Emma Sevilla

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.

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 Microcystisaeruginosa 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 Microcystisaeruginosa 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.

Differential oxidative stress responses to pure Microcystin-LR and Microcystin-containing and non-containing cyanobacterial crude extracts on Caco-2 cells

Cyanobacterial blooms are a worldwide problem due to the production of cyanotoxins such as microcystins (MCs), causing serious water pollution and public health hazard to humans and livestock. Oxidative stress plays a significant role in MCs toxicity. In the present work the differential oxidative stress responses to pure MCs, and Microcystin-containing and non-containing cyanobacterial crude extracts on the human colon carcinoma cell line Caco-2 has been studied for the first time. After exposure, cells were collected and the antioxidant enzymes activities superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) were measured. Moreover, lipid peroxidation (LPO) induction, reactive oxygen species (ROS) and reduced glutathione (GSH) content were also analyzed. The oxidative stress biomarkers that experienced higher alterations were ROS, CAT, SOD and GR activities. The MC containing cyanobacterial extract showed the higher toxic effects, followed by pure MC-LR. The non-MC containing cyanobacterial extract showed limited effects mainly in SOD activity, GSH content, and GP and GR activities only at the highest concentration used. These results suggest that MC-LR is the responsible of the oxidative stress responses observed in Caco-2 cells, but other compounds contained in the cyanobacterial extracts can contribute to the toxic effects.

New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress

Fur (ferric uptake regulator) is a prokaryotic transcriptional regulator that controls a large number of genes mainly related to iron metabolism. Several Fur homologues with different physiological roles are frequently found in the same organism. The genome of the filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 codes for three different fur genes. FurA is an essential protein involved in iron homoeostasis that also modulates dinitrogen fixation. FurA interacts with haem, impairing its DNA-binding ability. To explore functional differences between Fur homologues in Anabaena, factors affecting their regulation, as well as some biochemical characteristics, have been investigated. Although incubation of FurB with haem severely hinders its ability to interact with DNA, binding of haem to FurC could not be detected. Oxidative stress enhances the transcription of the three fur genes, especially that of furB and furC. In addition, overexpression of FurA and FurB in Escherichia coli increases survival when the cells are challenged with H(2)O(2) or Methyl Viologen (paraquat), a superoxide-anion-generating reagent. When present in saturating concentrations, FurB exhibits unspecific DNA-binding activity and protects DNA from cleavage produced by hydroxyl radicals or DNaseI. On the basis of these results, we suggest that, whereas at low concentrations FurB would act as a member of the Fur family, at saturating concentrations FurB protects DNA, showing a DNA-protection-during-starvation-like behaviour.

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.

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.

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.

Exploring the interaction of microcystin-LR with proteins and DNA.

The physiological role of microcystin-LR is still under discussion, and since binding of microcystin-LR to proteins different from their main cellular targets was described, we have performed experiments in order to explore this interaction. A non-specific interaction of microcystin-LR with a variety of soluble proteins in vitro is disrupted when using organic solvents such as methanol. The isoelectric point of proteins is not affected by their interaction with microcystin-LR, even though the presence of microcystin-LR alters the pool of peptides obtained by tryptic digestions. Under the conditions tested, microcystin-LR does not exhibit affinity for DNA. Although it is unlikely that the non-specific binding of microcystin-LR to proteins has a physiological meaning, one must be aware of the fact that determinations of the toxin extracted from any biological sample may be affected by the presence of proteins in the extracts. Consequently, we strongly recommend use organic solvents and to lyophilise the tissue samples to guarantee the accessibility of these organic solvents to microcystin-LR when performing experiments with tissue or cell extracts.

Pivotal Role of Iron in the Regulation of Cyanobacterial Electron Transport.

Iron-containing metalloproteins are the main cornerstones for efficient electron transport in biological systems. The abundance and diversity of iron-dependent proteins in cyanobacteria makes those organisms highly dependent of this micronutrient. To cope with iron imbalance, cyanobacteria have developed a survey of adaptation strategies that are strongly related to the regulation of photosynthesis, nitrogen metabolism and other central electron transfer pathways. Furthermore, either in its ferrous form or as a component of the haem group, iron plays a crucial role as regulatory signalling molecule that directly or indirectly modulates the composition and efficiency of cyanobacterial redox reactions. We present here the major mechanism used by cyanobacteria to couple iron homeostasis to the regulation of electron transport, making special emphasis in processes specific in those organisms.

FurA from Anabaena PCC 7120: New insights on its regulation and the interaction with DNA

Fur (ferric uptake regulator) proteins are global regulatory proteins involved in the maintenance of iron homeostasis. They recognize specific DNA sequences denoted iron boxes. It is assumed that Fur proteins act as classical repressors. Under iron‐rich conditions, Fur dimers complexed with ferrous ions bind to iron boxes, preventing transcription. In addition to iron homeostasis, Fur proteins control the concerted response to oxidative and acidic stresses in heterotrophic prokaryotes. Our group studies the interaction between Fur proteins and target DNA sequences. Moreover, the regulation of FurA in the nitrogen‐fixing cyanobacterium Anabaena sp. PCC 7120, whose genome codes for three fur homologues has been investigated. We present an overview about the different factors involved in the regulation of FurA and analyze the parameters that influence FurA‐DNA interaction in the cyanobacterium Anabaena PCC 7120.