Proteins belonging to the FUR (ferric uptake regulator) family are the cornerstone of metalloregulation in most prokaryotes. Although numerous reviews have been devoted to these proteins, these reports are mainly focused on the Fur paralog that gives name to the family. In the last years, the increasing knowledge on the other, less ubiquitous members of this family has evidenced their importance in bacterial metabolism. As the Fur paralog, the major regulator of iron homeostasis, Zur, Irr, BosR and PerR are tightly related to stress defenses and host-pathogen interaction being in many cases essential for virulence. Furthermore, the Nur and Mur paralogs largely contribute to control nickel and manganese homeostasis, which are cofactors of pivotal proteins for host colonization and bacterial redox homeostasis. The present review highlights the main features of FUR proteins that differ to the canonical Fur paralog either in the coregulatory metal, such as Zur, Nur and Mur, or in the action mechanism to control target genes, such as PerR, Irr and BosR.

Iron is an essential nutrient for most living organisms. Due to the low solubility of ferric iron at physiological pH, the transition from an anaerobic atmosphere to the actual oxidant environment caused a dramatical decrease of iron bioavailability. Therefore, most organisms had to adapt their lifestyle to survive under an iron-depleted environment. In cyanobacteria, the electron transport chains involved in photosynthesis and respiration, as well as the enzymes involved in nitrogen metabolism have a high content of iron. Hence, cyanobacterial iron requirements are much higher than those of heterotrophic organisms. In this chapter, we revise different strategies developed by this important group of microorganisms to cope with iron deficiency, as well as the regulatory networks involved in the homeostasis of this indispensable element.

Summary: FurC (PerR) from Anabaena sp. PCC7120 was previously described as a key transcriptional regulator involved in setting off the oxidative stress response. In the last years, the cross‐talk between oxidative stress, iron homeostasis and nitrogen metabolism is becoming more and more evident. In this work, the transcriptome of a furC ‐overexpressing strain was compared with that of a wild‐type strain under both standard and nitrogen‐deficiency conditions. The results showed that the overexpression of furC deregulates genes involved in several categories standing out photosynthesis, iron transport and nitrogen metabolism. The novel FurC‐direct targets included some regulatory elements that control heterocyst development ( hetZ and asr1734 ), genes directly involved in the heterocyst envelope formation ( devBCA and hepC ) and genes which participate in the nitrogen fixation process ( nifHDK and nifH2 , rbrA rubrerythrin and xisHI excisionase). Likewise, furC overexpression notably impacts the mRNA levels of patA encoding a key protein in the heterocyst pattern formation. The relevance of FurC in these processes is bringing out by the fact that the overexpression of furC impairs heterocyst development and cell growth under nitrogen step‐down conditions. In summary, this work reveals a new player in the complex regulatory network of heterocyst formation and nitrogen fixation.

Zinc is required for the activity of many enzymes and plays an essential role in gene regulation and redox homeostasis. In Anabaena ( Nostoc ) sp. PCC7120, the genes involved in zinc uptake and transport are controlled by the metalloregulator Zur (FurB). Comparative transcriptomics of a zur mutant (Δ zur ) with the parent strain unveiled unexpected links between zinc homeostasis and other metabolic pathways. A notable increase in the transcription of numerous desiccation tolerance‐related genes, including genes involved in the synthesis of trehalose and the transference of saccharide moieties, among many others, was detected. Biofilm formation analysis under static conditions revealed a reduced capacity of Δ zur filaments to form biofilms compared to the parent strain, and such capacity was enhanced when Zur was overexpressed. Furthermore, microscopy analysis revealed that zur expression is required for the correct formation of the envelope polysaccharide layer in the heterocyst, as Δ zur cells showed reduced staining with alcian blue compared to Anabaena sp. PCC7120. We suggest that Zur is an important regulator of the enzymes involved in the synthesis and transport of the envelope polysaccharide layer, influencing heterocyst development and biofilm formation, both relevant processes for cell division and interaction with substrates in its ecological niche.

FurC (PerR, Peroxide Response Regulator) fromAnabaenasp. PCC 7120 (also known asNostocsp. PCC 7120) is a master regulator engaged in the modulation of relevant processes including the response to oxidative stress, photosynthesis and nitrogen fixation. Previous differential gene expression analysis of afurC-overexpressing strain (EB2770FurC) allowed the inference of a putative FurC DNA-binding consensus sequence. In the present work, more data concerning the regulon of the FurC protein were obtained through the searching of the putative FurC-box in the wholeAnabaenasp. PCC 7120 genome. The total amount of novel FurC-DNA binding sites found in the promoter regions of genes with known function was validated by electrophoretic mobility shift assays (EMSA) identifying 22 new FurC targets. Some of these identified targets display relevant roles in nitrogen fixation (hetRandhgdC) and carbon assimilation processes (cmpR,glgP1andopcA), suggesting that FurC could be an additional player for the harmonization of carbon and nitrogen metabolisms. Moreover, differential gene expression of a selection of newly identified FurC targets was measured by Real Time RT-PCR in thefurC-overexpressing strain (EB2770FurC) comparing toAnabaenasp. PCC 7120 revealing that in most of these cases FurC could act as a transcriptional activator.

Microcystins are toxins produced by some strains of cyanobacteria. Several methods have been developed to allow the quantification of microcystins, which are mainly endotoxins. Among those methods, the protein phosphatase inhibition assay is a good candidate as a screening method because of its sensitivity, simplicity and specificity. In this work a method for intracellular microcystin extraction in field water samples and lab cyanobacterial cultures prior to their analysis by protein phosphatase inhibition assay has been optimized. Microcystin-LR and Microcystis aeruginosa PCC 7806 were used as reference microcystin and strain, respectively, in order to optimize the protocol. The protocol consists on filtering the sample through a nylon filter of 0.8 μm, filter extraction with methanol 80% 0.1% trifluoroacetic acid (TFA) 0.1% tween 20, extract centrifugation and supernatant dilution (1/20). The establishment of an extraction protocol was carried out determining the extraction volume, time of extraction and number of extractions. The advantages of the method developed in this work are basically its simplicity and avoiding the use of specific and expensive equipment.