Phylogenetic position and inter-relationships of the osmotrophic euglenids based on SSU rDNA data, with emphasis on the Rhabdomonadales (Euglenozoa).

In order to reconstruct the evolution of euglenid flagellates, euglenozoan SSU rDNA data have been used to investigate phylogenetic relationships with a focus on osmotrophic taxa and especially on the Rhabdomonadales. The dataset consisting of the SSU rDNAs of osmotrophic, phagotrophic and phototrophic taxa was used in parsimony, maximum-likelihood and distance analyses. Five genera make up the Rhabdomonadales, all of them osmotrophic: Gyropaigne, Menoidium, Parmidium, Rhabdomonas and Rhabdospira. According to our analyses they form a strongly supported monophyletic assemblage which is characterized by a low sequence divergence compared to the euglenids in general. Closest relatives are the members of the osmotrophic genus Distigma. All primary osmotrophic species constitute a larger monophyletic group with the phototrophic euglenids and the phagotroph Peranema trichophorum. The combination of three rhabdomonadalian species Rhabdomonas gibba, Rhabdomonas spiralis and Rhabdospira spiralis with nearly identical SSU rDNA sequences is strongly recommended. The phagotroph Petalomonas cantuscygni branches at the bottom of the euglenid subtree with significantly weaker support. The inter-relationship of the three distinct euglenozoan taxa (euglenids, kinetoplastids and diplonemids) could not be convincingly resolved by this study.

A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects.

The gene cphA encoding cyanophycin synthetase was interrupted in Anabaena variabilis ATCC 29413 by insertional mutagenesis. The mutant lacked cyanophycin granules and the polar nodules of heterocysts. The mutant grew as fast as the wild-type irrespective of the nitrogen source at low light intensity whereas growth on N(2) was somewhat reduced in high light. It is concluded that cyanophycin metabolism and polar nodules are not essential for aerobic N(2) fixation.

Interrelation between cyanophycin synthesis, L-arginine catabolism and photosynthesis in the cyanobacterium Synechocystis sp. strain PCC 6803.

Ultrastructural and immunocytochemical investigations gave evidence that cyanophycin (multi-L-arginyl-poly-L-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 L-arginine or L-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of L-arginine or L-asparagine with nitrate only caused minor cyanophycin accumulation. Growth of Synechocystis PCC 6803 on L-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 L-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. L-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 L-arginine as sole N-source. In both cells types the L-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on L-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 L-arginine to the total nitrogen pool, and the intracellular L-arginine concentration is greatly influenced by the activity of the L-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, L-arginine catabolism, and in addition photosynthesis in Synechocystis PCC 6803.

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.

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.

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.

A new quantitative assay for the determination of complement activity.

The suitability of the algal assay and the hemolysis method for the determination of complement activity was investigated with regard to reproducibility and quantification. It was shown that the day-to-day reproducibility is higher in the algal assay than in the hemolysis method in test tubes. The lysis of algal cells depends on serum concentrations. The extensive linear relationship between cytotoxic activity and serum concentration prevails at lysis rates between 20 and 80%. Therefore, it is often sufficient to measure the lysis rate at one serum concentration only. The algal assay enables the investigation of changes in complement activity, even if the changes are not expected to be drastic.

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.

The effects of tumor sera on cell shape and photosynthesis of Euglena gracilis.

Cells of Euglena gracilis treated with human sera show a marked change in cell shape: Fully elongated cells have nearly totally been transformed to disk-shaped cells. This serum-mediated contraction is followed by irreversible cytolysis. Disintegration of chloroplast membranes leads to decreased photosynthetic O2 evolution. Sera from humans suffering from tumors reveal higher lytic activities than sera from individuals not suffering from tumors. Heating sera at 56 degrees C for 10 min or addition of EDTA destroyed or inhibited, respectively, the lytic activities completely. Polysaccharides transformed in polyanions by sulphatisation like dextransulphates or heparin seem to protect Euglena against serum activities. The effects described for human sera are believed to display the role of the complement pathway in the cytolysis of Euglena gracilis.

[The formation of serotonin in Juglans regia L]. / Die Bildung von Serotonin in Juglans regia L.

The serotonin content of growing fruits and of germinating seeds of Juglans regia has been studied. In the embryo 0.4-0.6 mg serotonin/g FW were found; in contrast no serotonin was detectable in the fleshy pericarp and in the seed coat. Serotonin was also not detectable in leaves, stems and roots of the adult plant. Most of the serotonin found in the embryo is formed after abscission of the seeds. During the synthesis of serotonin there are no dramatic changes in the chemical composition of the seeds (Tables 3-5).The formation of serotonin could be followed in isolated cotyledons and under sterile conditions. This serotonin formation is stimulated by exogenous tryptophan (Fig. 2). That tryptophan acts as a precursor of serotonin could be demonstrated with labelled DL-tryptophan (benzene ring (14)C) (U). The possibility of stimulating serotonin formation in isolated cotyledons by the addition of tryptophan is limited to a certain stage of development and cannot be observed with material from fully matured seeds (Fig. 3).No serotonin was found in callus tissue and adventitious roots formed by isolated cotyledons; all the serotonin remained in the cotyledons. This was also the case in young seedlings, in which only the cotyledons showed the characteristic high serotonin content, whereas leaves, stems and roots contained no serotonin (Table 6).From these data we conclude that serotonin formation in the embryo of Juglans regia is not a special type of nitrogen storage but a way of ammonia detoxification in which ammonia from protein amino acid degradation is incorporated into serotonin via tryptophan.