and chicken liver NADP-malate dehydrogenase. In the presence of 1·0 mM succinate, the reaction velocity averaged 5·53 ± 0·44 pmol min^?1 μg^?1 islet protein. The K_m for succinate was close to 0·4 mM and the enzymic activity was restricted to mitochondria. These kinetic results indicate that, under the present experimental conditions, the activity of succinate dehydrogenase does not vastly exceed that of either NAD-isocitrate dehydrogenase or the 2-ketoglutarate dehydrogenase complex, at least when the latter enzymes are activated by ADP and/or Ca²⁺. Nevertheless, the activity of succinate dehydrogenase is sufficient to account for the increase in O₂ uptake evoked in intact islets by the monomethyl ester of succinic acid. It could become a rate-limiting step of the Krebs cycle in models of B-cell dysfunction.     

Role of Copper Ions in the Regulation of L-Glutamate Biosynthesis

Cell-free extracts of Brevibacterium thiogenitalis culture grown in the presence of copper catalyzed the oxidation of NADH₂ and succinate through an electron transport chain which contained menaquinones and cytochromes a, b and c. On the other hand, extracts of cells grown in the absence of copper lacked cytochromes a and c, and contained cytochrome d.

These findings, as well as the results obtained in inhibition experiments, suggest that in copper-deficient cells the major part of NADH₂ was oxidized via a bypass in which the electrons were transferred directly from flavoprotein or cytochrome b to molecular oxygen.

Electron transport from these substrates to molecular oxygen resulted in ATP synthesis. The average P/O ratios in extracts of the copper-sufficient cells were 0.33 for generated NADH₂, 0.20 for added NADH₂, and 0.34 for succinate, and those in extracts of the copper-deficient cells were 0.15, 0.13 and 0.21, respectively. In addition, a linear relationship was found between the yield of L-glutamate from acetate and the P/Ο ratios with both NADH₂ and succinate as substrates.

From these results, it is reasonable to consider that the poor yield of L-glutamate from acetate in copper-deficient cells was due to a reduction in energy supply, which was caused by the low efficiency of oxidative phosphorylation.     

The highly toxic oxyanion tellurite (TeO<Stack><Subscript>3</Subscript><Superscript>2−</Superscript></Stack>) enters the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> via an as yet uncharacterized monocarboxylate transport system

The facultative phototroph Rhodobacter capsulatus takes up the highly toxic oxyanion tellurite when grown under both photosynthetic and respiratory growth conditions. Previous works on Escherichia coli and R. capsulatus suggested that tellurite uptake occurred through a phosphate transporter. Here we present evidences indicating that tellurite enters R. capsulatus cells via a monocarboxylate transport system. Indeed, intracellular accumulation of tellurite was inhibited by the addition of monocarboxylates such as pyruvate, lactate and acetate, but not by dicarboxylates like malate or succinate. Acetate was the strongest tellurite uptake antagonist and this effect was concentration dependent, being already evident at 1 μM acetate. Conversely, tellurite at 100 μM was able to restrict the acetate entry into the cells. Both tellurite and acetate uptakes were energy dependent processes, since they were abolished by the protonophore FCCP and by the respiratory electron transport inhibitor KCN. Interestingly, cells grown on acetate, lactate or pyruvate showed a high level resistance to tellurite, whereas cells grown on malate or succinate proved to be very sensitive to the oxyanion. Taking these data together, we propose that: (a) tellurite enters R. capsulatus cells via an as yet uncharacterized monocarboxylate(s) transporter, (b) competition between acetate and tellurite results in a much higher level of tolerance against the oxyanion and (c) the toxic action of tellurite at the cytosolic level is significantly restricted by preventing tellurite uptake.