M. Teresa Bes

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.

Selective oxidation: stabilisation by multipoint attachment of ferredoxin NADP+ reductase, an interesting cofactor recycling enzyme

Ferredoxin-NADP+ reductase (FNR, EC 1.18.1.2) is an enzyme that is able to catalyse the oxidation of NADPH + H+. A strategy to prepare industrial derivatives of this enzyme for use as an ‘NADP’ regenerating enzyme in oxidizing reactions is presented. The strategy is based on a strictly controlled process of multipoint covalent attachment between the enzyme, via its amino groups, and a pre-existing solid activated with a monolayer of simple aldehyde groups linked by a space-arm of moderate length to the surface of the support. Controlling the variables which may have an influence in the multi-interaction process, we have prepared a number of enzyme derivatives with very different activity/stability properties. The ‘optimum derivative’ was found to be much more stable than its corresponding soluble enzyme under all the denaturation conditions assayed (high temperatures, extreme pH, organic solvents, etc.). Because of the excellent properties of this enzyme derivative, we can regenerate NADP+ by using molecular oxygen directly as the oxidizing agent under a wide range of conditions. Coupling this oxidative system to other NADP-dependent redox enzymes, we should be able to develop a very specific and selective oxidative procedure under very mild oxidizing conditions.

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.

Amperometric enzyme electrode for NADP+ based on a ferrodoxin-NADP+ reductase and viologen-modified glassy carbon electrode

Abstract A mediated amperometric enzyme electrode sensitive to NADP+ was developed by co-immobilization of ferrodoxin-NADP+ reductase and viologen amino derivatives of different chain length (C3, C6 and C10) on the surface of a glassy carbon electrode. Glutaraldehyde cross-linking in the presence of bovine serum albumin was used for immobilization. Communication between the redox centre of the enzyme and the electrode through the immobilized mediator was proved, and a fast electrical response in the presence of NADP+ was obtained. The best results were achieved with a medium-sized viologen. The sensitivity and linear response range depended on the polymer thickness and the immobilization procedure affected the stability of the modified electrode.

FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120

Knowledge on the regulatory mechanisms controlling iron homeostasis in cyanobacteria is limited. In Anabaena sp. PCC 7120, the ferric uptake regulator FurA is a constitutive and essential protein whose expression is induced under iron deprivation. Our previous analyses have shown that this protein acts as a global transcriptional regulator, controlling the expression of several genes belonging to different functional categories, including schT, a gene coding for a TonB-dependent schizokinen transporter. In the present study we analysed the impact of FurA overexpression and iron availability on the transcriptional modulation of a broad range of Anabaena iron uptake, transport, storage and cellular iron utilization mechanisms, including enzymes involved in siderophore biosynthesis, TonB-dependent siderophore outer membrane transporters, siderophore periplasmic binding proteins, ABC inner membrane permeases, ferritin Dps family proteins, and enzymes involved in tetrapyrrole biosynthesis. By combining reverse transcription-PCR analyses, electrophoretic mobility shift assays and DNase I footprinting experiments, we defined a variety of novel direct iron-dependent transcriptional targets of this metalloregulator, including genes encoding at least five enzymes involved in the tetrapyrrole biosynthesis pathway. The results unravel the role of FurA as the master regulator of iron homeostasis in Anabaena sp. PCC 7120, providing new insights into the Fur regulons in cyanobacteria.

Unravelling the regulatory function of FurA in Anabaena sp. PCC 7120 through 2-D DIGE proteomic analysis

The Anabaena sp. PCC 7120 ferric uptake regulator FurA controls iron homeostasis and appears implicated in a broad regulatory network, whereas failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we comparatively analyzed the proteomes of a furA-overexpressing strain and its parental wild-type in conjunction with subsequent semi-quantitative RT-PCR and electrophoretic mobility shift assays, in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approach drove us to find 10 novel direct targets belonging to different functional categories including photosynthesis, energy metabolism, oxidative stress defences, redox regulation, signal transduction mechanisms, DNA replication, thiamine biosynthesis and heterocyst differentiation. Two peroxiredoxins and the thioredoxin reductase exhibited the most significant changes in both mRNA level and protein abundance under a FurA overexpression background, indicating a connection between iron metabolism, redox signalling and oxidative stress defences. A FurA box consensus sequence was ill-defined. The results suggest that particular DNA structures rather than a defined sequence govern FurA regulation of its target genes. Overall, the results provide new insights into the FurA regulon in Anabaena sp. PCC 7120.

2-oxoglutarate enhances NtcA binding activity to promoter regions of the microcystin synthesis gene cluster

The binding affinity of NtcA towards promoter regions of the microcystin gene cluster from Microcystis aeruginosa PCC 7806 has been analyzed by band‐shift assay (EMSA). The key nitrogen transcriptional regulator exhibits affinity for two fragments of the bidirectional mcyDA promoter, as well as for promoter regions of mcyE and mcyH. The presence of 2‐oxoglutarate increased by 2.5 fold the affinity of NtcA for the mcyA promoter region. The 2‐oxoglutarate effect peaked at 0.8 mM, a physiological concentration for this compound under nitrogen‐limiting conditions. The results suggest that the 2‐oxoglutarate level, as a signal of the C to N balance of the cells, regulates the microcystin gene cluster.

Overexpression of FurA in Anabaena sp. PCC 7120 reveals new targets for this regulator involved in photosynthesis, iron uptake and cellular morphology.

Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for in vitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator in vivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 7120.

Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120

Heme is an iron‐containing cofactor that aside from serving as the active group of essential proteins is a key element in the control of many molecular and cellular processes. In prokaryotes, the family of Fur (ferric uptake regulator) proteins governs processes essential for the survival of microorganims such as the iron homeostasis. We show that purified recombinant FurA from Anabaena sp. PCC 7120 interacts strongly with heme in the micromolar range and this interaction affects the in vitro ability of FurA to bind DNA, inhibiting that process in a concentration‐dependent fashion. Our results provide the first evidence of the possible involvement of heme in the regulatory function of cyanobacterial Fur.