José A. Hernández

Interaction of FurA from Anabaena sp. PCC 7120 with DNA: A Reducing Environment and the Presence of Mn2+ are Positive Effectors in the Binding to isiB and furA Promoters

The Fur (ferric uptake regulator) protein is a global regulator in most prokaryotes that controls a large number of genes. Fur is a classical repressor that uses ferrous iron as co-repressor and binds to specific DNA sequences (iron boxes) as a dimer. Three different genes coding for Fur homologues have been identified in Anabaena sp. PCC 7120. FurA controls the transcription of flavodoxin, the product of the isiB gene, and is moderately autoregulated. In this work, the promoter of the furA gene was defined and the FurA protected regions in the furA and isiB promoters were identified, showing that the binding sites for Anabaena FurA contain A/T-rich sequences with a variable arrangement compared to the conventional 19-base pair Fur consensus. The influence of different factors on the interaction between FurA and the promoters was evaluated in vitro. The affinity of FurA for the DNA targets was significantly affected by the redox status of this regulator and the presence of Mn2+. The optimal binding conditions were observed in the presence of both Mn2+ and DTT. Those results suggest that, in addition to iron availability, FurA–DNA interaction is modulated by redox conditions.

FurA modulates gene expression of alr3808, a DpsA homologue in Nostoc (Anabaena) sp. PCC7120.

The DNA‐binding protein from stationary phase (Dps) protein family plays an important role in protecting microorganisms from oxidative and nutritional stresses. In silico analysis of the promoter region of alr3808, a dpsA homologue from the cyanobacterium Nostoc sp. PCC7120 shows putative iron‐boxes with high homology with those recognized by FurA (ferric uptake regulator). Evidence for the modulation of dpsA by FurA was obtained using in vitro and in vivo approaches. SELEX linked to PCR was used to identify P dpsA as a FurA target. Concurrently, EMSA assays showed high affinity of FurA for the dpsA promoter region. DpsA expression analysis in an insertional mutant of the alr1690‐αfurA message (that exhibited an increased expression of FurA) showed a reduced synthesis of DpsA. These studies suggest that FurA plays a significant role in the regulation of the DpsA.

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.

Mutants of Anabaena sp. PCC 7120 lacking alr1690 and α-furA antisense RNA show a pleiotropic phenotype and altered photosynthetic machinery

Fur proteins are global regulators present in all prokaryotes. In Anabaena sp. PCC 7120 FurA controls iron uptake and modulates an important set of genes related primarily to photosynthesis, nitrogen metabolism and oxidative stress defense. Expression of furA is tuned by the cis-acting antisense alpha-furA RNA that is co-transcribed with the outer-membrane protein Alr1690. Disruption of the alpha-furA-alr1690 message produces the iron-deficient JAH3 mutant that lacks Alr1690 and shows enhanced expression of FurA. JAH3 cells present severe structural disorders related to the number, organization and density of photosynthetic membranes. Quantitative analysis of the fluorescence induction shows that the mutation affects the J-I and I-P phases and causes important alterations in the photosynthetic apparatus, leading to lower photosynthetic performance indexes. These results reveal that expression of the alpha-furA-alr1690 message is required for maintenance of a proper thylakoid arrangement, efficient regulation of iron uptake and optimal yield of the photosynthetic machinery.

Mesoscopic model and free energy landscape for protein-DNA binding sites: Analysis of cyanobacterial promoters

The identification of protein binding sites in promoter sequences is a key problem to understand and control regulation in biochemistry and biotechnological processes. We use a computational method to analyze promoters from a given genome. Our approach is based on a physical model at the mesoscopic level of protein-DNA interaction based on the influence of DNA local conformation on the dynamics of a general particle along the chain. Following the proposed model, the joined dynamics of the protein particle and the DNA portion of interest, only characterized by its base pair sequence, is simulated. The simulation output is analyzed by generating and analyzing the Free Energy Landscape of the system. In order to prove the capacity of prediction of our computational method we have analyzed nine promoters of Anabaena PCC 7120. We are able to identify the transcription starting site of each of the promoters as the most populated macrostate in the dynamics. The developed procedure allows also to characterize promoter macrostates in terms of thermo-statistical magnitudes (free energy and entropy), with valuable biological implications. Our results agree with independent previous experimental results. Thus, our methods appear as a powerful complementary tool for identifying protein binding sites in promoter sequences.

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.