An ancient bacterial zinc acquisition system identified from a cyanobacterial exoproteome.

Bacteria have developed fine-tuned responses to cope with potential zinc limitation. The Zur protein is a key player in coordinating this response in most species. Comparative proteomics conducted on the cyanobacterium Anabaena highlighted the more abundant proteins in a zur mutant compared to the wild type. Experimental evidence showed that the exoprotein ZepA mediates zinc uptake. Genomic context of the zepA gene and protein structure prediction provided additional insights on the regulation and putative function of ZepA homologs. Phylogenetic analysis suggests that ZepA represents a primordial system for zinc acquisition that has been conserved for billions of years in a handful of species from distant bacterial lineages. Furthermore, these results show that Zur may have been one of the first regulators of the FUR family to evolve, consistent with the scarcity of zinc in the ecosystems of the Archean eon.

Unbalancing Zur (FurB)-mediated homeostasis in Anabaena sp. PCC7120: Consequences on metal trafficking, heterocyst development and biofilm formation.

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 (Δzur) 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 Δzur 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 Δzur 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) from Anabaena sp. PCC7120: a versatile transcriptional regulator engaged in the regulatory network of heterocyst development and nitrogen fixation.

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.

Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog.

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.

Regulation by FurC in Anabaena Links the Oxidative Stress Response to Photosynthetic Metabolism.

The FUR (Ferric Uptake Regulator) family in Anabaena sp. PCC 7120 consists of three paralogs named FurA (Fur), FurB (Zur) and FurC (PerR). furC seems to be an essential gene in the filamentous nitrogen-fixing strain Anabaena sp. PCC 7120, suggesting that it plays a fundamental role in this organism. In order to better understand the functions of FurC in Anabaena, the phenotype of a derivative strain that overexpresses this regulator (EB2770FurC) has been characterized. The furC-overexpressing variant presented alterations in growth rate, morphology and ultrastructure, as well as higher sensitivity to peroxide than Anabaena sp. PCC 7120. Interestingly, the overexpression of furC led to reduced photosynthetic O2 evolution, increased respiratory activity, and had a significant influence in the composition and efficiency of both photosystems. Comparative transcriptional analyses, together with electrophoretic mobility shift assays allowed the identification of different genes directly controlled by FurC, and involved in processes not previously related to PerR proteins, such as the cell division gene ftsZ and the major thylakoid membrane protease ftsH. The rise in the transcription of ftsH in EB2770FurC cells correlated with reduced levels of the D1 protein, which is involved in the PSII repair cycle. Deregulation of the oxidative stress response in EB2770FurC cells led to the identification of novel FurC targets involved in the response to H2O2 through different mechanisms. These results, together with the effect of furC overexpression on the composition, stability and efficiency of the photosynthetic machinery of Anabaena, disclose novel links between PerR proteins, cell division and photosynthesis in filamentous cyanobacteria.

Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120.

FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB-DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB-DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.

Microcystin-LR Binds Iron, and Iron Promotes Self-Assembly.

The microcystin-producing Microcystis aeruginosa PCC 7806 and its close strain, the nonproducing Microcystis aeruginosa PCC 7005, grow similarly in the presence of 17 µM iron. Under severe iron deficient conditions (0.05 µM), the toxigenic strain grows slightly less than in iron-replete conditions, while the nonproducing microcystin strain is not able to grow. Isothermal titration calorimetry performed at cyanobacterial cytosol or meaningful environmental pHs values shows a microcystin-LR dissociaton constant for Fe2+ and Fe3+ of 2.4 µM. Using atomic force microscopy, 40% of microcystin-LR dimers were observed, and the presence of iron promoted its oligomerization up to six units. Microcystin-LR binds also Mo6+, Cu2+, and Mn2+. Polymeric microcystin binding iron may be related with a toxic cell colony advantage, providing enhanced iron bioavailability and perhaps affecting the structure of the gelatinous sheath. Inside cells, with microcystin implicated in the fitness of the photosynthetic machinery under stress conditions, the toxin would be involved in avoiding metal-dependent Fenton reactions when photooxidation causes disassembly of the iron-rich photosystems. Additionally, it could be hypothesized that polymerization-depolymerization dynamics may be an additional signal that could trigger changes (for example, in the binding of microcystin to proteins).

The FurA regulon in Anabaena sp. PCC 7120: in silico prediction and experimental validation of novel target genes.

In the filamentous cyanobacterium Anabaena sp. PCC 7120, the ferric uptake regulator FurA functions as a global transcriptional regulator. Despite several analyses have focused on elucidating the FurA-regulatory network, the number of target genes described for this essential transcription factor is limited to a handful of examples. In this article, we combine an in silico genome-wide predictive approach with experimental determinations to better define the FurA regulon. Predicted FurA-binding sites were identified upstream of 215 genes belonging to diverse functional categories including iron homeostasis, photosynthesis and respiration, heterocyst differentiation, oxidative stress defence and light-dependent signal transduction mechanisms, among others. The probabilistic model proved to be effective at discerning FurA boxes from non-cognate sequences, while subsequent electrophoretic mobility shift assay experiments confirmed the in vitro specific binding of FurA to at least 20 selected predicted targets. Gene-expression analyses further supported the dual role of FurA as transcriptional modulator that can act both as repressor and as activator. In either role, the in vitro affinity of the protein to its target sequences is strongly dependent on metal co-regulator and reducing conditions, suggesting that FurA couples in vivo iron homeostasis and the response to oxidative stress to major physiological processes in cyanobacteria.

The Pkn22 Ser/Thr kinase in Nostoc PCC 7120: role of FurA and NtcA regulators and transcript profiling under nitrogen starvation and oxidative stress.

BACKGROUND: The filamentous cyanobacterium Nostoc sp. strain PCC 7120 can fix N2 when combined nitrogen is not available. Furthermore, it has to cope with reactive oxygen species generated as byproducts of photosynthesis and respiration. We have previously demonstrated the synthesis of Ser/Thr kinase Pkn22 as an important survival response of Nostoc to oxidative damage. In this study we wished to investigate the possible involvement of this kinase in signalling peroxide stress and nitrogen deprivation. RESULTS: Quantitative RT-PCR experiments revealed that the pkn22 gene is induced in response to peroxide stress and to combined nitrogen starvation. Electrophoretic motility assays indicated that the pkn22 promoter is recognized by the global transcriptional regulators FurA and NtcA. Transcriptomic analysis comparing a pkn22-insertion mutant and the wild type strain indicated that this kinase regulates genes involved in important cellular functions such as photosynthesis, carbon metabolism and iron acquisition. Since metabolic changes may lead to oxidative stress, we investigated whether this is the case with nitrogen starvation. Our results rather invalidate this hypothesis thereby suggesting that the function of Pkn22 under nitrogen starvation is independent of its role in response to peroxide stress. CONCLUSIONS: Our analyses have permitted a more complete functional description of Ser/Thr kinase in Nostoc. We have decrypted the transcriptional regulation of the pkn22 gene, and analysed the whole set of genes under the control of this kinase in response to the two environmental changes often encountered by cyanobacteria in their natural habitat: oxidative stress and nitrogen deprivation.

Zur (FurB) is a key factor in the control of the oxidative stress response in Anabaena sp. PCC 7120.

Iron and zinc are necessary nutrients whose homeostasis is tightly controlled by members of the ferric uptake regulator (FUR) superfamily in the cyanobacterium Anabaena sp. PCC7120. Although the link between iron metabolism and oxidative stress management is well documented, little is known about the connection between zinc homeostasis and the oxidative stress response in cyanobacteria. Zinc homeostasis in Anabaena is controlled by Zur, also named FurB. When overexpressed in Escherichia coli, Zur (FurB) improved cell survival during oxidative stress. In order to investigate the possible correlation between Zur and the oxidative stress response in Anabaena, zur deletion and zur-overexpressing strains have been constructed, and the consequences of Zur imbalance evaluated. The lack of Zur increased sensitivity to hydrogen peroxide (H2 O2 ), whereas an excess of Zur enhanced oxidative stress resistance. Both mutants displayed pleiotropic phenotypes, including alterations on the filament surfaces observable by scanning electron microscopy, reduced content of endogenous H2 O2 and altered expression of sodA, catalases and several peroxiredoxins. Transcriptional and biochemical analyses unveiled that the appropriate level of Zur is required for proper control of the oxidative stress response and allowed us to identify major antioxidant enzymes as novel members of the Zur regulon.

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.

γ-Lindane Increases Microcystin Synthesis in Microcystis aeruginosa PCC7806.

HCH factories, and the waste dumpsites associated to its production, have become a global environmental concern, and their runoff could pollute ground and surface waters with high levels of the pollutant. In this study, the influence of lindane (γ-HCH) on microcystin production has been investigated in Microcystis aeruginosa PCC7806. This toxic cyanobacterium is highly tolerant to γ-lindane (20 mg/L), and produces more toxin (microcystin) in the presence of the pollutant. Microcystis degrades γ-lindane and presence of γ-lindane induces genes involved in its own degradation (nirA). RT-PCRsq has been used to monitor changes in levels of transcripts encoded by the mcy operon (mcyD, mcyH and mcyJ), responsible for the microcystin synthesis machinery, as well as other genes involved in its transcriptional regulation, such as ntcA and fur family members. The presence of lindane in the culture media induces mcyD expression, as well as ntcA gene transcription, while other genes, such as mcyH, (putative ABC transporter), are downregulated. The amount of microcystin found in the cells and the culture media is higher when M. aeruginosa is treated with γ-lindane than in control cells. The results suggest that in a lindane polluted environment, Microcystis toxic strains may enhance their microcystin synthesis.

The FUR (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators.

Control of metal homeostasis is essential for life in all kingdoms. In most prokaryotic organisms the FUR (ferric uptake regulator) family of transcriptional regulators is involved in the regulation of iron and zinc metabolism through control by Fur and Zur proteins. A third member of this family, the peroxide-stress response PerR, is present in most Gram-positives, establishing a tight functional interaction with the global regulator Fur. These proteins play a pivotal role for microbial survival under adverse conditions and in the expression of virulence in most pathogens. In this paper we present the current state of the art in the knowledge of the FUR family, including those members only present in more reduced numbers of bacteria, namely Mur, Nur and Irr. The huge amount of work done in the two last decades shows that FUR proteins present considerable diversity in their regulatory mechanisms and interesting structural differences. However, much work needs to be done to obtain a more complete picture of this family, especially in connection with the roles of some members as gas and redox sensors as well as to fully characterize their participation in bacterial adaptative responses.

FurC (PerR) contributes to the regulation of peptidoglycan remodeling and intercellular molecular transfer in the cyanobacterium Anabaena sp. strain PCC 7120.

Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment of Anabaena, the analysis of the extracellular metabolites and proteins of a furC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction of Anabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome of furC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins and amiC gene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, including amiC1 and amiC2, could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in the furC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments in furC-overexpressing cells, namely 1,6-anhydro-N-acetyl-ß-D-muramic acid (anhydroMurNAc) and its associated disaccharide (ß-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.IMPORTANCECyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions. Anabaena sp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator in Anabaena, which modulates the response to several stresses. Here, we show that furC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer.

Functional characterization of Fur from the strict anaerobe Clostridioides difficile provides insight into its redox-driven regulatory capacity.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H2O2 produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization.

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through ß-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

Responses of Anabaena sp. PCC7120 to lindane: Physiological effects and differential expression of potential lin genes.

Lindane (γ-HCH) is an organochlorine pesticide that causes huge environmental concerns worldwide due to its recalcitrance and toxicity. The use of the cyanobacterium Anabaena sp. PCC 7120 in aquatic lindane bioremediation has been suggested but information relative to this process is scarce. In the present work, data relative to the growth, pigment composition, photosynthetic/respiration rate, and oxidative stress response of Anabaena sp. PCC 7120 in the presence of lindane at its solubility limit in water are shown. In addition, lindane degradation experiments revealed almost a total disappearance of lindane in the supernatants of Anabaena sp. PCC 7120 culture after 6 days of incubation. The diminishing in lindane concentration was in concordance with an increase in the levels of trichlorobenzene inside the cells. Furthermore, to identify potential orthologs of the linA, linB, linC, linD, linE, and linR genes from Sphingomonas paucimobilis B90A in Anabaena sp. PCC 7120, a whole genome screening was performed allowing the identification of five putative lin orthologs (all1353 and all0193 putative orthologs of linB, all3836 putative orthologs of linC, and all0352 and alr0353 putative orthologs of linE and linR, respectively) which could be involved in the lindane degradation pathway. Differential expression analysis of these genes in the presence of lindane revealed strong upregulation of one of the potential lin genes of Anabaena sp. PCC 7120.

Expanding the FurC (PerR) regulon in Anabaena (Nostoc) sp. PCC 7120: Genome-wide identification of novel direct targets uncovers FurC participation in central carbon metabolism regulation.

FurC (PerR, Peroxide Response Regulator) from Anabaena sp. PCC 7120 (also known as Nostoc sp. 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 a furC-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 whole Anabaena sp. 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 (hetR and hgdC) and carbon assimilation processes (cmpR, glgP1 and opcA), 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 the furC-overexpressing strain (EB2770FurC) comparing to Anabaena sp. PCC 7120 revealing that in most of these cases FurC could act as a transcriptional activator.

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.

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.