Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

Summary In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

Contribution of organic acids to the acidification of the rhizosphere of maize seedlings

The participation of organic acids in the process of soil acidification was related to other H⁺ pumping processes. The ratio between efflux of organic acids and proton secretion of maize roots was determined with the use of a pH-stat combined with a collecting system for organic acids. Changes in the composition of carboxylic acids influenced by nitrogen supply were monitored by HPLC and via enzymatic conversion. The following substances were found to be secreted by maize roots: glycolate, glyoxylate, fumarate, 2-oxoglutarate and oxalate. Malate, however, could not be detected. There is no organic acid dominantly secreted by the roots, but changes are observed during aging which might result from deficiencies of nutrients e.g. P. Fertilization of N-deficient plants with urea leads to a significant change in the composition of acids secreted. In this case, oxalate was additionally detected with a concomitant increase in glyoxylate, indicating important changes in metabolism. Acidification of the rhizosphere is predominantly maintained by secretion of protons, not by efflux of organic acids, which contributed 0.2 to 0.3% to this process only. The role of organic acids in nutrient uptake is discussed.     

Diurnal periodicity of chalcone-synthase activity during the development of oat primary leaves

Chalcone-synthase (CHS) activity was followed during the development of primary leaves of oat (Avena sativa L.) seedlings grown under different illumination conditions. Continuous darkness and continuous light resulted in similar time courses of enzyme activity. The maximum of CHS activity in etiolated leaves was delayed by 1 d and reached about half the level of that of light-grown leaves. In seedlings grown under defined light-dark cycles a diurnal rhythm of CHS activity and its protein level was observed which followed the rhythm of CHS-mRNA translational activity (Knogge et al. 1986). This rhythm persisted in continuous light after a short-term pre-exposure to the light-dark cycle but not in continuous darkness.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonio lyase Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged (G.W., We 630/9-7; We 630/10-1). Thanks are given to Dr. St. Kellam (Department of Plant Microbiological Sciences, University of Canterbury, New Zealand) for correcting the English.     

Modified structure and kinetics of cytochrome-c oxidase in fibroblasts from patients with Leigh syndrome

In this study we compared the properties of cytochrome-c oxidase (COX) in cultured fibroblasts from two patients with Leigh Syndrome with COX from control fibroblasts. The fibroblasts from patients showed decreased growth reates and elevated lactate production. COX activity of patients fibroblasts was about 25% of control. Kinetic studies with isolated mitochondria showed a higher K_m for cytochrome c and a markedly reduced molecular turnover of COX from patients, indicating a different structure of the enzyme. A biphasic change of COX activity was obtained by titration of dodecylmaltoside solubilized mitochondria from control fibroblasts with increasing concentrations of anions. With patient mitochondria we found only the inhibiting phase of COX activity and, in contrast to control mitochondria, irreversible inhibition of COX activity by guanidinium chloride. ELISA titrations with monoclonal antibodies to subunit II, IV, Vab, VIac and VIIab indicated a normal amount of mitochondrial coded subunit II, but a reduced amound of nuclear coded subunits. The data indicate incompletely assembled nuclear coded subunits of COX from patient fibroblasts.     

Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5

Transgenic tomato plants expressing antisense RNA to a ripening-related cDNA clone (pTOM5) had yellow ripening fruit and pale coloured flowers. Carotenoid levels in fruit of these plants were reduced by up to 97%. In order to determine the step of carotenoid biosynthesis which was blocked, a cell-free system active in the synthesis of carotenoid intermediates was prepared. Incubations with radiolabelled carotenoid precursors led to the identification of the block between GGDP and phytoene. Analysis of carotenoids in different tissues of transgenic and control plants indicated that although ripe fruit and flower carotenoid levels were reduced in the modified plants, leaf carotenoid levels were not decreased. This implies that the pTOM5 gene product is not involved in carotenoid synthesis in the leaf.     

Influence of salt concentration and temperature on the fatty acid compositions of Ectothiorhodospira and other halophilic phototrophic purple bacteria

Influences of the salt concentration on the fatty acid composition of Ectothiorhodospira species and other phototrophic purple bacteria have been analysed. Major fatty acids in bacteria of the genera Rhodobacter, Rhodopseudomonas, Chromatium, and Ectothiorhodospira were straight chain saturated and monounsaturated C-16 and C-18 fatty acids. Salt-dependent responses of all investigated bacteria revealed relations to their salt optima. Minimum values of C-16 and saturated fatty acids and maximum values of C-18 and unsaturated fatty acids were found at or close to the salt optima. Responses of Ectothiorhodospira mobilis upon changes in salinity were nearly identical, whether cells were grown in batch culture or in continuous culture with identical dilution rates at all salt concentrations. With increasing temperature, the fatty acid composition of Ectothiorhodospira mobilis and Ectothiorhodospira halophila strains showed decreasing portions of C-18 and of unsaturated fatty acids, while the contents of C-16 and saturated fatty acids increased. The results are discussed with respect to bilayer stabilisation and membrane fluidity.Abbreviations PC phosphatidylcholine - PG phosphatidylglycerol - CL cardiolipin - PE phosphatidylethanolamine