Succinate Transport in Escherichia coli Mutants Defective in Succinate Metabolism

To investigate the operation of a succinate transport system in Escherichia coli, mutants defective in succinate metabolism were isolated. Although the metabolic blocks in the mutant cells were not complete, the succinate transport assays became possible.

Pyruvate, lactate or many other carbon sources stimulated succinate uptake, and the uptake was strongly inhibited by some electron transport inhibitors, uncouplers of oxidative phosphorylation and sulfhydryl reagents. The mutant strains accumulated succinate into the cells against a concentration gradient when suitable energy sources were supplied.

Presence of glucose in the medium strongly repressed the formation of the succinate transport system. The optimum pH for the succinate uptake was between 7.8 and 8.0.     

Effects of Ipomeamarone on Respiratory Enzyme System in Mitochondria

Ipomeamarone inhibited oxidation and phosphorylation in tightly coupled rat liver mitochondria. The inhibition of the oxygen uptake was higher when either β-hydroxybutyrate or α-ketoglutarate was supplied as the substrate than when succinate was used. In mitochondrial preparations which had been uncoupled, inhibitions of the electron transport chain from β-hydroxybutyrate to cytochrome c, and of the enzymes succinate cytochrome c oxidoreductase and β-hydroxybutyrate dehydrogenase were observed. Ipomeamarone inhibited also the ATP-inorganic phosphate exchange reaction, but did not act as an uncoupler; it repressed 2, 4-dinitropheaol-induced oxygen uptake.     

DctA- and Dcu-independent transport of succinate in Escherichia coli: contribution of diffusion and of alternative carriers

Quintuple mutants of Escherichia coli deficient in the C(4)-dicarboxylate carriers of aerobic and anaerobic metabolism (DctA, DcuA, DcuB, DcuC, and the DcuC homolog DcuD, or the citrate/succinate antiporter CitT) showed only poor growth on succinate (or other C(4)-dicarboxylates) under oxic conditions. At acidic pH (pH 6) the mutants regained aerobic growth on succinate, but not on fumarate. Succinate uptake by the mutants could not be saturated at physiological succinate concentrations (< or =5 mM), in contrast to the wild-type, which had a K(m) for succinate of 50 microM and a V(max) of 35 U/g dry weight at pH 6. At high substrate concentrations, the mutants showed transport activities (32 U/g dry weight) comparable to that of the wild-type. In the wild-type using DctA as the carrier, succinate uptake had a pH optimum of 6, whereas succinate uptake in the mutants was maximal at pH 5. In the mutants succinate uptake was inhibited competitively by monocarboxylic acids. Diffusion of succinate or fumarate across phospholipid membranes (liposomes) was orders of magnitude slower than the transport in the wild-type or the mutants. The data suggest that mutants deficient in DctA, DcuA, DcuB, DcuC, DcuD (or CitT) contain a carrier, possibly a monocarboxylate carrier, which is able to transport succinate, but not fumarate, at acidic pH, when succinate is present as a monoanion. Succinate uptake by this carrier was inhibited by addition of an uncoupler. Growth by fumarate respiration (requiring fumarate/succinate antiport) was also lost in the quintuple mutants, and growth was not restored at pH 6. In contrast, the efflux of succinate produced during glucose fermentation was not affected in the mutants, demonstrating that, for succinate efflux, a carrier different from, or in addition to, the known Dcu and CitT carriers is used.     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 3. Effect on Uptake of Potassium, Magnesium and Inorganic Phosphate

A venacin, the resistance factor in oat roots against Ophiobolus graminis var. graminis, and a related triterpeneglycoside, aescin, inhibited the uptake of K⁺ and Mg²⁺ in the fungal mycelium both in phosphate and succinate buffers. The uptake of the cations in Neurospora crassa was similarly inhibited when the inhibitors were dissolved in phosphate or acetatebuffer, while no decrease in the uptake of K⁺ and Mg²⁺ was observed when the inhibitors were dissolved in succinate buffer. The uptake of cations in Aspergillus niger and Pythium irregulare was more or less unaffected by aescin. The uptake of inorganic phosphate was in no case inhibited, but some decrease of the accumulation of inorganic phosphate in Ophiobolus graminis and Ncurospora crassa due to inhibitor treatment in phosphate buffer was observed. No accumulation of Ca²⁺ was observed in any of the tested fungi.     

Improved succinate production from galactose-rich feedstocks by engineered Escherichia coli under anaerobic conditions

It is of great economic interest to produce succinate from low-grade carbon sources, which can make it more economically competitive against petrochemical-based succinate. Galactose sugars constitute a significant fraction of the soluble carbohydrate in a meal from agricultural sources which is considered a low value or waste byproduct of oilseed processing. To improve the galactose utilization, the effect of galR and glk on sugars uptake was investigated by deactivation of each gene in three previously engineered host strains. As expected, glk plays an important role in glucose uptake, while, the effect of deactivation of galR is highly dependent on the strength of the downstream module (succinate production module). A new succinate producer FZ661T was constructed by enhancement of the succinate producing module and manipulation of the gal operon. The succinate productivity reached 4.57 g/L/hr when a mixed sugar feedstock was used as a carbon source in shake-flask fermentation, up to 812 mM succinate was accumulated in 80 hr in fed-batch fermentation. When SoyMolaGal hydrolysate was used as a carbon source, 628 mM (74 g/L) succinate was produced within 72 hr. In this study, we demonstrate that FZ661T can produce succinate quickly with relatively high yield, giving it the potential for industrial application.     

Diauxic growth of Agrobacterium tumefaciens 15955 on succinate and mannopine.

Diauxic growth was observed upon incubation of Agrobacterium tumefaciens 15955 on a mixture of succinate and mannopine as the carbon source. Diauxic growth was also observed when either fumarate or L-malate was mixed with mannopine. No diauxie was detectable when A. tumefaciens 15955 was grown on a mixture of mannopine and glucose, fructose, sucrose, or L-arabinose. Preferential utilization of succinate was observed in the initial growth phase of diauxie, whereas the final growth phase occurred at the expense of mannopine. Cells harvested during the initial growth phase exhibited a capacity for uptake of [14C]succinate but not of [14C] mannopine. A capacity for [14C]mannopine uptake was expressed during the final growth phase. Extracts from cells grown on a mixture of succinate and mannopine exhibited a low level of mannopine cyclase activity in the initial phase of diauxie. This activity increased substantially in the final phase of growth. Added succinate had no effect on the rate of [14C]mannopine uptake or mannopine cyclase activities of cells previously grown on mannopine. Diauxie was also observed during growth of strain 15955 on a mixture of succinate and octopine.     

Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas ruminantium.

When Selenomonas ruminantium HD4 was grown in a chemostat, maximal succinate production and the highest molar growth yield values were both observed at a dilution rate of roughly 0.2 h-1. To determine the possible relationship between succinate efflux and high molar growth yields, the generation of a membrane potential by succinate efflux was studied in whole cells and vesicles (inside-out and right-side-out) prepared from S. ruminantium. Washed whole cells took up succinate in the absence of an exogenous energy supply; uptake was completely abolished by brief treatment with dinitrophenol or with nigericin and valinomycin. High levels of sodium ions (with respect to the intracellular sodium concentration in the assay buffer had a stimulatory effect on succinate uptake. When succinate was added to inside-out vesicles, a membrane potential (inside positive) was generated, as indicated by fluorescence quenching of the anionic lipophilic dye Oxonol V. Fluorescence quenching was sensitive to uncoupling by gramicidin D but only partially sensitive to the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In right-side-out vesicles, succinate uptake could be driven by an artificially imposed sodium gradient but not by a potassium diffusion potential; imposition of both a sodium gradient and potassium diffusion potential resulted in improved succinate uptake. The generation of a membrane potential (inside negative) upon succinate efflux was demonstrated directly in right-side-out vesicles when succinate-loaded vesicles were diluted into succinate-free buffer, and the lipophilic cationic probe tetraphenylphosphonium accumulated in the vesicles. Results indicate that an electrogenic succinate-sodium symporter is present in S. ruminantium. Transport of succinate out of the cell via the symporter might be responsible for the high molar growth yields obtained by this organism when it is grown at dilution rates where maximal succinate production occurs.     

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.     

Some Reactions of Isolated Corn Mitochondria Influenced by Juglone

The effects of juglone on the uptake of O₂ by excised corn roots (Zea mays L., Wf9 cms- T × M14) and isolated corn mitochondria arc reported. The O₂ uptake by excised corn roots, as measured by an O₂ electrode, was inhibited more than 90% after a one-hour treatment of 500 μM juglone. Lesser inhibitions were observed with 50 μM and 250 μM juglone. In a KC1 reaction medium in the absence of inorganic phosphate (Pi), juglone stimulated the rate of O₂ uptake by isolated mitochondria oxidizing NADH, succinate, or malate + pyruvate. In the presence of Pi, juglone concentrations of 3 μM and greater inhibited the state 3 oxidation rates of succinate and malate + pyruvate, lowered respiratory control and ADP/O ratios obtained from the oxidation of NADH, malate + pyruvate, or succinate, and reduced the coupled deposition of calcium phosphate within isolated mitochondria driven, by the oxidation of malate + pyruvate. The inhibition of state 3 O₂ uptake by isolated mitochondria, an oxidative state in which electron transfer is coupled to ATP production, is seen to correlate with the inhibition affected by juglone when applied to tissues in vivo.     

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.     

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

Two succinate uptake systems inBradyrhizobium japonicum

Succinate uptake inBradyrhizobium japonicum 61-A-101 has a biphasic kinetic, indicating the presence of a high-and low-affinity uptake system. The apparent K_M data are 2.4 M (high-affinity system) and 172 M (low-affinity system). ForEnterobacter aerogenes NCTC 10006, only one uptake system for succinate was found with an apparent K_M of 80 M. InBradyrhizobium japonicum andEnterobacter aerogenes, succinate uptake is carrier mediated and constitutive. 2,4-Dinitrophenol and cyanide severely inhibit uptake, whereas arsenate inhibits only to a lesser extent. In both strains, fumarate is also transported by the succinate uptake system. Glucose has significant effects on succinate uptake or metabolism inEnterobacter aerogenes, but not inBradyrhizobium japonicum.     

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.     

Bistability of the <Emphasis Type="Italic">lac</Emphasis> Operon During Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> on Lactose and Lactose + Glucose

The lac operon of Escherichia coli can exhibit bistability. Early studies showed that bistability occurs during growth on TMG/succinate and lactose + glucose, but not during growth on lactose. More recently, studies with lacGFP-transfected cells show bistability during growth on TMG/succinate, but not during growth on lactose and lactose + glucose. In the literature, these results are invariably attributed to variations in the destabilizing effect of the positive feedback generated by induction. Specifically, during growth on TMG/succinate, lac induction generates strong positive feedback because the permease stimulates the accumulation of intracellular TMG, which in turn, promotes the synthesis of even more permease. This positive feedback is attenuated during growth on lactose because hydrolysis of intracellular lactose by β-galactosidase suppresses the stimulatory effect of the permease. It is attenuated even more during growth on lactose + glucose because glucose inhibits the uptake of lactose. But it is clear that the stabilizing effect of dilution also changes dramatically as a function of the medium composition. For instance, during growth on TMG/succinate, the dilution rate of lac permease is proportional to its activity, e, because the specific growth rate is independent of e (it is completely determined by the concentration of succinate). However, during growth on lactose, the dilution rate of the permease is proportional to e ² because the specific growth rate is proportional to the specific lactose uptake rate, which in turn, proportional to e. We show that: (a) This dependence on e ² creates such a strong stabilizing effect that bistability is virtually impossible during growth on lactose, even in the face of the intense positive feedback generated by induction. (b) This stabilizing effect is weakened during growth on lactose + glucose because the specific growth rate on glucose is independent of e, so that the dilution rate once again contains a term that is proportional to e. These results imply that the lac operon is much more prone to bistability if the medium contains carbon sources that cannot be metabolized by the lac enzymes, e.g., succinate during growth on TMG/succinate and glucose during growth on lactose + glucose. We discuss the experimental data in the light of these results.     

Identification of the membrane protein SucE and its role in succinate transport in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Succinic acid is excreted during anaerobiosis by many bacteria, and manifold applications are known making the biotechnological production of succinate attractive. Although the pathways for succinate formation are known, succinate export is not understood in most of the succinate producing bacteria. Here, we present a bioinformatic approach for identification of a putative succinate export system in Corynebacterium glutamicum. The subsequent screening revealed that a mutant in the gene cg2425 is impaired in succinate production or transport under anaerobic conditions. A function of the Cg2425 protein as import system was excluded. In contrast, a role of the Cg2425 protein as succinate export system was indicated by accumulation of increased amounts of internal succinate under anaerobic conditions in a Cg2425-dependent manner and a concomitant impairment of external succinate accumulation. In conclusion, we propose that Cg2425 participates in succinate export in C. glutamicum and suggest the name SucE for the protein.     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

Comparison of exogenous energy sources for in vitro maintenance of follicle cell-freeXenopus laevis oocytes

Summary The purpose of these experiments was to determine which exogenous energy sources are suitable for isolated follicle cell-free oocytes from the frog,Xenopus laevis. In order to compare prospective energy sources, follicle cell-free oocytes from 0.4 to 1.3 mm in diameter were incubated in a 1 mM concentration of each of a variety of energy sources and scored daily for the maintenance of morphological characteristics. Vitellogenic oocytes placed in succinate or fumarate deteriorated at the same time as those in saline alone. Oocytes incubated in oxaloacetate (OAA) appeared to remain in the best morphological condition, followed by oocytes maintained in pyruvate or glucose. Fully grown oocytes were tested at various times of incubation for their ability to respond to progesterone by undergoing germinal vesicle breakdown. These experiments showed that oocytes placed in OAA or pyruvate retained the ability to respond to progesterone longer than those in the other energy sources. Increased respiratory rates were stimulated in isolated oocyte mitochondria by succinate as well as pyruvate and OAA. However, oocytes incubated in labelled pyruvate evolved 80 to 140 times as much labelled CO₂ as oocytes incubated in labelled glucose or succinate. In addition, it was found that the rate of uptake of pyruvate is 20 to 25 times greater than the rate of uptake of glucose or succinate. It is concluded from these experiments that OAA and pyruvate are the most effective exogenous energy sources for the in vitro maintenance ofXenopus oocytes. One possible explanation for the ineffectiveness of glucose or succinate as exogenous energy sources is a restriction in their uptake into the oocytes. Research supported by grants 10179 from the Research Foundation of the City University of New York and BMS 74-18790 from the National Science Foundation.