The expression of ferredoxin-NADP+ reductase (FNR) from Anabaena sp. PCC 7119 in heterocysts and vegetative cells has been quantified. Specific reductase activity in heterocysts was approximately 10 times higher than in vegetative cells, corresponding to the increased FNR protein content. This was confirmed by immunoquantification of the FNR protein from whole filaments of Anabaena sp. PCC 7120 grown in media with and without combined nitrogen. Transcription of the petH gene was markedly enhanced in the absence of combined nitrogen. This suggests that the increased RNA level is mainly responsible for the up-regulation of FNR in heterocysts. As has been observed for nif genes, iron deficiency also increased transcription of petH. Characterization of the FNR purified from isolated heterocysts showed no detectable differences from the enzyme from vegetative cells. Although nitrogen stress was a key regulatory factor, localization of the petH gene in the genomic map of Anabaena PCC 7120 showed that this gene is not physically associated with the nif cluster.
In Anabaena sp. strain PCC 7120, vegetative cell ferredoxin synthesis under iron starvation was repressed 25-fold, whereas heterocyst ferredoxin synthesis decreased only 2.8-fold. Induction of flavodoxin under iron depletion was independent of the availability of combined nitrogen. Under iron stress but in the presence of combined nitrogen, fdxH and nijH genes were transcriptionally active; although excision of the 11-kb element seemed to be completed, nitrogenase activity and the fdH gene product were not detectable.
An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.
In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP(+) reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP(+), using thylakoids from vegetative cells.
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