Genetic regulation and physiology of cyanobacteria

 

 

Cyanobacteria are microorganisms that perform oxygenic photosynthesis and are able to colonize the most extreme habitats. Due to their

abundance and ubiquity, cyanobacteria play a basic role in the overall carbon and nitrogen cycle and they constitute the basis of the ocean trophic

chain. Some cyanobacterial species are able to fix atmospheric nitrogen and have been used in the fertilizer production. Likewise, strategies to use

cyanobacteria as biotechnological choice in the biodiesel production or in the removal of heavy metals from sewage are under development.

However, cyanobacteria can also be harmful. Due to the growing water reservoir eutrophization, it is becoming frequent the appearance of blooms

from these microorganisms in water intended for consumption or recreative uses. Several cyanobacterial species proliferating in these blooms can

produce toxins that have deleterious effects on the human health as well as in animals. Although at present the factors unleashing the cyanotoxin

synthesis are unknown, iron availability seems to be a determinant factor.

 

In our group, we investigate the regulation of iron metabolism in cyanobacteria and its relationship with nitrogen metabolism, oxidative stress and

cyanotoxin production.

 

 

 

FUNCTIONAL CHARACTERIZATION OF Fur (FERRIC UPTAKE REGULATOR) IN CYANOBACTERIA

 

Fur (ferric uptake regulator) proteins constitute a family of transcription global regulators present in most prokaryotic organisms. Originally, Fur was

identified as an iron homeostasis regulator that using iron as co-repressor bound to nucleotide sequences (iron boxes) placed on the promoters of

the controlled genes. At present, it is established that its mechanism implies higher complexity, this protein controlling a high number of genes

involved in intermediary metabolism, virulence factor production or the defense against different type of stresses. In pathogenic bacteria, Fur

activates the production of a number of toxins as response to iron deficiency. In cyanobacteria, Fur is involved in the microcystin synthesis

regulation, this one being the most ubiquitous cianotoxin responsible of serious sanitary and environmental problems.

 

On the other hand, Fur might modulate the ability of fixing nitrogen by filamentous cyanobacteria, the most used organisms in paddy fields

fertilization as well as in other cultures studied at the experimental level.

 

In order to puzzle out the role of Fur in cyanobacteria, as well as its action mechanism, our research focus on the next aspects:

 

 

Identification and biochemical characterization of the Fur proteins in cyanobacteria.

 

With this aim, the nitrogen-fixing filamentous strain Anabaena PCC 7120 and the toxic unicellular cyanobacterium Microcystis aeruginosa PCC 7806

have been used as models. Three Fur paralogues have been identified in both cyanobacteria that have been called FurA, FurB and FurC.

Anabaena sp. PCC 7120

Microcystis aeruginosa PCC 7806

 

 

Fur-DNA interaction

It has been proposed that Fur proteins work as classical repressors using Fe2+ as co-repressor to bind their DNA targets, also called "iron-boxes".

However, there are many other factors affecting que quality of Fur-DNA interaction, such as the redox state of cysteine residues, as well as the

participation of other efectors such as heme or the presence of other transcription factors.

 

In collaboration with Drs. Adrián Velázquez and Olga Abián (BIFI), two metal-binding sites have been identified in FurA from de Anabaena sp. PCC

7120 using isothermal titration calorimetry (ITC). One of them corresponds to the co-repressor binding site, while the second one could play a

structural role. However, unlike Fur from some heterotrophic bacteria that contain one structural Zn2+ per monomer, no Zn2+ could be detected in

FurA from Anabaena.

 

There are several efectors modulating FurA-DNA interaction, such as the redox state of the environment, the presence of heme, as well as other

transcription factors (see below). Binding of FurA and FurB to DNA is also modified in the presence of FurC paralogue.

 

Role of cysteines and histidines in Fur proteins

Fur proteins are identified by the presence of the motif HHXHXXCXXC. FurA from Anabaena sp. PCC 7120 contains 12 histidine residues, 5 of them in

the Fur signature, and 5 cysteines. Four of them, C101, C104, C141 and C144 are present as CXXC motifs in the C-terminal domain of the protein.

We are investigating the role of histidines and cysteines present in FurA in:

 

  • FurA-DNA interaction
  • Dimer formation and oligomerization of FurA
  • Heme-binding
  • Binding to co-repressor

 

Regulation of Fur proteins

Influence of iron and nitrogen availability, oxidative stress challenge and saline and osmotic stresses have been monitorized in Anabaena sp. PCC 7120

using reporter vectors containing the promoter regions of every Fur paralogue fused to GFP (collaboration with Prof. P. Wolk from MSU DOE, Plant

Research Laboratory and Department of Plant Biology, Michigan State University).

 

 

 

  • Two NtcA-binding sites have been identified in the PfurA. EMSA and footprinting assays show that NtcA binds to the promoter region
  • of FurA, likely activating its expression in the heterocyst.
  • Expression of Fur proteins in Anabaena is triggered by ROS (López-Gomollón et al. Biochemical Journal, 2010).
  • FurB recognizes in vitro the furA promoter, while FurC modulates the DNA-binding activity of the other Fur paralogues (Hernández et al. FEMS, 2004)
  • The presence of anti-furA RNAs seems to be a general feature of cyanobacterial genomes. Our group has identified the occurrence of cis-acting anti-furA RNAs in Anabaena PCC 7120, Synechocystis PCC 6803 and the toxic strain Microcystis aeruginosa PCC 7806. (Hernández et al. J.Mol.Biol. 2006, Sevilla et al., 2010). In Anabaena, α-furA is encoded in a dicistron with the membrane protein Alr1690. Delection of the alr1690-α-furA message has pleiotropic effects and causes severe alterations in the photosynthetic machinery (Hernández et al. J Plant Physiol. 2010).

 

 

Ultrastructure of Anabaena sp. PCC 7120 (up) and the alr1690-αfurA delection mutant (down).

 

 

 

Therefore,


Regulation of FurA takes place at every step of the flow of genetic information.

 

 

 


 

Regulatory networks operated by FurA

We have demonstrated the participation of FurA in the modulation of nitrogen metabolism through the cross-talk with the master regulator of

nitrogen control NtcA. (López-Gomollón et al., Microbiology, 2007).

 

Since furA is an essential gene for Anabaena under standard culture conditions, we have generated a FurA overexpressing strain to gain new insights

into the participation of FurA in cyanobacterial metabolic and regulatory networks.

 

Identification of the FurA regulon

We are involved in the identification of FurA-regulated genes in cyanobacteria with special emphasis in the analysis of genes involved in

cyanobacterial-specific processes such as photosynthesis, heterocysts metabolism or cyanotoxin synthesis. Computational, genetic, biochemical and

proteomic approaches are currently being used to identify the FurA regulon.