An Internal Promoter Drives the Expression of a Truncated Form of CCC1 Capable of Protecting Yeast from Iron Toxicity.

In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.

Yeast AP-1 like transcription factors (Yap) and stress response: a current overview.

Yeast adaptation to stress has been extensively studied. It involves large reprogramming of genome expression operated by many, more or less specific, transcription factors. Here, we review our current knowledge on the function of the eight Yap transcription factors (Yap1 to Yap8) in Saccharomyces cerevisiae, which were shown to be involved in various stress responses. More precisely, Yap1 is activated under oxidative stress, Yap2/Cad1 under cadmium, Yap4/Cin5 and Yap6 under osmotic shock, Yap5 under iron overload and Yap8/Arr1 by arsenic compounds. Yap3 and Yap7 seem to be involved in hydroquinone and nitrosative stresses, respectively. The data presented in this article illustrate how much knowledge on the function of these Yap transcription factors is advanced. The evolution of the Yap family and its roles in various pathogenic and non-pathogenic fungal species is discussed in the last section.

Transcriptional regulation of FeS biogenesis genes: A possible shield against arsenate toxicity activated by Yap1.

In the eukaryotic model yeast Saccharomyces cerevisiae, arsenic (As) detoxification is regulated by two transcriptional factors, Yap8 and Yap1. Yap8 specifically controls As extrusion from the cell, whether Yap1 avoids arsenic-induced oxidative damages. Accordingly, cells lacking both Yap1 and Yap8 are more sensitive to arsenate than cells lacking each regulator individually. Strikingly enough, the same sensitivity pattern was observed under anoxia, suggesting that Yap1 role in As detoxification might not be restricted to the regulation of the oxidative stress response. This finding prompted us to study the transcriptomic profile of wild-type and yap1 mutant cells exposed to arsenate. Interestingly, we found that, under such conditions, several genes involved in the biogenesis of FeS proteins were upregulated in a Yap1-dependent way. In line with this observation, arsenate treatment decreases the activity of the mitochondrial aconitase, Aco1, an FeS cluster-containing enzyme, this effect being even more pronounced in the yap1 mutant. Reinforcing the relevance of FeS cluster biogenesis in arsenate detoxification, the overexpression of several ISC and CIA machinery genes alleviates the deleterious effect of arsenate caused by the absence of Yap1 and Yap8. Altogether our data suggest that the upregulation of FeS biogenesis genes regulated by Yap1 might work as a cellular shield against arsenate toxicity.

Ace1 prevents intracellular copper accumulation by regulating Fet3 expression and thereby restricting Aft1 activity.

In the yeast Saccharomyces cerevisiae Aft1, the low iron-sensing transcription factor is known to regulate the expression of the FET3 gene. However, we found that a strain-lacking FET3 is more sensitive to copper excess than a strain-lacking AFT1, and accordingly, FET3 expression is not fully compromised in the latter. These findings suggest that, under such conditions, another regulator comes into play and controls FET3 expression. In this work, we identify Ace1, the regulator of copper detoxification genes, as a regulator of FET3. We suggest that the activation of FET3 by Ace1 prevents the hyper activation of Aft1, possibly by assuring the adequate functioning of mitochondrial iron-sulfur cluster biogenesis. While reinforcing the link between iron and copper homeostasis, this work unveils a novel protection mechanism against copper toxicity mediated by Ace1, which relies in the activation of FET3 and results in the restriction of Aft1 activity as a means to prevent excessive copper accumulation.

Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes.

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Electron transfer pathways of formate-driven H2 production in Desulfovibrio.

The potential of sulfate-reducing bacteria (SRB) as biocatalysts for H2 production from formate was recently demonstrated, but the electron transfer pathways involved were not described. In the present work, we analyzed the H2 production capacity of five Desulfovibrio strains: Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio fructosivorans, and Desulfovibrio gigas. D. vulgaris showed the highest H2 productivity (865 mL Lmedium (-1)), and D. gigas the lowest one (374 mL Lmedium (-1) of H2). The electron transfer pathways involved in formate-driven H2 production by these two organisms were further investigated through the study of deletion mutants of hydrogenases (Hases) and formate dehydrogenases (Fdhs). In D. vulgaris, the periplasmic FdhAB is the key enzyme for formate oxidation and two pathways are apparently involved in the production of H2 from formate: a direct one only involving periplasmic enzymes and a second one that involves transmembrane electron transfer and may allow energy conservation. In the presence of selenium, the Hys [NiFeSe] Hase is the main periplasmic enzyme responsible for H2 production, and the cytoplasmic Coo Hase is apparently involved in the ability of D. vulgaris to grow by converting formate to H2, in sparging conditions. Contrary to D. vulgaris, H2 production in D. gigas occurs exclusively by the direct periplasmic route and does not involve the single cytoplasmic Hase, Ech. This is the first report of the metabolic pathways involved in formate metabolism in the absence of sulfate in SRB, revealing that the electron transfer pathways are species-specific.

The C-terminal region of the Hot1 transcription factor binds GGGACAAA-related sequences in the promoter of its target genes.

Response to hyperosmotic stress in the yeast Saccharomyces cerevisiae involves the participation of the general stress response mediated by Msn2/4 transcription factors and the HOG pathway. One of the transcription factors activated through this pathway is Hot1, which contributes to the control of the expression of several genes involved in glycerol synthesis and flux, or in other functions related to adaptation to adverse conditions. This work provides new data about the interaction mechanism of this transcription factor with DNA. By means of one-hybrid and electrophoretic mobility assays, we demonstrate that the C-terminal region, which corresponds to amino acids 610-719, is the DNA-binding domain of Hot1. We also describe how this domain recognizes sequence 5'-GGGACAAA-3' located in the promoter of gene STL1. The bioinformatics analysis carried out in this work allowed the identification of identical or similar sequences (with up to two mismatches) in the promoter of other Hot1 targets, where central element GGACA was quite conserved among them. Finally, we found that small variations in the sequence recognized by Hot1 may influence its ability to recognize its targets in vivo.

An HcpR paralog of Desulfovibrio gigas provides protection against nitrosative stress.

Desulfovibrio gigas belongs to the group of sulfate reducing bacteria (SRB). These ubiquitous and metabolically versatile microorganisms are often exposed to reactive nitrogen species (RNS). Nonetheless, the mechanisms and regulatory elements involved in nitrosative stress protection are still poorly understood. The transcription factor HcpR has emerged as a putative regulator of nitrosative stress response among anaerobic bacteria. HcpR is known to orchestrate the expression of the hybrid cluster protein gene, hcp, proposed to be involved in cellular defense against RNS. According to phylogenetic analyses, the occurrence of hcpR paralog genes is a common feature among several Desulfovibrio species. Within the D. gigas genome we have identified two HcpR-related sequences. One of these sequences, hcpR1, was found in the close vicinity of the hcp gene and this finding prompted us to proceed with its functional characterization. We observed that the growth of a D. gigas strain lacking hcpR1 is severely impaired under nitrosative stress. An in silico search revealed several putative targets of HcpR1 that were experimentally validated. The fact that HcpR1 regulates several genes encoding proteins involved in nitrite and nitrate metabolism, together with the sensitive growth phenotype to NO displayed by an hcpR1 mutant strain, strongly supports a relevant role of this factor under nitrosative stress. Moreover, the finding that several Desulfovibrio species possess HcpR paralogs, which have been transmitted vertically in the evolution and diversification of the genus, suggests that these sequences may confer adaptive or survival advantage to these organisms, possibly by increasing their tolerance to nitrosative stress.

Inhibition of Yap2 activity by MAPKAP kinase Rck1 affects yeast tolerance to cadmium.

Yap2 is a cadmium responsive transcription factor that interacts with MAPK-activated protein (MAPKAP) kinase Rck1. We show that Rck1 deletion confers protection against cadmium toxicity and that the mechanism underlying this observation relies on Yap2. Rck1 removal from the yeast genome potentiates Yap2 activity by increasing protein half-life and delaying its nuclear export. As a consequence, several Yap2 antioxidant targets are over-activated by a mechanism that also requires Yap1. Several genes of the cell wall integrity (CWI) pathway are upregulated under cadmium stress in a Yap2 dependent way. We showed that deletion of CWI genes renders yeast cells more sensitive to cadmium. These findings led us to suggest that in response to cadmium stress Yap2 may serve a dual purpose: oxidative stress attenuation and cell wall maintenance.

E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif.

Adaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation of arsenic detoxification machinery by the Yap8 transcription factor. Yap8 is targeted by the ubiquitin proteasome system for degradation under physiological conditions, yet it escapes proteolysis in arsenic-injured cells by a mechanism that remains to be elucidated. Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels. Under these conditions, Ufd2 interacts with Yap8 mediating its stabilization, thereby controlling expression of ACR3 and capacity of cells to adapt to arsenic injury. We also show that Ufd2 U-box domain, which is associated to the ubiquitination activity of specific ubiquitin ligases, is dispensable for Yap8 stability and has no role in cell tolerance to arsenic stress. Thus, our data disclose a novel Ufd2 role beyond degradation. This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation.

Repression of the Low Affinity Iron Transporter Gene FET4: A NOVEL MECHANISM AGAINST CADMIUM TOXICITY ORCHESTRATED BY YAP1 VIA ROX1.

Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake.

Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus.

Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.

Yap1 mediates tolerance to cobalt toxicity in the yeast Saccharomyces cerevisiae.

BACKGROUND: Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1 mediates tolerance to cobalt toxicity. METHODS: Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. RESULTS: Cobalt activates Yap1 under aerobiosis and anaerobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we show that Yap1 is a repressor of the low affinity iron transporter, FET4, which is also known to transport cobalt. CONCLUSIONS: Cobalt activates Yap1 that alleviates the oxidative damage caused by this metal. Yap1 partially controls cobalt cellular uptake via the regulation of PHO84. Although FET4 repression by Yap1 has no effect on cobalt uptake, it may be its first line of defense against other toxic metals. GENERAL SIGNIFICANCE: Our results emphasize the important role of Yap1 in mediating cobalt-induced oxidative damages and reveal new routes for cell protection provided by this regulator.

Two residues in the basic region of the yeast transcription factor Yap8 are crucial for its DNA-binding specificity.

In Saccharomyces cerevisiae, the transcription factor Yap8 is a key determinant in arsenic stress response. Contrary to Yap1, another basic region-leucine zipper (bZIP) yeast regulator, Yap8 has a very restricted DNA-binding specificity and only orchestrates the expression of ACR2 and ACR3 genes. In the DNA-binding basic region, Yap8 has three distinct amino acids residues, Leu26, Ser29 and Asn31, at sites of highly conserved positions in the other Yap family of transcriptional regulators and Pap1 of Schizosaccharomyces pombe. To evaluate whether these residues are relevant to Yap8 specificity, we first built a homology model of the complex Yap8bZIP-DNA based on Pap1-DNA crystal structure. Several Yap8 mutants were then generated in order to confirm the contribution of the residues predicted to interact with DNA. Using bioinformatics analysis together with in vivo and in vitro approaches, we have identified several conserved residues critical for Yap8-DNA binding. Moreover, our data suggest that Leu26 is required for Yap8 binding to DNA and that this residue together with Asn31, hinder Yap1 response element recognition by Yap8, thus narrowing its DNA-binding specificity. Furthermore our results point to a role of these two amino acids in the stability of the Yap8-DNA complex.

Molecular characterization of TaSTOP1 homoeologues and their response to aluminium and proton (H(+)) toxicity in bread wheat (Triticum aestivum L.).

BACKGROUND: Aluminium (Al) toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. Being a trait governed by multiple genes, the identification and characterization of novel transcription factors (TFs) regulating the expression of entire response networks is a very promising approach. Therefore, the aim of the present study was to clone, localize, and characterize the TaSTOP1 gene, which belongs to the zinc finger family (Cys2His2 type) transcription factor, at molecular level in bread wheat. RESULTS: TaSTOP1 loci were cloned and localized on the long arm of homoeologous group 3 chromosomes [3AL (TaSTOP1-A), 3BL (TaSTOP1-B) and 3DL (TaSTOP1-D)] in bread wheat. TaSTOP1 showed four potential zinc finger domains and the homoeologue TaSTOP1-A exhibited transactivation activity in yeast. Expression profiling of TaSTOP1 transcripts identified the predominance of homoeologue TaSTOP1-A followed by TaSTOP1-D over TaSTOP1-B in root and only predominance of TaSTOP1-A in shoot tissues of two diverse bread wheat genotypes. Al and proton (H(+)) stress appeared to slightly modulate the transcript of TaSTOP1 homoeologues expression in both genotypes of bread wheat. CONCLUSIONS: Physical localization of TaSTOP1 results indicated the presence of a single copy of TaSTOP1 on homoeologous group 3 chromosomes in bread wheat. The three homoeologues of TaSTOP1 have similar genomic structures, but showed biased transcript expression and different response to Al and proton (H(+)) toxicity. These results indicate that TaSTOP1 homoeologues may differentially contribute under Al or proton (H(+)) toxicity in bread wheat. Moreover, it seems that TaSTOP1-A transactivation potential is constitutive and may not depend on the presence/absence of Al at least in yeast. Finally, the localization of TaSTOP1 on long arm of homoeologous group 3 chromosomes and the previously reported major loci associated with Al resistance at chromosome 3BL, through QTL and genome wide association mapping studies suggests that TaSTOP1 could be a potential candidate gene for genomic assisted breeding for Al tolerance in bread wheat.

Roles of HynAB and Ech, the only two hydrogenases found in the model sulfate reducer Desulfovibrio gigas.

Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism.

Relationship between ethanol and oxidative stress in laboratory and brewing yeast strains.

Ethanol is a chemical stress factor that inhibits cellular growth and determines metabolic changes leading to reduction of cell viability during fermentation and yeast storage. To determine the effect of time, temperature and ethanol during storage of brewing yeasts we have monitored viability of cells stored for 72 h, at 6 °C or 12 °C, in the presence of various ethanol concentrations. Under the conditions tested, 6 °C is the most favourable temperature to store brewing yeast creams emphasizing the importance of a tight temperature control in the storage vessels. Because W210 is less resistant to storage in the presence of ethanol than W34/70, the optimal storage parameters obtained under our laboratory conditions vary significantly. The ale strain is sensitive to storage under ethanol concentrations higher than 5% (v/v) for more than 48 h at 6 °C whereas at the same temperature the lager strain tolerates ethanol up to 7.5% (v/v) for 72 h. Also, the viability assays indicate that the antioxidant protein Yap1 is an important factor to storage resistance of BY4741 laboratory strain. To investigate the molecular mechanisms underlying tolerance of brewing yeast strains to ethanol, we have performed phenotypic analysis, localization studies and have monitored the activation of antioxidant and protection genes as well as the intracellular contents of glycogen and trehalose. Overall, our data suggest that the ale strain W210 has a defective antioxidant defence system and that ethanol may induce the antioxidant defences as well as glycogen and trehalose protection mechanisms in laboratory and brewing yeast strains.

Role of NorR-like transcriptional regulators under nitrosative stress of the δ-proteobacterium, Desulfovibrio gigas.

NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. gigas mutants Δroo and ΔnorR1-like is impaired. In order to confirm that D. gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ΔnorR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ΔnorR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. gigas subjected to nitrosative stress. It is identified in δ-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.

Yeast protective response to arsenate involves the repression of the high affinity iron uptake system.

Arsenic is a double-edge sword. On the one hand it is powerful carcinogen and on the other it is used therapeutically to treat acute promyelocytic leukemia. Here we report that arsenic activates the iron responsive transcription factor, Aft1, as a consequence of a defective high-affinity iron uptake mediated by Fet3 and Ftr1, whose mRNAs are drastically decreased upon arsenic exposure. Moreover, arsenic causes the internalization and degradation of Fet3. Most importantly, fet3ftr1 mutant exhibits increased arsenic resistance and decreased arsenic accumulation over the wild-type suggesting that Fet3 plays a role in arsenic toxicity. Finally we provide data suggesting that arsenic also disrupts iron uptake in mammals and the link between Fet3, arsenic and iron, can be relevant to clinical applications.