Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose

Diauxic growth was observed in rice (Oryza sativa L.) suspension cells growing on acetate (10 mM) and glucose (10 mM). Cells used acetate during the first growth phase and the acetate level in the medium was rapidly decreased, whereas the level of glucose remained essentially unchanged. After acetate was depleted from the medium, cells started to use glucose, forming the second growth phase. It appears that uptake of [14C]glucose was repressed during the first growth phase and became active during the second growth phase. In contrast, uptake of [14C]acetate occurred actively throughout the diauxic growth. By further demonstrating the specific induction of isocitrate lyase (EC 4.1.3.1), a glyoxylate cycle enzyme, and hexokinase (EC 2.7.1.1), a glycolysis enzyme, during the first and second growth phases, respectively, it was clearly shown that rice cells use acetate first and do not use both carbon sources simultaneously. This kind of diauxic growth pattern has been observed in bacteria. To our knowledege, this study is the first report demonstrating the presence of diauxic growth in plant cells.     

Evidence for an operative glyoxylate cycle in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Both key enzymes for the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), were purified and characterized from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Whereas the former enzyme was copurified with the aconitase, the latter enzyme could be enriched to apparent homogeneity. Amino acid sequencing of three internal peptides of the isocitrate lyase revealed the presence of highly conserved residues. With respect to cofactor requirement and quarternary structure the crenarchaeal malate synthase might represent a novel type of this enzyme family. High activities of both glyoxylate cycle enzymes could already be detected in extracts of glucose grown cells and both increased about two-fold in extracts of acetate grown cells.     

Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates.

Alanine: glyoxylate aminotransferase (EC 2.6.1.44), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized. The enzyme had Mr about 80 000, with two identical subunits. It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor. The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively. The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively). The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells. When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period. Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.     

Glyoxylate cycle in Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was grown in media containing acetate, glutamate, and peptone as carbon sources. The component enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, were present under these conditions throughout the growth cycles. Highest specific activities for each enzyme were found in media with acetate as the carbon source. In an enriched peptone medium containing glucose, neither activity was detected until glucose was exhausted from the medium. Treatment of acetate-grown cells with glucose resulted in a rapid decline in the specific activities of both enzymes. The importance of this cycle in acetate-grown cells was indicated by the ability of itaconic acid (100 mM) to inhibit the growth of M. racemosus in acetate but not glutamate media. Itaconate was also shown to be a potent inhibitor of isocitrate lyase activity in vitro.     

Serine: glyoxylate, alanine:glyoxylate, and glutamate:glyoxylate aminotransferase reactions in peroxisomes from spinach leaves

Two different aminotransferases, that have glyoxylate as the amino acceptor, have specific activities of 1 to 2 mumol . min-1 . mg of protein-1 in the isolated peroxisomal fraction from spinach leaves. Their properties were evaluated after separation on a hydroxylapatite column. Both enzymes had a Km for glyoxylate of 0.15 mM and an amino acid Km of 2 to 3 mM. Reactions proceeded by a Ping Pong Bi Bi mechanism. Serine:glyoxylate aminotransferase was relatively specific for both substrates and could only be slightly reversed with 100 mM glycine, although the Ki of glycine was 33 mM. The glutamate:glyoxylate amino-transferase protein was equally active in catalyzing an alanine:glyoxylate aminotransferase reaction, but the reverse reactions with 100 mM glycine were hardly measureable, although the Ki (glycine) was 8.7 mM. Protection against hydroxylamine inhibition from reaction with pyridoxal phosphate was used to investigate the specificity of amino acid binding. Substrate amino acids protected at about the same concentration as their Km, while glycine protected at its Ki concentration. Thus, the nearly irreversible catalysis with glycine is not due to a failure to bind glycine. The significance of a peroxisomal alanine:glyoxylate aminotransferase activity has not been incorporated into schemes for the oxidative photosynthetic carbon cycle.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

Studies on the Respiratory Physiology of Euglena gracilis Cultured on Acetate or Glucose

SYNOPSIS. The glyoxylate cycle operates at a high level in Euglena gracilis when acetate is the only carbon source, and at a low level when glucose is the only carbon source, as indicated by activities of malate synthase. Altho glucose causes a moderate repression of some of the enzymes of the glyoxylate cycle, it neither represses nor inhibits malate synthase. The specific activity of the malic enzyme was about 5-fold greater in acetate-grown Euglena than in glucose-grown cells, but the absolute rate of CO₂ fixation was about twice as great in cells grown on glucose. The respiratory quotient was unity regardless of substrate.     

Effect of different carbon sources on the regulation of carbohydrate metabolism inSaccharomyces cerevisiae

The carbohydrate metabolism ofSaccharomyces cerevisiae is strongly influenced by the concentration and the nature of the carbon source. As long as glucose is present in the growth medium, the cells possess a predominantly glycolytic pathway of degradation and low levels of α-glucosidase and of those enzymes of the citric-acid cycle, the respiratory chain, and the glyoxylate cycle, which are localized in the mitochondria. After the depletion of glucose the level of these enzymes rises considerably. As long as the carbon source can be demonstrated in the medium, maltose-grown cells have a greater oxidative activity and a higher level of these enzymes than glucose-grown cells, unlike glucose-grown cells they easily adapt to ethanol and acetate. Catabolite repression is suggested as an important factor in the regulation of synthesis of enzymes of the citric-acid cycle, the glyoxylate cycle and the respiratory chain. There is an obvious correlation between the regulation of α-glucosidase and of the enzymes of oxidative carbohydrate metabolism.     

Evidence for the presence of the glyoxylate cycle in Chloroflexus

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present in cell-free extracts of the phototrophic, green, thermophilic bacterium Chloroflexus aurantiacus grown with acetate as the sole organic carbon source.The optimum temperature of these enzymes was 40° C, and their specific activities were high enough to account for the observed growth rate. Lower levels of the enzymes were found in extracts from cells grown on a complete medium.Itaconate was shown to inhibit isocitrate lyase from C. aurantiacus 96% at a concentration of 0.25 mM and also had a profound effect on the growth of the organism on acetate, 0.25 mM inhibiting completely. Itaconate also inhibited the growth when added to the complex medium, but in this case much higher concentrations were required.     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Changes in Activity of Glyoxylate Cycle Enzymes During Myxospore Development in Myxococcus xanthus

Activities of the glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) were assayed in extracts prepared at different stages of myxospore formation in liquid cultures of Myxococcus xanthus. Activities of both enzymes attained peak values during conversion of rods to spheres. Isocitrate lyase activity decreased after reaching its peak value. Malate synthase activity also declined but at a much slower rate. The loss of isocitrate lyase activity could be prevented by the addition of chloramphenicol to cultures early in myxospore formation (during the initial rise in enzyme activity), but not by such addition at later stages of myxospore formation. The increase in glyoxylate cycle enzymes was not observed in a mutant unable to form myxospores in liquid culture under conditions suitable for morphological conversion of the wild type, or in wild-type cells incubated in the absence of an inducer for myxospore formation. It is concluded that the changes in the glyoxylate cycle enzymes represent regulatory phenomena associated with the development of the myxospore.     

Fermentation characteristics of Fusariumoxysporum grown on acetate

In this study, the growth characteristics of Fusariumoxysporum were evaluated in minimal medium using acetate or different mixtures of acetate and glucose as carbon source. The minimum inhibitory concentration (MIC) of acetic acid that F.oxysporum cells could tolerate was 0.8%w/v while glucose was consumed preferentially to acetate. The activity of isocitrate lyase was high when cells were grown on acetate and acetate plus glucose indicating an activation of the glyoxylate cycle. Investigation of the metabolic fingerprinting and footprinting revealed higher levels of intracellular and extracellular TCA cycle intermediates when F.oxysporum cells were grown on mixtures of acetate and glucose compared to growth on only glucose. Our data support the hypothesis that a higher flux through TCA cycle during acetate consumption could significantly increase the pool of NADH, resulting in the activation of succinate-propionate pathway which consumes reducing power (NADH) via conversion of succinate to propionyl-CoA and produce propionate.     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

Constitutive uptake and degradation of fatty acids by Yersinia pestis.

Yersinia pestis was found to utilize palmitic acid as a primary carbon and energy source. No inhibition of growth by palmitic acid was observed. Comparison of palmitic acid uptake by cells pregrown either with or without palmitic acid demonstrated that fatty acid uptake was constitutive. High basal levels of two enzymes of beta-oxidation, beta-hydroxyacyl-coenzyme A dehydrogenase and thiolase, and the two enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, were found in cells grown in defined medium with glucose. Elevated levels of all four enzymes were found when cells were grown with acetate as a primary carbon and energy source, and even higher levels were observed when palmitic acid was provided as a primary carbon and energy source. High-pressure liquid chromatography was used to demonstrate that, in the presence of glucose, uniformly labeled [14C]palmitic acid was converted to intermediates of the tricarboxylic acid cycle and glyoxylate shunt. Pregrowth with palmitic acid was not required for this conversion. Strains lacking the 6- or the 47-megadalton plasmid did not take up [3H]palmitic acid but did possess levels of enzyme activity comparable to those observed in the wild-type strain.