Browsing by Author "Mbeunkui, Flaubert"

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    The effects of low nitrate levels on the freshwater cyanobacterium Synechocystis sp. strain PCC 6803: construction of a bioreporter assay and molecular characterization by transcriptome and proteome analysis
    (2003) Mbeunkui, Flaubert; Schmid, Rolf D. (Prof. Dr.)
    Since a few decades the so-called blue algal blooms came to public awareness and their occurrence was more frequently reported. These blooms stem from the mass proliferation of some cyanobacterial species and they occur both in the sea as well as in fresh water. The exact reasons for this phenomenon have not been finally clarified, but nutrient availability, limitation and excess, has a proven influence. Some bioavailability patterns of P, N, S and Fe strongly promote cyanobacterial proliferation. Due to the negative effects of these blooms, i.e. severe neuro- and hepato-toxin release, a deeper understanding, prediction and monitoring are desirable. It was the aim of this work to develop and use biotechnological tools to fulfill this requirement. In order to avoid the problems associated with water blooms and to understand the behavior of these microorganisms at low nutrient concentration, an assay as an early warning system for monitoring of water blooms formation at low nitrate concentration was developed; and the analysis of the physiological change at the level of the transcriptome and proteome was performed. Starting with a cyanobacterial reporter strain, Synechocystis sp. strain PCC 6803 harboring PnblA::luxAB-kmR in its genome, a luminescent reporter assay for the detection of nitrate bioavailability was constructed. In this construction, luxAB gene encoding the luciferase, from the luminescent bacterium Vibrio harveyi was fused with the kanamycin resistance gene (kmR), leading to a luxAB-kmR gene complex. This gene complex was then fused with the nblA1 gene of Synechocystis and inserted in its chromosomal DNA. This reporter strain was designated N1LuxKm. The expression of the luxAB gene was induced by nitrate deficiency and was quantified by the bioluminescence emission. By means of immobilization of N1LuxKm in microtiter plates, the sensor was storable for about one month and showed a dose-dependent luminescence signal in a concentration range of 4-100 µM nitrate after a sample incubation time of 10 h under continuous illumination (50 µE.m-2.s-1 of white light). Combined with ecological and physiological data this sensor could be used as an early warning system for water blooms. In order to further understand cellular processes resulting from nitrate starvation and their influence on cyanobacterial blooms, the proteome dynamics of Synechocystis sp. PCC 6803 was analyzed through 2D gel electrophoresis, MALDI-TOF/MS of trypsin-digested protein fragments and N-terminal amino acid sequencing. This simultaneous analysis of total gene expression at the level of protein represents one of the premiere strategies for studying biological systems and understanding the relationship between various expressed genes and gene products. This approach allowed the identification of four proteins which were up-regulated under nitrate starvation conditions, namely two isoforms of "the nitrogen regulatory protein P-II" encoded by glnB gene; "the carbon dioxide concentrating mechanism protein" and the plastocyanin encoded by ccmK and petE genes respectively. The information gained with proteomics was confirmed and extended by RNA expression analysis related to nitrate depletion using oligonucleotide sequences immobilized on microarrays. Total RNAs were reverse transcribed to fluorescent-labeled cDNAs, then hybridized to the immobilized probes. The difference in the abundance of the transcripts was recorded through the difference in the fluorescence emission. All the genes, which encoded the proteins, identified with proteomics were up-regulated. nblA gene used for the construction of the reporter strain and the ntcA gene (found in the literature to be induced under nitrate deficiency) were also up-regulated whereas those encoding some units of the phycobilisomes were constantly expressed.