RESUMEN
After the Great Oxidation Event (GOE), iron availability was greatly decreased, and photosynthetic organisms evolved several alternative proteins and mechanisms. One of these proteins, plastocyanin, is a type I blue-copper protein that can replace cytochrome c6 as a soluble electron carrier between cytochrome b6f and photosystem I. In most cyanobacteria, expression of these two alternative proteins is regulated by copper availability, but the regulatory system remains unknown. Herein, we provide evidence that the regulatory system is composed of a BlaI/CopY-family transcription factor (PetR) and a BlaR-membrane protease (PetP). PetR represses petE (plastocyanin) expression and activates petJ (cytochrome c6), while PetP controls PetR levels in vivo. Using whole-cell extracts, we demonstrated that PetR degradation requires both PetP and copper. Transcriptomic analysis revealed that the PetRP system regulates only four genes (petE, petJ, slr0601, and slr0602), highlighting its specificity. Furthermore, the presence of petE and petRP in early branching cyanobacteria indicates that acquisition of these genes could represent an early adaptation to decreased iron bioavailability following the GOE.
Asunto(s)
Citocromos c/metabolismo , Péptido Hidrolasas/metabolismo , Plastocianina/metabolismo , Synechocystis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cobre/farmacología , Epistasis Genética/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Regulón/genética , Synechocystis/efectos de los fármacosRESUMEN
In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.
Asunto(s)
Diatomeas , Deficiencias de Hierro , Humanos , Complejo de Proteína del Fotosistema II/metabolismo , Diatomeas/metabolismo , Citocromos c/metabolismo , Clorofila/metabolismo , Oxígeno/metabolismo , Hierro/metabolismo , Agua/metabolismoRESUMEN
Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.
Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/enzimología , Disulfuros/química , Flavina-Adenina Dinucleótido/química , Oxidorreductasas/química , Synechocystis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Membrana Celular/química , Membrana Celular/enzimología , Cristalografía por Rayos X , Cianobacterias/genética , Disulfuros/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Synechocystis/genética , Reductasa de Tiorredoxina-Disulfuro/química , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismoRESUMEN
Organisms are constantly exposed to a wide range of environmental changes, including both short-term changes during their lifetime and longer-term changes across generations. Stress-related gene expression programmes, characterized by distinct transcriptional mechanisms and high levels of noise in their expression patterns, need to be balanced with growth-related gene expression programmes. A range of recent studies give fascinating insight into cellular strategies for keeping gene expression in tune with physiological needs dictated by the environment, promoting adaptation to both short- and long-term environmental changes. Not only do organisms show great resilience to external challenges, but emerging data suggest that they also exploit these challenges to fuel phenotypic variation and evolutionary innovation.
Asunto(s)
Adaptación Biológica/genética , Evolución Biológica , Ambiente , Regulación de la Expresión Génica/fisiología , Animales , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Crecimiento y Desarrollo/genética , Crecimiento y Desarrollo/fisiología , Homeostasis/genética , Homeostasis/fisiología , Humanos , Modelos Biológicos , Estrés Fisiológico/genética , Factores de Tiempo , Transcripción Genética/fisiologíaRESUMEN
Schizosaccharomyces pombe Clp1 is a Cdc14-family phosphatase that reverses mitotic Cdk1 phosphorylation. Despite evolutionary conservation, Clp1 's mammalian orthologs do not share this function. Rather, higher eukaryotic Cdc14 enzymes act in DNA repair, ciliogenesis, and gene regulation. To examine if Clp1 regulates gene expression, we compared the transcriptional profiles of cells lacking Clp1 function to that of wildtype. Because clp1∆ cells are sensitive to the actin depolymerizing drug, LatrunculinA, we also investigated whether a transcriptional response was involved. Our results indicate that Clp1 does not detectably affect gene expression and highlight the organism-specific functions of this conserved phosphatase family.
RESUMEN
Histone lysine acetylation has emerged as a key regulator of genome organization. However, with a few exceptions, the contribution of each acetylated lysine to cellular functions is not well understood because of the limited specificity of most histone acetyltransferases and histone deacetylases. Here we show that the Mst2 complex in Schizosaccharomyces pombe is a highly specific H3 lysine 14 (H3K14) acetyltransferase that functions together with Gcn5 to regulate global levels of H3K14 acetylation (H3K14ac). By analyzing the effect of H3K14ac loss through both enzymatic inactivation and histone mutations, we found that H3K14ac is critical for DNA damage checkpoint activation by directly regulating the compaction of chromatin and by recruiting chromatin remodeling protein complex RSC.
Asunto(s)
Acetiltransferasas/metabolismo , Cromatina/metabolismo , Daño del ADN/fisiología , ADN de Hongos/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Acetilación , Acetiltransferasas/genética , Cromatina/genética , ADN de Hongos/genética , Histonas/genética , Mutación , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.
Asunto(s)
Arseniato Reductasas/química , Proteínas Bacterianas/química , Glutarredoxinas/química , Synechocystis/enzimología , Arseniato Reductasas/genética , Arseniatos/metabolismo , Biocatálisis , Clonación Molecular , Cisteína/química , Glutatión/química , Datos de Secuencia Molecular , Oxidación-Reducción , Homología de Secuencia de AminoácidoRESUMEN
Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the P(I)-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Cobre/toxicidad , Transducción de Señal/efectos de los fármacos , Synechocystis/efectos de los fármacos , Synechocystis/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cobre/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Periplasma/efectos de los fármacos , Periplasma/metabolismo , Plastocianina/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Tilacoides/efectos de los fármacos , Tilacoides/metabolismoRESUMEN
In recent years, a plethora of new synthetic biology tools for use in cyanobacteria have been published; however, their reported characterizations often cannot be reproduced, greatly limiting the comparability of results and hindering their applicability. In this interlaboratory study, the reproducibility of a standard microbiological experiment for the cyanobacterial model organism Synechocystis sp. PCC 6803 was assessed. Participants from eight different laboratories quantified the fluorescence intensity of mVENUS as a proxy for the transcription activity of the three promoters PJ23100, PrhaBAD, and PpetE over time. In addition, growth rates were measured to compare growth conditions between laboratories. By establishing strict and standardized laboratory protocols, reflecting frequently reported methods, we aimed to identify issues with state-of-the-art procedures and assess their effect on reproducibility. Significant differences in spectrophotometer measurements across laboratories from identical samples were found, suggesting that commonly used reporting practices of optical density values need to be supplemented by cell count or biomass measurements. Further, despite standardized light intensity in the incubators, significantly different growth rates between incubators used in this study were observed, highlighting the need for additional reporting requirements of growth conditions for phototrophic organisms beyond the light intensity and CO2 supply. Despite the use of a regulatory system orthogonal to Synechocystis sp. PCC 6803, PrhaBAD, and a high level of protocol standardization, â¼32% variation in promoter activity under induced conditions was found across laboratories, suggesting that the reproducibility of other data in the field of cyanobacteria might be affected similarly.
Asunto(s)
Synechocystis , Reproducibilidad de los Resultados , Biomasa , Synechocystis/genética , Genes Reporteros , Regiones Promotoras GenéticasRESUMEN
The cyanobacterium Synechocystis sp. PCC 6803 possesses an arsenic resistance operon that encodes, among others, an ArsH protein. ArsH is a flavin mononucleotide (FMN)-containing protein of unknown function and a member of the family of NADPH-dependent FMN reductases. The nature of its final electron acceptor and the role of ArsH in the resistance to arsenic remained to be clarified. Here we have expressed and purified Synechocystis ArsH and conducted an intensive biochemical study. We present kinetic evidence supporting a quinone reductase activity for ArsH, with a preference for quinones with hydrophobic substituents. By using steady-state activity measurements, as well as stopped-flow and laser-flash photolysis kinetic analyses, it has been possible to establish the mechanism of the process and estimate the values of the kinetic constants. Although the enzyme is able to stabilize the anionic semiquinone form of the FMN, reduction of quinones involves the hydroquinone form of the flavin cofactor, and the enzymatic reaction occurs through a ping-pong-type mechanism. ArsH is able to catalyze one-electron reactions (oxygen and cytocrome c reduction), involving the FMN semiquinone form, but with lower efficiency. In addition, arsH mutants are sensitive to the oxidizing agent menadione, suggesting that ArsH plays a role in the response to oxidative stress caused by arsenite.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , NAD(P)H Deshidrogenasa (Quinona)/química , Synechocystis/enzimología , Arsénico/toxicidad , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/enzimología , Escherichia coli/genética , FMN Reductasa/química , FMN Reductasa/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , Operón/genética , Oxidación-Reducción , Synechocystis/genéticaRESUMEN
To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression in the model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1(+), that encodes a putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with the copper permease Ctr4, it contains four putative copper-binding motifs that are typically found in members of the copper transporter family of copper transporters. Similarly to the ctr4(+) gene, the transcription of mfc1(+) was induced by low concentrations of copper. However, its temporal expression profile during meiosis was distinct to ctr4(+). Whereas Ctr4 was observed at the plasma membrane shortly after induction of meiosis, Mfc1 appeared later in precursor vesicles and, subsequently, at the forespore membrane of ascospores. Using the fluorescent copper-binding tracker Coppersensor-1 (CS1), labile cellular copper was primarily detected in the forespores in an mfc1(+)/mfc1(+) strain, whereas an mfc1Δ/mfc1Δ mutant exhibited an intracellular dispersed punctate distribution of labile copper ions. In addition, the copper amine oxidase Cao1, which localized primarily in the forespores of asci, was fully active in mfc1(+)/mfc1(+) cells, but its activity was drastically reduced in an mfc1Δ/mfc1Δ strain. Furthermore, our data showed that meiotic cells that express the mfc1(+) gene have a distinct developmental advantage over mfc1Δ/mfc1Δ mutant cells when copper is limiting. Taken together, the data reveal that Mfc1 serves to transport copper for accurate and timely meiotic differentiation under copper-limiting conditions.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Cobre/metabolismo , Meiosis/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencias de Aminoácidos , Proteínas de Transporte de Catión/genética , Cationes/metabolismo , Membrana Celular/genética , Transporte Iónico/fisiología , Proteínas SLC31 , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) are two essential activities in the Calvin-Benson-Bassham cycle that catalyze two irreversible reactions and are key for proper regulation and functioning of the cycle. These two activities are codified by a single gene in all cyanobacteria, although some cyanobacteria contain an additional gene coding for a FBPase. Mutants lacking the gene coding for SBP/FBPase protein are not able to grow photoautotrophically and require glucose to survive. As this protein presents both activities, we have tried to elucidate which of the two are required for photoautrophic growth in Synechocystis sp PCC 6803. For this, the genes coding for plant FBPase and SBPase were introduced in a SBP/FBPase mutant strain, and the strains were tested for growth in the absence of glucose. Ectopic expression of only a plant SBPase gene did not allow growth in the absence of glucose although allowed mutation of both Synechocystis' FBPase genes. When both plant FBPase and SBPase genes were expressed, photoautrophic growth of the SBP/FBPase mutants was restored. This complementation was partial as the strain only grew in low light, but growth was impaired at higher light intensities. Redox regulation of the Calvin-Benson-Bassham cycle is essential to properly coordinate light reactions to carbon fixation in the chloroplast. Two of the best characterized proteins that are redox-regulated in the cycle are FBPase and SBPase. These two proteins are targets of the FTR-Trx redox system with Trx f being the main reductant in vivo. Introduction of the TrxF gene improves growth of the complemented strain, suggesting that the redox state of the proteins may be the cause of this phenotype. The redox state of the plant proteins was also checked in these strains, and it shows that the cyanobacterial redox system is able to reduce all of them (SBPase, FBPase, and TrxF) in a light-dependent manner. Thus, the TrxF-FBPase-SBPase plant chloroplast system is active in cyanobacteria despite that these organisms do not contain proteins related to them. Furthermore, our system opens the possibility to study specificity of the Trx system in vivo without the complication of the different isoforms present in plants.
RESUMEN
Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.
Asunto(s)
Arseniato Reductasas/metabolismo , Arseniatos/metabolismo , Proteínas Bacterianas/metabolismo , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Synechocystis/metabolismo , Secuencia de Aminoácidos , Arseniato Reductasas/química , Arseniato Reductasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Northern Blotting , Clonación Molecular , Datos de Secuencia Molecular , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Synechocystis/genética , Tiorredoxinas/metabolismoRESUMEN
The slr1192 (adhA) gene from Synechocystis sp. strain PCC 6803 encodes a member of the medium-chain alcohol dehydrogenase/reductase family. The gene product AdhA exhibits NADP-dependent alcohol dehydrogenase activity, acting on a broad variety of aromatic and aliphatic primary alcohols and aldehydes but not on secondary alcohols or ketones. It exhibits superior catalytic efficiency for aldehyde reduction compared to that for alcohol oxidation. The enzyme is a cytosolic protein present in photoautotrophically grown Synechocystis cells. The expression of AdhA is enhanced upon the exposure of cells to different environmental stresses, although it is not essential for survival even under such stress conditions. The induction of the expression of the adhA gene is dependent on the Hik34-Rre1 two-component system, as it is severely impaired in mutant strains lacking either the histidine kinase Hik34 or the response regulator Rre1. In vitro DNA-protein interaction analysis reveals that the response regulator Rre1 binds specifically to the promoter region of the adhA gene.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Synechocystis/enzimología , Synechocystis/metabolismo , Alcohol Deshidrogenasa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Northern Blotting , Western Blotting , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Mutación , Regiones Promotoras Genéticas/genética , Synechocystis/genéticaRESUMEN
Cyanobacteria form a diverse group of oxygenic photosynthetic prokaryotes considered to be the antecessor of plant chloroplast. They contain four different thioredoxins isoforms, three of them corresponding to m, x and y type present in plant chloroplast, while the fourth one (named TrxC) is exclusively found in cyanobacteria. TrxC has a modified active site (WCGLC) instead of the canonical (WCGPC) present in most thioredoxins. We have purified it and assayed its activity but surprisingly TrxC lacked all the classical activities, such as insulin precipitation or activation of the fructose-1,6-bisphosphatase. Mutants lacking trxC or over-expressing it were generated in the model cyanobacterium Synechocystis sp. PCC 6803 and their phenotypes have been analyzed. The ΔtrxC mutant grew at similar rates to WT in all conditions tested although it showed an increased carotenoid content especially under low carbon conditions. Overexpression strains showed reduced growth under the same conditions and accumulated lower amounts of carotenoids. They also showed lower oxygen evolution rates at high light but higher Fv'/Fm' and Non-photochemical-quenching (NPQ) in dark adapted cells, suggesting a more oxidized plastoquinone pool. All these data suggest that TrxC might have a role in regulating photosynthetic adaptation to low carbon and/or high light conditions.
RESUMEN
Plastocyanin (petE) plays an essential role in photosynthesis as an electron carrier between cytochrome b6 f and photosystem I, and in some cyanobacteria it can be replaced by the haem-containing protein, cytochrome c6 (petJ). In Synechocystis sp. PCC 6803, transcription of petE and petJ is activated and repressed, respectively, by Cu. Here, we show that Ni can act similarly to Cu in inducing petE and repressing petJ, thus leading to a partial switch between cytochrome c6 and plastocyanin. Transcription of these genes is only altered by Ni in Cu-depleted medium, and none of the Ni-dependent transcription factors described in Synechocystis, NrsR and InrS seem to be involved in this regulation. Finally, we show that plastocyanin is essential for growth under conditions of excess Ni.
Asunto(s)
Citocromos c6/genética , Níquel/metabolismo , Plastocianina/genética , Synechocystis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citocromos c6/metabolismo , Regulación Bacteriana de la Expresión Génica , Plastocianina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Transcripción GenéticaRESUMEN
Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux-resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (KD ~3 × 10(-16) ). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Cobre/farmacología , Synechocystis/metabolismo , Secuencia de Aminoácidos , Anabaena/genética , Anabaena/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Cobre/metabolismo , Farmacorresistencia Bacteriana/genética , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis Insercional , Operón , Synechocystis/genética , Transcripción GenéticaRESUMEN
Cyanobacteria represent the largest and most diverse group of prokaryotes capable of performing oxygenic photosynthesis and are frequently found in environments contaminated with heavy metals. Several studies have been performed in these organisms in order to better understand the effects of metals such as Zn, Cd, Cu, Ni and Co. In Synechocystis sp. PCC 6803, genes involved in Ni, Co, Cu and Zn resistance have been reported. However, proteomic studies for the identification of proteins modulated by heavy metals have not been carried out. In the present work, we have analyzed the proteomic pattern alterations of the cyanobacterium Synechocystis sp. PCC 6803 in response to Ni, Co and Cd in order to identify the metabolic processes affected by these metals. We show that some proteins are commonly regulated in response to the different metal ions, including ribulose1,5-bisphosphate carboxylase and the periplasmic iron-binding protein FutA2, while others, such as chaperones, were specifically induced by each metal. We also show that the main processes affected by the metals are carbon metabolism and photosynthesis, since heavy metals affect proteins required for the correct functioning of these activities. BIOLOGICAL SIGNIFICANCE: This is the first report on the proteomic profile of Synechocystis sp. PCC 6803 wild type and mutant strains for the identification of proteins affected by the heavy metals Ni, Co and Cd. We have identified proteins commonly responsive to all three metals and also chaperones specifically modulated by each metal. Our data also supports previous studies that suggest the existence of additional sensor systems for Co.
Asunto(s)
Cadmio/química , Cobalto/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Níquel/química , Synechocystis/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Citosol/metabolismo , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Homeostasis , Mutación , Proteoma/metabolismoRESUMEN
Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As(III)] and arsenate [As(V)]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.
Asunto(s)
Arsénico/química , Synechocystis/efectos de los fármacos , Synechocystis/genética , Arseniato Reductasas/metabolismo , Arseniatos/química , Arsenitos/química , Transporte Biológico , Cobre/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genoma Bacteriano/efectos de los fármacos , Glutatión/metabolismo , Metales/química , Mutación , Níquel/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Estrés Oxidativo , Fenotipo , Azufre/químicaRESUMEN
Target of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising â¼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.