RESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Clostridioides difficile , Regulación Bacteriana de la Expresión Génica , Hierro , Oxidación-Reducción , Regiones Promotoras Genéticas , Proteínas Represoras , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Clostridioides difficile/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Hierro/metabolismo , Regiones Promotoras Genéticas/genética , Estrés Oxidativo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacologíaRESUMEN
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.
Asunto(s)
Anabaena , Nostoc , Regulón/genética , Nostoc/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Transcripción/genética , Anabaena/genética , Anabaena/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Fruit-tree rootstock selection is a challenge under a scenario of growing environmental stresses in which the soil and climate are greatly affected. Salinization is an increasing global process that severely affects soil fertility. The selection of rootstocks with the ability to tolerate salt stress is essential. Excised root cultures may be an excellent experimental approach to study stress physiology and a predictive tool to assess possible tolerance. In this study, we show how protein changes in response to salt stress evaluated in excised root cultures of Prunus cerasus (moderate salt-sensitive cultivar) could be representative of these changes in the roots of whole plants. The 2D electrophoresis of root extracts and subsequent spot identification by MALDI-TOF/TOF-MS show 16 relevant proteins differentially expressed in roots as a response to 60 mM NaCl. Cytoplasmic isozyme fructose 1,6-bisphosphate aldolase shows relevant changes in its relative presence of isoforms as a response to saline stress, while the total level of enzymes remains similar. Ferredoxin-NADP+ reductase increases as a response to salinity, even though the measured activity is not significantly different. The observed changes are congruent with previous proteomic studies on the roots of whole plants that are involved in protection mechanisms against salt stress.
RESUMEN
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.
Asunto(s)
Anabaena , Fijación del Nitrógeno , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Fijación del Nitrógeno/genéticaRESUMEN
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.
Asunto(s)
Bacterias , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas , Interacciones Huésped-Patógeno , Hierro/metabolismo , Proteínas Represoras , Bacterias/genética , Bacterias/metabolismo , Bacterias/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
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.
Asunto(s)
Anabaena/metabolismo , Anabaena/fisiología , Proteínas Bacterianas/metabolismo , Fotosíntesis/fisiología , Anabaena/genética , Proteínas Bacterianas/genética , División Celular/fisiología , Estrés Oxidativo/fisiología , Fotosíntesis/genéticaRESUMEN
In the nitrogen-fixing heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, the ferric uptake regulator FurA plays a global regulatory role. Failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we developed a selectively regulated furA expression system by the replacement of furA promoter in the Anabaena sp. chromosomes with the Co2+/Zn2+ inducible coaT promoter from Synechocystis sp. PCC 6803. By removing Co2+ and Zn2+ from the medium and shutting off furA expression, we showed that FurA was absolutely required for cyanobacterial growth. RNA-seq based comparative transcriptome analyses of the furA-turning off strain and its parental wild-type in conjunction with subsequent electrophoretic mobility shift assays and semi-quantitative RT-PCR were carried out in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approaches led us to identify 15 novel direct iron-dependent transcriptional targets belonging to different functional categories including detoxification and defences against oxidative stress, phycobilisome degradation, chlorophyll catabolism and programmed cell death, light sensing and response, heterocyst differentiation, exopolysaccharide biosynthesis, among others. Our analyses evidence novel interactions in the complex regulatory network orchestrated by FurA in cyanobacteria.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Anabaena/citología , Anabaena/efectos de los fármacos , Anabaena/genética , Proteínas Bacterianas/genética , Proliferación Celular/efectos de los fármacos , Cobalto/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Fenotipo , Transcriptoma/efectos de los fármacos , Zinc/farmacologíaRESUMEN
The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase.
Asunto(s)
Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/metabolismo , Mitocondrias/enzimología , Secuencia de Aminoácidos , Factor Inductor de la Apoptosis/genética , Dominio Catalítico , Secuencia Conservada , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , NAD/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
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.
Asunto(s)
Anabaena/metabolismo , Anabaena/fisiología , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/fisiología , Anabaena/genética , Catalasa/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Oxidación-Reducción , Estrés Oxidativo/genética , Peroxirredoxinas/metabolismo , Regulón , Superóxido Dismutasa/metabolismo , Zinc/metabolismoRESUMEN
UNLABELLED: Cyanobacterial FurA works as a global regulator linking iron homeostasis to photosynthetic metabolism and the responses to different environmental stresses. Additionally, FurA modulates several genes involved in redox homeostasis and fulfills the characteristics of a heme-sensor protein whose interaction with this cofactor negatively affects its DNA binding ability. FurA from Anabaena PCC 7120 contains five cysteine residues, four of them arranged in two redox CXXC motifs. AIMS: Our goals were to analyze in depth the putative contribution of these CXXC motifs in the redox properties of FurA and to identify potential interacting partners of this regulator. RESULTS: Insulin reduction assays unravel that FurA exhibits disulfide reductase activity. Simultaneous presence of both CXXC signatures greatly enhances the reduction rate, although the redox motif containing Cys(101) and Cys(104) seems a major contributor to this activity. Disulfide reductase activity was not detected in other ferric uptake regulator (Fur) proteins isolated from heterotrophic bacteria. In vivo, FurA presents different redox states involving intramolecular disulfide bonds when is partially oxidized. Redox potential values for CXXC motifs, -235 and -238 mV, are consistent with those reported for other proteins displaying disulfide reductase activity. Pull-down and two-hybrid assays unveil potential FurA interacting partners, namely phosphoribulokinase Alr4123, the hypothetical amidase-containing domain All1140 and the DNA-binding protein HU. INNOVATION: A novel biochemical activity of cyanobacterial FurA based on its cysteine arrangements and the identification of novel interacting partners are reported. CONCLUSION: The present study discloses a putative connection of FurA with the cyanobacterial redox-signaling pathway.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Secuencias de Aminoácidos , Anabaena/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras , Citoplasma/metabolismo , Disulfuros/metabolismo , Activación Enzimática , Glutatión/metabolismo , Mutación , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
In Anabaena sp. PCC 7120, FurA is a global transcriptional regulator whose expression is strongly induced by NtcA in proheterocysts and remains stably expressed in mature heterocysts. In the present study, overexpression of furA partially suppressed heterocyst differentiation by impairing morphogenesis at an early stage. Recombinant purified FurA specifically bound in vitro to the promoter regions of ntcA, while quantitative RT-PCR analyses indicated that furA overexpression strongly affected the transient increase of ntcA expression that occurs shortly after nitrogen step-down. Overall, the results suggest a connection between iron homeostasis and heterocyst differentiation via FurA, by modulating the expression of ntcA.
Asunto(s)
Anabaena/citología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Secuencia de Bases , Diferenciación Celular , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasas/química , Homeostasis , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
A real-time RT-PCR analysis of the transcriptional response to phosphate availability of the mcyD gene and microcystin-LR synthesis in Microcystis aeruginosa PCC7806 revealed that no significant changes were observed in the relative quantification of mcyD under excess phosphate (N/P = 1:1), whereas in deficiency of this nutrient (N/P = 40:1), a steady increase of mcyD during the exponential growth phase was detected, showing a maximal level on the 7th day of growth with a 6.8-fold increase over the control cells. The microcystin content in phosphate deficient cells correlates with the trend of mcyD transcription observed. Also, in this work we demonstrate that under phosphate deficiency conditions with a ratio of 40:1 N/P, the growth of M. aeruginosa PCC7806 was not affected when compared to control and phosphate excess samples. When blooms occur, the nutrients become exhausted and therefore phosphate availability will be scarce. In such a complex scenario, microcystin synthesis could be a response to phosphate deficiency, among other stress parameters.
Asunto(s)
Microcistinas/metabolismo , Microcystis/metabolismo , Nitrógeno/metabolismo , Fosfatos/deficiencia , Fosfatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microcistinas/genética , Microcystis/genética , Operón/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Anabaena/genética , Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Homeostasis/genética , Hierro/metabolismo , Regulón/fisiología , Tetrapirroles/biosíntesis , Sitios de Unión , Transporte Biológico/genética , Hemo/biosíntesis , Hemo/metabolismo , Ácidos Hidroxámicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Regiones Promotoras Genéticas , Sideróforos/biosíntesis , Sideróforos/metabolismoRESUMEN
The transcriptional repressor Fur (ferric uptake regulator) is one of the most important switches regulating prokaryotic iron metabolism. Cyanobacterial FurA binds heme in the micromolar concentration range and this interaction negatively affects its in vitro DNA binding ability in a concentration-dependent manner. Using site-directed mutagenesis along with difference absorption UV-visible, circular dichroism and electronic paramagnetic resonance spectroscopies, we further analyse the nature of heme binding in FurA. Our data point to Cys141, within a Cys-Pro motif, as an axial ligand of the Fe(III) high-spin heme. In the Fe(II) oxidation state, the heme shows low-spin form with an electronic absorption spectrum typical of six-coordinated low-spin heme proteins with a Soret absorption maximum blue-shifted by 25 nm in relation to typical low-spin thiolate-ligated Fe(II) heme proteins. Moreover, the ferrous C141S mutant shows Soret, α and ß bands almost identical to those observed for ferrous wild-type heme-FurA, indicating that the heme in ferrous C141S is in the same six-coordinated heme ligation as the ferrous native form. Therefore, Cys141 is not a ligand of the Fe(II) heme centre, suggesting a redox-dependent ligand switch undergone by this regulator. Our results indicate that the binding of heme to FurA exhibits the same physicochemical features as previously described for heme sensor proteins.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Hemo/metabolismo , Anabaena/genética , Proteínas Bacterianas/genética , Dicroismo Circular , Mutagénesis Sitio-Dirigida , Espectrofotometría UltravioletaRESUMEN
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.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Microcistinas/biosíntesis , Microcystis/metabolismo , Fotosíntesis/fisiología , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón , Luz , Microcistinas/genética , Microcistinas/toxicidad , Microcystis/genética , Microcystis/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Estrés Oxidativo/efectos de la radiación , Fotosíntesis/efectos de la radiación , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Cetoglutáricos/farmacología , Microcistinas/biosíntesis , Microcystis/metabolismo , Familia de Multigenes/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Sitios de Unión , Carbono/metabolismo , Microcystis/efectos de los fármacos , Microcystis/genética , Datos de Secuencia Molecular , Familia de Multigenes/efectos de los fármacos , Nitrógeno/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
Ferric uptake regulation (Fur) proteins are prokaryotic transcriptional regulators that integrate signaling of iron metabolism and oxidative stress responses with several environmental stresses. In photosynthetic organisms, Fur proteins regulate many genes involved in photosynthesis, nitrogen metabolism and other key processes. Also, Fur triggers the expression of virulence factors in many bacterial pathogens, and Fur from Microcystis aeruginosa has been shown to bind promoter regions of the microcystin synthesis gene cluster. In this work, we studied transcriptional responses of fur genes under different light intensities and oxidative stress. An antisense of fur, the α-fur RNA, plays an important role in regulating fur expression under oxidative stress, affecting levels of Fur protein in cells. Importantly, an active photosynthetic electron chain is required for the expression of the fur gene.
Asunto(s)
Proteínas Bacterianas/metabolismo , Luz , Microcystis/efectos de los fármacos , Microcystis/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Peróxido de Hidrógeno/farmacología , Immunoblotting , Microcystis/genética , Estrés Oxidativo/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Hierro/metabolismo , Proteoma/biosíntesis , Regulón/fisiología , Anabaena/genética , Proteínas Bacterianas/genética , Proteoma/genética , Proteómica/métodosRESUMEN
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.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Fotosíntesis , Anabaena/enzimología , Anabaena/fisiología , Proteínas Bacterianas/genética , Catalasa/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Genes de Plantas , Regiones Promotoras Genéticas , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismoRESUMEN
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 Microcystis aeruginosa 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 Microcystis aeruginosa 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.