An active photosynthetic electron transfer chain required for mcyD transcription and microcystin synthesis in Microcystis aeruginosa PCC7806.

In this study, quantitative real time RT-PCR has been used to monitor changes in the levels of transcripts encoding mcyD in Microcystis aeruginosa PCC7806 under oxidative agents and different conditions of light intensity. Microcystin content has also been determined in the same stressed cell aliquots. Our results corroborate the fact that changes in light intensities are able to induce mcyD gene transcription, but our data show that this is an early and short-term event. mcyD transcription requires an active photosynthetic electron transfer chain and the increased transcript level as a consequence of light is not related to oxidative stress. Indeed, oxidative stress leads to a general trend of a decrease of mcyD trancript. Microcystin amount found in the cells follows a tendency consistent with the mcyD transcript level. In summary, the data indicate that the synthesis of microcystin is dependent on photosynthesis, and also show that oxidative stress decreases the microcystin synthesis in toxigenic Microcystis.

Identification of three novel antisense RNAs in the fur locus from unicellular cyanobacteria.

The interplay between Fur (ferric uptake regulator) proteins and small, non-coding RNAs has been described as a key regulatory loop in several bacteria. In the filamentous cyanobacterium Anabaena sp. PCC 7120, a large dicistronic transcript encoding the putative membrane protein Alr1690 and an α-furA RNA is involved in the modulation of the global regulator FurA. In this work we report the existence of three novel antisense RNAs in cyanobacteria and show that a cis α-furA RNA is conserved in very different genomic contexts, namely in the unicellular cyanobacteria Microcystis aeruginosa PCC 7806 and Synechocystis sp. PCC 6803. Syα-fur RNA covers only part of the coding sequence of the fur orthologue sll0567, whose flanking genes encode two hypothetical proteins. Transcriptional analysis of fur and its adjacent genes in Microcystis unravels a highly compact organization of this locus involving overlapping transcripts. Maα-fur RNA spans the whole Mafur CDS and part of the flanking dnaJ and sufE sequences. In addition, Mafur seems to be part of a dicistronic operon encoding this regulator and an α-sufE RNA. These results allow new insights into the transcriptomes of two unicellular cyanobacteria and suggest that in M. aeruginosa PCC 7806, the α-fur and α-sufE RNAs might participate in a regulatory connection between the genes of the dnaJ-fur-sufE locus.

Microcystin-LR synthesis as response to nitrogen: transcriptional analysis of the mcyD gene in Microcystis aeruginosa PCC7806.

The influence of environmental factors on microcystin production by toxic cyanobacteria has been extensively studied. However, the effect of nitrogen on the synthesis of this toxin remains unclear because of the literature contradictory data. The aim of this work was to determine how nitrate affects the transcriptional response of mcyD gene and the microcystin-LR synthesis in Microcystis aeruginosa PCC 7806. For first time real time RT-PCR has been used to investigate the effect of nitrogen availability. Our results show that, under laboratory conditions, an excess of nitrate triggers Microcystis aeruginosa growth without increasing the synthesis of microcystin-LR per cell. The concentration of microcystin in the cultures correlates with mcyD gene expression, being both parameters independent of nitrate availability. Analysis of the bidirectional promoter mcy unravels that the transcription start points of mcyA and mcyD genes did not change under different nitrate regimes. The effect of nitrate inputs in the development of toxic blooms is primarily due to the increased growth rate and population, not to the induction of the mcy operon.

Iron availability affects mcyD expression and microcystin-LR synthesis in Microcystis aeruginosa PCC7806.

Microcystins are toxins produced by cyanobacteria that entail serious health and environmental problems. They are cyclic heptapeptides synthesized via a mixed polyketide synthase/non-ribosomal peptide synthetase system called microcystin synthetase. Environmental and nutritional factors that trigger microcystin synthesis are still debated and this work deals with the study of the influence of iron nutritional status on the microcystin synthesis. The results indicate that iron deficiency could be one of the inducing factors of the microcystin synthesis. For the first time, increased transcription of an essential mcy gene and correlative microcystin synthesis has been established. Real-time PCR analysis of mcyD, and microcystin-LR synthesis were studied on Microcystis aeruginosa PCC7806 grown in iron-replete and iron-deplete media. Iron starvation causes an increase of mcyD transcription, correlative to the increase of microcystin-LR levels. Four transcription start points were identified for mcyD and two for mcyA, and they are not changed as a consequence of iron deficiency.

Identification of a Ferric uptake regulator from Microcystis aeruginosa PCC7806.

Ferric uptake regulator (Fur) proteins are widely recognized as repressors that in many prokaryotes regulate a large number of genes involved in iron homeostasis and oxidative stress response. In our study, we were able to identify the complete sequence of the fur gene from Microcystis aeruginosa using inverse-polymerase chain reaction. DNA sequence analysis confirmed the presence of a 183 amino-acid open reading frame that showed high identity with Fur proteins reported for cyanobacteria. The recombinant Fur protein has been purified and electrophoretical mobility shift assays shown to be active. Mn2+ and dithiothreitol enable Fur to bind to its promoter, with dithiothreitol being more potent. The expression of Fur in Microcystis was induced about twofold in iron-deficient conditions.

Fur from Microcystis aeruginosa binds in vitro promoter regions of the microcystin biosynthesis gene cluster.

Promoter regions of the mcy operon from Microcystis aeruginosa PCC7806, which is responsible for microcystin synthesis in this organism, exhibit sequences that are similar to the sequences recognized by Fur (ferric uptake regulator). This DNA-binding protein is a sensor of iron availability and oxidative stress. In the presence of Fe(2+), a dimer of Fur binds the iron-boxes in their target genes, repressing their expression. When iron is absent the expression of those gene products is allowed. Here, we show that Fur from M. aeruginosa binds in vitro promoter regions of several mcy genes, which suggests that Fur might regulate, among other factors, microcystin synthesis. The binding affinity is increased by the presence of metal and DTT, suggesting a response to iron availability and redox status of the cell.

Expression of fur and its antisense α-fur from Microcystis aeruginosa PCC7806 as response to light and oxidative stress.

Ferric uptake regulation (Fur) proteins are prokaryotic transcriptional regulators that integrate signaling of iron metabolism and oxidative stress responses with several environmental stresses. In photosynthetic organisms, Fur proteins regulate many genes involved in photosynthesis, nitrogen metabolism and other key processes. Also, Fur triggers the expression of virulence factors in many bacterial pathogens, and Fur from Microcystis aeruginosa has been shown to bind promoter regions of the microcystin synthesis gene cluster. In this work, we studied transcriptional responses of fur genes under different light intensities and oxidative stress. An antisense of fur, the α-fur RNA, plays an important role in regulating fur expression under oxidative stress, affecting levels of Fur protein in cells. Importantly, an active photosynthetic electron chain is required for the expression of the fur gene.