Hans Georg Ruppel

Phylogenetic analyses of various euglenoid taxa (euglenozoa) based on 18s rdna sequence data

18S rRNA genes (SSU rDNA) of five newly sequenced species were used as molecular markers to infer phylogenetic relationships within the euglenoids. Two members of the order Euglenales (Lepocinclis ovata Playfair, Phacus similis Christen), two of the order Eutreptiales (Distigma proteus Ehrenberg,, D. curvata Pringsheim) and Gyropaigne lefévrei Bourelly et Georges of the order Rhabdomonadales were used in parsimony, maximum likelihood, and distance analyses. All trees derived from SSU rRNA data strongly supported the monophyletic origin of the Euglenozoa, with kinetoplastids as sister clade to the euglenoids and Petalomonas cantuscygni Cann et Pennick diverging at the base of the monophyletic euglenoid lineage. The data also supported the theory that phagotrophic euglenoids arose prior to osmotrophs and phototrophs. A lineage of Peranema trichophorum Ehrenberg and all sequenced Euglenales formed a sister clade to the osmotrophs. This suggests that the evolution of phototrophy within the euglenoids radiated from a single event.

ISOLATION AND CHARACTERIZATION OF PARAMYLON SYNTHASE FROM EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The aim of this study was to isolate and characterize the paramylon synthesizing enzyme from Euglena gracilis Klebs. A method for enzyme solubilization with high synthase activity using the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate is presented. Fractionated purification showed that the main enzyme activity was associated with the paramylon granula fraction, isolated from heterotrophically grown cells of E. gracilis. Further purification by sucrose density centrifugation resulted in a large enzyme complex with an apparent molar mass of 670 kDa (native). The complex remained active throughout the isolation procedures and produced beta‐1,3‐glucan in vitro. Two polypeptides of 37 and 54 kDa could be identified by photoaffinity labeling with [32P]‐UDP‐glucose as substrate after SDS‐PAGE.

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Abstract. Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635–2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion.

Interrelation between Cyanophycin Synthesis, L-Arginine Catabolism and Photosynthesis in the Cyanobacterium Synechocystis Sp. Strain PCC 6803

ʟ Ultrastructural and imm unocytochemical investigations gave evidence that cyanophycin (multi--arginyl-poly-ʟ-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on ʟ-arginine or ʟ-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of ʟ-arginine or ʟ-asparagine with nitrate only caused minor cyanophycin accum ulation. Growth of Synechocystis PCC 6803 on ʟ-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on ʟ-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. ʟ-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on ʟ-arginine as sole N-source. In both cells types the ʟ-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on ʟ-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of ʟ-arginine to the total nitrogen pool, and the intracellular ʟ-arginine concentration is greatly influenced by the activity of the ʟ-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, ʟ-arginine catabolism , and in addition photosynthesis in Synechocystis PCC 6803.

Isolation, partial characterization and localization of a dihydrolipoamide dehydrogenase from the cyanobacterium Synechocystis PCC 6803.

A dihydrolipoamide dehydrogenase (LPD; dihydrolipoamide:NAD oxidoreductase, EC 1.8.1.4.) activity has been detected in the cyanobacterium Synechocystis PCC 6803. The enzyme was isolated from the membraneous fraction after detergent solubilization and shown to be homogenous on the basis of SDS-PAGE and N-terminal sequencing. The isolated enzyme had a specific activity of 75 U (mg protein)(-1) and was shown to be a homodimer with an apparent molecular mass of 104 kDa for the dimer and 55 kDa for the subunits. The enzyme contains 1.75 mol noncovalently bound FAD (mol enzyme)(-1) suggesting that each subunit contains 1 mol FAD and that the FAD is fairly tightly associated with the enzyme. N-terminal sequencing gave a contiguous amino acid sequence of 17 residues and showed that the N-terminus of the LPD from Synechocystis PCC 6803 has significant homologies to other LPDs sequenced so far. Immunoblot experiments indicated that the enzyme is mainly present in the membrane fraction, and immunocytochemical investigations gave evidence that the LPD in Synechocystis PCC 6803 is located in the periplasma space between the cytoplasma membrane and the peptidoglycan layer. This is the first report on an extracellular, membrane-bound LPD in a cyanobacterium.

Immunocytological and chemical studies on the stromacentre-forming protein from Avena plastids

The stromacentre (SC), a particular structure in the plastids of Avena, was isolated from etioplasts of Avena sativa by density gradient centrifugation and then analyzed and compared with ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) from A. sativa, with pyrenoids of Chlorella vulgaris and with the “stromacentre” of Opuntia. Purified SC-elements consisted of protein subunits with a relative molecular weight of 63 kDa, different from the isolated RuBPCase of A. sativa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After peptide mapping, the proteolytic cleavage patterns of the 63-kDa protein were also found to be different from those of the RuBPCase. Antibodies against SC-elements, RuBPCase, and the large subunit of RuBPCase were produced. Ouchterlony double immunodiffusion tests did not give crossreactions between the SC-elements and RuBPCase or the large subunit of this enzyme. Immunogold labelling of ultrathin sections showed that antibodies against the SC-elements marked the stromacentre in Avena, but not the pyrenoids in Chlorella. Antibodies against the large subunit of RuBPCase, however, did not label the SC, but labelled the stroma of the plastids in Avena and the pyrenoids of Chlorella. In Opuntia, a comparable structure described as an SC was not labelled by any of the antisera. Immunoelectrophoretical investigations demonstrated a strong correlation between the presence of the 63-kDa protein and the occurrence of the SC in different Avena species with and without SC.

Occurrence of Secondary Carotenoids in PS I Complexes Isolated from Eremosphaera viridis De Bary (Chlorophyceae)

Abstract Photosystem I complexes of Eremosphaera viridis De Bary (Chlorophyceae, Chlorococcales) were isolated and partially characterized. In the isolated PS I complexes, peptides of 64-60, 26, 23, 20, 15, 11 and 8.5 kDa could be detected. When Eremosphaera was grown under regular conditions the pigment composition of the isolated PS I complexes was similar to that found in PS I complexes from other green algae. However, when Eremosphaera was grown under nitrogen deficient conditions, PS I complexes contained the secondary carotenoids canthaxanthin and traces of astaxanthin and echinenone in addition to β-carotene, violaxanthin and lutein. The results presented indicate that the secondary carotenoids are associated with the LHC I of PS I. To our knowledge this represents the first report about the association of secondary carotenoids with light harvesting pigment protein complexes of green algae.

Über nucleinsäuren in chloroplasten von Allium porrum und Antirrhinum majus

Abstract 1. 1. Chloroplasts have been isolated after the Behrens -method from adult leaves of Antirrhinum majus and from green leaf sections of Allium porrum. 2. 2. After the combined treatment of the chloroplasts with dodecylsulfate phenol a RNA fraction has been isolated from the aqueous phase. The RNA nucleotide composition is very similar to that of the ribosomes, obtained from the ribosome fraction of the same leaves. 3. 3. After the determination of the total nucleotide content of the plastids about 60% of the nucleotides remained in the phenolic phase. The base composition of the nucleotide fraction in the phenolic phase was different from that of the RNA, obtained from the aqueous phase. 4. 4. The total nucleotide content of the chloroplasts is about 1% of the dry weight of the chloroplasts. 5. 5. After the hydrolysis of the chloroplasts with concentrated formic acid no thymine could be found among the other bases. Consequently, there was no indication for the occurrence of DNA in chloroplasts from adult leaves of higher plants.

Cell Surface Glycoconjugates of Euglena gracilis (Euglenozoa): Modifications under Potassium and Magnesium Deficiency

Abstract Biochemical and ultrastructural examinations on the pellicle of autotrophically grown Euglena gracilis were carried out after three days under potassium and magnesium deficiency. Cell-surface changes were detected by lectin assay. Compared to cells grown in complete medium, deficient cells become larger in shape, accompanied by rising carbohydrate, chlorophyll and protein content, bind more and other lectin molecules: an increase of mainly galactose and N-acetylgalactosamine receptors was observed. Investigations with the mucilage stains alcian blue and ruthenium red indicated that mucilaginous material is released under deficient conditions, whereas the control cells show a strong precipitate of these stains well inside the cells beneath the pellicle.

PHENOLIC CONSTITUENTS OF GALACTITES TOMENTOSA (ASTERACEAE)

The Asteracea Galactites tomentosa. known as a plant causing allergic contact reactions, was investigated for new constituents in leaves and stems. Chlorogenic acid and 3,5-dicaffeoylquinic acid could be detected by HPLC in tissues and trichome preparations as well