Purification and characterization of aconitase isoforms from etiolated pumpkin cotyledons

Although aconitase (EC 4.2.1.3) is involved in the glyoxylate cycle, which is localized in glyoxysomes, we detected very low aconitase activity in glyoxysomal fractions after sucrose gradient centrifugation of extracts prepared from etiolated pumpkin ( Cucurbita sp.) colyledons. Two aconitase isoforms were purified to homogeneity, albeit in low yield, by hydrophobic interaction, hydroxylapatite and anion exchange chromatography. They were designated Aco I and Aco II; both were shown to be monomeric proteins of M₁ 100 000 or 98 000 by gel filtration and SDS-PAGE analysis, respectively; isoelectric points were 5.0 and 4.8, respectively. Kinetic studies revealed similarities between Aco I and Aco II. A third aconitase isoform (Aco III) was revealed but not purified to homogeneity.     

Increased expression of transglutaminase 2 drives glycolytic metabolism in renal carcinoma cells

Transglutaminase 2 (TGase 2) expression and glycolysis are increased in most renal cell carcinoma (RCC) cell lines compared to the HEK293 kidney cell line. Although increased glycolysis and altered tricarboxylic acid cycle are common in RCC, the detailed mechanism by which this phenomenon occurs remains to be elucidated. In the present study, TGase 2 siRNA treatment lowered glucose consumption and lactate levels by about 20–30 % in RCC cells; conversely, high expression of TGase 2 increased glucose consumption and lactate production together with decreased mitochondrial aconitase (Aco 2) levels. In addition, TGase 2 siRNA increased mitochondrial membrane potential and ATP levels by about 20–30 % and restored Aco 2 levels in RCC cells. Similarly, Aco 2 levels and ATP production decreased significantly upon TGase 2 overexpression in HEK293 cells. Therefore, TGase 2 leads to depletion of Aco 2, which promotes glycolytic metabolism in RCC cells.     

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.     

Developmental Block in Citric Acid Cycle Mutants of Bacillus subtilis

Mutants deficient in different enzymes of the citric acid cycle can be subdivided into two groups according to the frequency at which they produce heat-resistant spores in nutrient sporulation medium. However, the majority of cells can develop in this medium only to the axial filament stage I of sporulation; aconitase and isocitrate dehydrogenase mutants need the addition of glutamate to reach this stage.     

Gene flow analysis demonstrates that Phytophthora fragariae var. rubi constitutes a distinct species, Phytophthora rubi comb. nov

Isozyme analysis and cytochrome oxidase sequences were used to examine whether differentiation of P. fragariae var. fragariae and P. fragariae var. rubi at the variety level is justified. In isozyme studies six strains of both P. fragariae varieties were analyzed with malate dehydrogenase (MDH), glucose phosphate isomerase (GPI), aconitase (ACO), isocitrate dehydrogenase (IDH) and phosphogluconate dehydrogenase (PGD), comprising altogether seven putative loci. Five unique alleles (Mdh-1(A), Mdh-2(B), Gpi(A), Aco(B) and Idh-1(B)) were found in strains of P. fragariae var. fragariae, whereas five unique alleles (Mdh-1(B), Mdh-2(A), Gpi(B), Aco(A) and Idh-1(A)) were present in strains of P. fragariae var. rubi. It was inferred from these data that there is no gene flow between the two P. fragariae varieties. Cytochrome oxidase I (Cox I) sequences showed consistent differences at 15 positions between strains of Fragaria and Rubus respectively. Based on isozyme data, cytochrome oxidase I sequences, and previously published differences in restyriction enzyme patterns of mitochondrial DNA, sequences of nuclear and mitochondrial genes, AFLP patterns and pathogenicity, it was concluded that both specific pathogenic varieties of P. fragariae are reproductively isolated and constitute a distinct species. Consequently strains isolated from Rubus idaeus are assigned to Phytophthora rubi comb. nov.     

Production and characterization of monoclonal antibodies specific for Shewanella colwelliana exopolysaccharide.

Six monoclonal antibodies were produced to whole cells of Shewanella colwelliana (Aco1 to Aco6) and two (Aco22 to Aco23) to purified exopolysaccharide (EPS). Aco1, -4 to -6, -22, and -23 bound to both the cell surface and the purified EPS, while Aco2 and -3 bound to cells only. The EPS of S. colwelliana was antigenically unique from those of nine other species of marine bacteria that were tested. Mapping studies revealed that all of the EPS-specific monoclonal antibodies bound to the same epitope. This EPS epitope was sensitive to cleavage of ester bonds, but neither pyruvate, acetate, nor terminal nonreducing sugars were required for antigenicity. When S. colwelliana was grown on rich media, most of its EPS was loosely associated with the cell surface.     

Sporulation of Tricarboxylic Acid Cycle Mutants of Bacillus subtilis

A mutant of Bacillus subtilis 168 lacking aconitase (EC 4.2.1.3) was found to be blocked at stage 0 or I of sporulation. Although adenosine triphosphate levels, which normally decrease in tricarboxylic acid cycle mutants at the completion of exponential growth, could be maintained at higher levels by feeding metabolizable carbon sources, this did not permit the cells to progress further into the sporulation sequence. When post-exponential-phase cells of mutants blocked in the first half of the tricarboxylic acid cycle were resuspended with an energy source in culture fluid from post-exponential-phase wild-type B. subtilis or Escherichia coli, good sporulation occurred. The spores produced retained the mutant genotype and were heat stable but lost refractility and heat stability several hours after their production.     

Studies on two isozymes of aconitase from Bacillus cereus T. III. Enzymatic properties.

The present communication describes a comparative study of some enzymatic properties of an early and a late aconitase (EC.4.2.1.3) present in $\text{Bacillus}\text{cereus}$ T cells of 5 and 12 hr culture age, respectively. The activity of both enzymes increased linearly with increase in enzyme concentration. They demonstrated similar pH *7.5) and temperature (30 C) optima, but differed in their activation energy and affinity for substrate. Late aconitase had higher activation energy (16,100 cal) as compared to early aconitase (9,200 cal). Early aconitase showed a Km value of 100 × 10^?4M for sodium citrate and 33.3 × 10^?4M for isocitrate. Late aconitase exhibited 5 to 7 times greater affinity for citrate and isocitrate yielding Km values 14 × 10^?4M and 7 × 10^?4M, respectively. On the basis of available evidence, it is suggested that early and late aconitase present in 5 and 12 hr aged cells of $\text{Bacillus}\text{cereus}$ T behave as isozymes, and may be designated as aconitase (EC.4.2.1.3) isozyme I and aconitase (EC.4.2.1.3) isozyme II, respectively. The significance of their plausible role during growth and sporulation has been discussed.     

Sporulation properties of cytochrome a-deficient mutants of Bacillus subtilis

Three classes of cytochrome a-deficient mutants of Bacillus subtilis have been found to be asporogenic or oligosporogenic. All three classes showed declines in adenosine 5'-triphosphate (ATP) concentrations during early sporulation, at a time when ATP levels in wild-type strains are constant. Class III mutants were found to be deficient in aconitase and isocitric dehydrogenase, and showed reduced maximum growth in nutrient sporulation medium. These mutants also suffered the most rapid decline in ATP concentration in early sporulation, and exhibited neither the biphasic oxygen consumption curve nor the increase in pH normally observed at the end of logarithmic growth in nutrient sporulation medium. Nicotinamide adenine dinucleotide oxidase activities of purified membrane preparations were approximately normal for mutants in all classes, except for two of the class II mutants and one class III mutant. Neither cytochrome a nor cytochrome c appears to be an obligatory intermediate in cyanide-sensitive nicotinamide adenine dinucleotide oxidation in B. subtilis.     

Induction of citric acid cycle enzymes during initiation of sporulation by guanine nucleotide deprivation

In Bacillus subtilis, conditions causing partial deprivation of guanine nucleotides initiated sporulation and caused the synthesis of citrate synthase, aconitase, and alpha-ketoglutarate dehydrogenase. Alpha-ketoglutarate dehydrogenase could also be induced by acetate, and the specific activity of this enzyme was elevated in mutants that had high intracellular acetyl coenzyme A concentrations because they lacked citrate synthase activity. After deprivation of guanine nucleotides, the intracellular concentration of acetyl coenzyme A also increased, which explained the induction of alpha-ketoglutarate dehydrogenase. Furthermore, the decreases in alpha-ketoglutarate and L-malate concentrations observed during this deprivation accounted for the observed increases in citrate synthase activity (which was repressed by alpha-ketoglutarate and malate) and aconitase activity (which was repressed by alpha-ketoglutarate).     

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells

Introduction

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.

Objectives

The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.

Methods

To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.

Results

Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.

Conclusion

Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

     

Influence of temperature on biomass, iron metabolism and some related bioindicators in tomato and watermelon plants

Tomato, Lycopersicon esculentum L. cv. RX-335, and watermelon plants, Citrullus lanatus [Thom.] Mansf. cv. F-90 were grown under controlled conditions at three different temperatures (10 degrees, 25 degrees and 35 degrees C) for 30 days. The aim of the experiment was to analyse the effect of the different temperatures on Fe uptake and distribution, as well as the behaviour of the main bioindicators of this element. Thus, we analysed the total and free Fe concentrations and H2O2 concentrations, as well as enzymatic activities of Fe-chelate reductase (FeCH-R), aconitase (Aco), guaiacol peroxidase (GPX), catalase (CAT), and Fe-superoxide dismutase (FeSOD), and the dry weight of the plants. The effect caused by each temperature varied according to the species of plant. Our results indicate that heat stress appears in tomato plants when grown at 35 degrees C (above the optimal temperature for growth), while in watermelon plants, which need more heat than do tomatoes, cold stress appears at 10 degrees C (below the optimal temperature for growth). Despite these differences between the two species, the results under conditions of thermal stress were the same: 1) decreased shoot weight, 2) reduced Fe uptake, 3) depressed activities of FeCH-R, Aco, GPX, CAT and 4) boosted SOD activity. In short, our results appear to indicate that, whether heat in tomato plants or cold stress in watermelon plants, Fe uptake was diminished, as were the enzymatic activities related to the levels of this micronutrient in the plant. The high FeSOD activity in these plants could be explained by a defensive response to heat or cold stress.     

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.     

Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.     

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.