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Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165-170 nmol min-1 (mg protein)-1] were found in cells grown on acetate or beta-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50-80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent Km of isocitrate lyase for D,L-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 microM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the Vmax nor the D,L-isocitrate Km of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum.

Propionibacterium freudenreichii strain DSM 20271 was grown in a mineral medium containing 0.1% (w/v) yeast extract. Acetate was oxidized by growing cells with potassium hexacyanofen-ate as electron acceptot, which was oxidized by a three-electrode poised-potential system at a redox potential of +510 mV. Growth with acetate under these conditions followed linear rather than exponential kinetics, whereas growth with other substrates such as lactate under the same conditions was exponential. Cell-free extracts of P. freudenreichii cells grown with acetate contained all enzymes of the classical citric acid cycle except 2-oxoglutarate-oxidizing activity. No activity of anaplerotic reactions such as isocitrate lyase or malate synthase was found. Instead, moderate activities of glutamate decarboxylase, 4-aminobutyrate:2-oxoglutarate aminotransferase, and succinate semialdehyde dehydrogenase were detected. In short-term radiolabeling experiments with U-~4C-acetate, 4-aminobutyrate was identified as a major early intermediate in acetate oxidation by these cells. These findings allow the construction of a modified citric acid cycle that compensates the lack of 2-oxoglutarate dehydrogenase by a subcycle through glutamate, 4-aminobutyrate, and succinate semialdehyde. Lack of anaplerotic reactions explains subexponential growth kinetics during growth with acetate.

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min-1·mg-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min-1·mg-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a Rf value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg2+ and cysteine.

The physiology of Thermus aquaticus strain Z05 was investigated. Substantial evidence for gene and enzyme regulation in the central metabolism of this extreme thermophile was found. Two anaplerotic pathways were detected: (1) phosphoenolpyruvate carboxylase; (2) a glyoxylate shunt which proved to be essential for growth on pyruvate as well as acetate. The synthesis of isocitrate lyase and malate synthase were found to depend on a common control mechanism. Pronounced regulatory effects were observed on the activity of malic enzyme, pyruvate kinase and phosphoenolpyruvate carboxylase. The data could be fitted together into a picture of the metabolism during glycolysis and gluconeogenesis which shows how variations of enzyme levels and activities correlate with the apparent needs of the cell. Our results call attention to a peculiar metabolic analogy between T. aquaticus and Acinetobacter

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.

During growth of the yeasts Candida utilis and Hansenula polymorpha in mineral media containing ethanol as a carbon source and ammonium sulphate as a nitrogen source, the specific activities of isocitrate lyase and malate synthase were significantly increased when compared to glucose/ammonium sulphate-grown cells. In addition to the enhanced levels of these glyoxylate cycle enzymes, an increase in the specific activities of D-amino acid oxidase, amine oxidase or urate oxidase was observed when ammonium sulphate in the ethanol medium was replaced by D-alanine, methyl- or ethylamine, or uric acid. The subcellular localization of these enzymes was investigated by cell fractionation studies involving homogenization of protoplasts followed by differential and sucrose gradient centrifugation. In ethanol/ammonium sulphate-grown cells, isocitrate lyase and malate synthase cosedimented in a fraction together with catalase and part of the malate dehydrogenase. Electron microscopy revealed that this fraction consisted of microbodies which must be regarded as glyoxysomes. Two other glyoxylate cycle enzymes, citrate synthase and aconitase together with the other part of malate dehydrogenase, cosedimented with cytochrome c oxidase, a mitochondrial marker enzyme. In ethanol/D-alanine-, ethanol/methylamine- or ethanol/ethylamine-grown C. utilis and ethanol/uric acid-grown H. polymorpha, a peroxisomal enzyme, i.e. D-amino acid oxidase, amine oxidase or uric acid oxidase cosedimented with the glyoxysomal key enzymes. Cytochemical staining experiments demonstrated that in these variously-grown cells the activities of the oxidases were confined to the microbody-matrix; this also contained malate synthase activity. Transfer of C. utilis cells from glucose/ammonium sulphate- into ethanol/ammonium sulphate-containing media resulted in an increase in the original size and volume fraction of the microbodies. A further increase was observed when ammonium sulphate was replaced by methylamine. Essentially similar results were obtained with H. polymorpha cells. In neither of the two organisms indications of de novo synthesis of microbodies was obtained during transfer experiments. Invariably the microbodies developing in cells placed in the new environment originated from organelles already present in the inoculum cells by import of the substrate-specific enzyme protein(s). The combined results of biochemical, cytochemical and electron microscopical experiments showed that in the yeasts studied under appropriate conditions glyoxysomal and peroxisomal enzyme activities were localized in one and the same microbody, rather than in separate organelles.

The production of poly (A)+ RNA was determined over the cell-cycle in Euglena cultures division-synchronized by 14 h light periods alternating with 10 h dark periods. The amount of poly (A)+ RNA per culture aliquot was constant from the beginning until near the end of the light phase of the cell-cycle when it increased in a sharp burst after the time of DNA replication. Poly (A)+ RNA from all stages of the cell-cycle promoted protein synthesis with equal efficiency in a mRNA-dependent reticulocyte lysate translation system. Malate synthase mRNA was detected using malate synthase antibody to precipitate nascent malate synthase protein in the translation system, binding the IgG-antigen complex to Staphylococcus aureus, concentrating, eluting the IgG-antigen complex and analysing the products by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Malate synthase mRNA was absent from phototrophic cells and was only produced in darkened cultures following the addition of acetate.

Malate synthase, one of the key enzymes in the glyoxylate cycle, was purified from peroxisomes of alkane-grown yeast, Candida tropicalis. The enzyme was mainly localized in the matrix of peroxisomes, judging from subcellular fractionation followed by exposure of the organelles to hypotonic conditions. The molecular mass of this peroxisomal malate synthase was determined to be 250,000 daltons by gel filtration on a Sepharose 6B column as well as by ultracentrifugation. On sodium dodecylsulfate/polyacrylamide slab-gel electrophoresis, the molecular mass of the subunit of the enzyme was demonstrated to be 61,000 daltons. These results revealed that the native form of this enzyme was homo-tetrameric. Peroxisomal malate synthase showed the optimal activity pH at 8.0 and absolutely required Mg2+ for enzymatic activity. The Km values for Mg2+, acetyl-CoA and glyoxylate were 4.7 mM, 80 microM and 1.0 mM, respectively.

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.

When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found. The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway. Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: 1. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light.