Letteratura scientifica selezionata sul tema "Nitrogen catabolite repression"

Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.

Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.

We describe the experimental methodology that led to the discovery of the creA gene in Aspergillus nidulans. This gene codes for a transcriptional repressor mediating carbon catabolite repression in many pathways in this organism. We compare both the mode and the mechanism of action in two pathways subject to CreA-mediated repression. The genes comprising the ethanol regulon are subject to carbon catabolite repression independently of the nitrogen source, while the genes involved in proline utilization are repressed by glucose only when a repressing nitrogen source is also present. In the ethanol regulon, CreA drastically represses the expression of the positive regulatory gene alcR, thus preventing the expression of the structural genes. Direct repression of the structural genes is also existant. In the proline utilization pathway, repression operates directly at the level of the structural genes. In the ethanol regulon, CreA prevents the self-induction of alcR and the induction of the structural genes by competing with the binding of the AlcR protein. In proline gene cluster, CreA does not interfere with induction mediated by PrnA but with the activity of an unknown and more general transcription factor. Key words: carbon catabolite repression, ascomycetes, Zn fingers.

The paper of Arst and Cove (Mol. Gen. Genet. 126: 111 – 141, 1973) on "Nitrogen metabolite repression in Aspergillus nidulans" has influenced studies and perceptions of gene regulation in filamentous fungi during the past 21 years. Here I attempt to appraise the contributions of that paper and assess its role in further developments. Nitrogen metabolite repression, carbon catabolite repression, pathway-specific and integrated induction, as-acting regulatory mutations, a useful class of growth inhibitors, and a homologous Neurospora crassa gene are all discussed. Key words: Aspergillus nidulans, carbon catabolite repression, nitrogen metabolite repression.

ABSTRACT Bacillus subtilis cells cannot sporulate in the presence of catabolites such as glucose. During the analysis of Tn10-generated mutants, we found that deletion of the C-terminal region of the tnrA gene, which encodes a global regulator that positively regulates a number of genes in response to nitrogen limitation, results in a catabolite-resistant sporulation phenotype. Analyses of nrg-lacZ and nasB-lacZ, which are activated by TnrA under nitrogen limitation, showed that C-terminally truncated TnrA activates nitrogen-regulated genes constitutively. The relief of catabolite repression of sporulation may result from the uncontrolled expression of the TnrA-regulated genes.

Yeast can use a wide variety of nitrogen compounds. However, the ability to synthesize enzymes and permeases for catabolism of poor nitrogen sources is limited in the presence of a rich one. This general mechanism of transcriptional control is called nitrogen catabolite repression. Poor nitrogen sources, such as leucine, γ-aminobutyric acid (GABA), and allantoin, enable growth after the synthesis of pathway-specific catabolic enzymes and permeases. This synthesis occurs only under conditions of nitrogen limitation and in the presence of a pathway-specific signal. In this work we studied the temporal order in the induction of AGP1, BAP2, UGA4, and DAL7, genes that are involved in the catabolism and use of leucine, GABA, and allantoin, three poor nitrogen sources. We found that when these amino acids are available, cells will express AGP1 and BAP2 in the first place, then DAL7, and at last UGA4. Dal81, a general positive regulator of genes involved in nitrogen utilization related to the metabolisms of GABA, leucine, and allantoin, plays a central role in this coordinated regulation.

ABSTRACT The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of gram-positive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ΔhprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ΔhprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.

Nitrogen Catabolite Repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae reduces the expression of genes encoding components involved in the utilization of poor nitrogen sources when rich ones are available. Expression of NCR-sensitive genes is controlled by the negative regulator Ure2 and four DNA-binding GATA-like transcription factors: two activators (Gln3 and Gat1) and two repressors (Dal80 and Gzf3). In the presence of preferred nitrogen sources, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner, whereas upon growth under non-preferred nitrogen conditions, the GATA activators relocate to the nucleus and mediate the transcription of NCR-sensitive genes. Even though the Target of Rapamycin Complex 1 (TORC1) as well as several phosphatases are involved in regulating Gln3 and Gat1 subcellular localization, a detailed mechanistic understanding of the NCR process is still lacking.

In the first part of this work, we have shown that class C and D VPS (vacuolar protein sorting) components, involved in Golgi-to-vacuole vesicular trafficking, are required for intact Gat1 and Gln3 nuclear localization in response to TORC1-inhibiting rapamycin treatment or upon shifting cells from rich to poor nitrogen conditions. The requirements of Vps proteins for Gln3 function are media-specific: a requirement after rapamycin treatment was observed in minimal but not in rich medium. Moreover, we have seen that a significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. These observations support the view that GATA factor regulation in response to nitrogen signals seems to occur at intracellular compartments.

In a second step, we confirmed an important role for the anabolic glutamate dehydrogenase (Gdh1) within NCR, through the control of Gat1 function. However, since we observed a strong correlation between the anabolic activity of Gdh1 and its NCR regulatory capacity, we do not exclude that an alteration of Gdh1-substrates or any other metabolite could be responsible for the phenotype exhibited by gdh1 mutants. We also showed that there is no simple and direct link between the intracellular levels of glutamine/glutamate (reported in the literature as signals for NCR), TORC1 activity and NCR. In conclusion, the mechanisms regulating the perception of the quality of the nitrogen sources are still not fully understood.

Several screens for multi-copy suppression of mutated phenotypes were conducted during this work and led to the identification of several elements (URE2, BAP2, STP2, GZF3 and KDX1) that could interfere with NCR-sensitive gene expression. Among these, the gene encoding the Kdx1 kinase was identified in two independent screens.

In the last part of this work, we uncovered a role for leucine in NCR signaling. We showed that the addition of leucine in the culture medium could impair Gat1-dependent expression of certain NCR genes, while leucine starvation had no effect at this level. The repressive effect of leucine appeared to involve elements of the SPS signaling pathway which is required for the induction of genes encoding amino acid transporters in response to extracellular amino acids. The mechanism(s) by which leucine regulates Gat1 function is still not fully clear and requires further investigation:La levure Saccharomyces cerevisiae adapte l’expression de ses gènes selon la disponibilité en azote dans son environnement au moyen d’un contrôle majeur appelé répression catabolique azotée (NCR, pour « nitrogen catabolite repression ». L’expression des gènes NCR est contrôlée par un régulateur négatif de type prion (Ure2) et quatre facteurs de transcription de type GATA :deux activateurs, Gat1 et Gln3 et deux répresseurs, Dal80 et Gzf3. Bien que le complexe TORC1 et les phosphatases qu’il régule soient impliquées dans la régulation NCR, le mécanisme précis par lequel la NCR se produit est loin d’être compris.

Dans la première partie de ce travail, nous avons montré que les composants VPS (vacuolar protein sorting) de classe C et D, impliqués dans le trafic vésiculaire entre le Golgi et la vacuole, sont requis pour que Gat1 et Gln3 rejoignent le noyau en réponse à un traitement par la rapamycine, un inhibiteur de TORC1. En accord avec cette observation, nous avons montré que Gat1, comme Gln3, est associé aux membranes intracellulaires légères.

Dans un second temps, nous avons confirmé un rôle important pour la glutamate déshydrogénase anabolique (Gdh1) au sein de la NCR, par l’intermédiaire du contrôle de la fonction de Gat1. Cependant, étant donné qu’il semble exister une forte corrélation entre l’activité anabolique de Gdh1 et sa capacité à réguler la NCR, nous n’excluons pas qu’une altération des substrats de Gdh1 ou de tout autre métabolite pourrait être responsable du phénotype observé du mutant gdh1. Nous avons également montré qu’il n’existait pas de lien simple et direct entre niveaux intracellulaires de glutamine/glutamate, activité de TORC1 et signalisation NCR. En conclusion, les mécanismes conditionnant la perception de la qualité de l’aliment azoté sont encore méconnus à ce jour.

Plusieurs cribles de suppression multicopie ont été menés pendant ce travail et ont conduit à l’identification de plusieurs éléments pouvant éventuellement intervenir dans la voie de signalisation NCR. Parmi ceux-ci, le gène codant pour la kinase KDX1 a été identifié à deux reprises. Nous avons caractérisé en détail le rôle qu’elle joue dans la régulation des gènes NCR.

Dans la dernière partie de ce travail, nous avons montré que l’addition de leucine dans le milieu de culture pouvait affecter l’expression Gat1-dépendante de certains gènes NCR, alors que par ailleurs une carence en leucine est sans effet à ce niveau. Cet effet de répression par la leucine semble nécessiter des éléments de la voie de signalisation SPS, requise pour l’induction, en réponse aux acides aminés extracellulaires, de gènes codant pour des transporteurs d’acides aminés.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

The process of specific gene transcription by RNA polymerase II (Pol II) is initiated by the

binding of specific transcription factors to DNA. A global understanding of the mechanisms of gene

transcriptional regulation of Saccharomyces cerevisiae goes through the description of the targets and

the behavior of those transcription factors.

The GATA factors are specific transcription factors intervening in the regulation of Nitrogen

Catabolite Repression (NCR)-sensitive genes, a mechanism encompassing the transcriptional

regulations leading to the preferential use of good nitrogen sources of the growth medium of yeast in

the presence of less good nitrogen sources. Those 4 GATA factors involved in NCR comprise 2

activators (Gat1 and Gln3) and 2 repressors (Gzf3 and Dal80).

Generally speaking, the promoters of genes have always been described like the main place for

the integration of the transcription regulation signals relayed by the general and specific transcription

factors and the chromatin remodeling factors. Furthermore, the GATA factors have been described as

integrating the external signals of nitrogen availability thanks to their specific DNA binding to

consensus GATA sequences in the promoter of NCR-sensitive genes. The results presented here

introduce many nuances to the model, notably implying new proteins but also new regions in the

regulation process of the NCR-sensitive gene regulation. Indeed, the first goal of this work is to

discover and understand the mechanisms of NCR-sensitive gene regulation that will explain the

variations in their expression levels in the presence of various nitrogen sources and their dependency

towards the GATA factors.

Strikingly, it appeared that GATA factor positioning was not limited to the promoter, but

occurred also in the transcribed region. It seems that the transcription factors may have been driven

by the general transcription machinery (Pol II). The binding of a chromatin remodeling complex, RSC,

has also been demonstrated in the coding region of NCR-sensitive genes. Moreover, the binding of the

histone acetyltransferase complex, SAGA, recruited by the GATA activators, was highlighted along

NCR-sensitive genes. The SAGA complex was also implied in their transcriptional regulation.

Finally, a ChIP-sequencing experiment revealed an unsuspected number and diversification of

targets of the GATA factors in yeast, which were not limited to NCR-sensitive genes.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Thesis (MSc)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: The wine yeast Saccharomyces cerevisiae has the ability to utilize several different nitrogenous compounds to fulfill its metabolic requirements. Based upon different growth rates of the yeast in a particular nitrogen source, nitrogen compounds have been classified as either good or poor nitrogen sources. In an environment which contains different quality nitrogen sources, such as grape must, the yeast first utilizes good and then the poor nitrogen sources. This discrimination between good and poor nitrogen sources is referred to as nitrogen catabolite repression (NCR). Examples of good nitrogen sources are ammonia, glutamine and asparagine. Nitrogen sources such as allantoin, y-aminobutyrate (GABA), arginine and proline are poor quality nitrogen sources. Several regulatory proteins, Ure2p, Gln3p, Da180p,Gat1pand Deh1p, mediate NCR in S. cerevisiae. These trans-acting factors regulate transcription of NCR sensitive genes. All these proteins, except Ure2p, bind cis-acting elements in the promoters of genes that are responsible for degradation of poor nitrogen sources. Gln3p is an activator of NCR sensitive genes in the absence of good nitrogen sources. The predominant mechanism by which NCR functions is by using Ure2p to inactivate the activator Gln3p in the presence of a good nitrogen source. Several research groups have studied the Ure2p, mainly due to its prion-like characteristics. The Ure2p has two domains: a prion inducing domain located in the N-terminal region and a NCR regulatory domain located in the C-terminal domain. The aims of this study were (i) to determine the part of the C-terminal domain which is responsible for NCR, (ii) to establish if ure2 deletion mutants produce less ethyl carbamate during wine fermentations and (iii) if NCR functions in industrial yeast strains. Nested deletions of the URE2 gene revealed that the NCR regulatory domain resides in the last ten amino acids of the Ure2p. This was established by Northern blot analysis on the NCR sensitive genes DAL5, CAN1, and GAP1 genes. Ethyl carbamate in wine is produced by spontaneous chemical reaction between urea and ethanol in wine. Urea is produced by S. cerevisiae during the metabolism of arginine. Arginine is degraded to ornithine and urea by arginase, the product of the CAR1 gene. Degradation of urea by S. cerevisiae is accomplished by urea amidolyase, a bi-functional enzyme and product of the DUR1,2 gene which is subject to NCR. This study investigated if a ure2 mutant strain produced less ethyl carbamate during wine fermentations. Wine fermentations were conducted with diploid laboratory strains: a ure2 mutant strain and its isogenic wild type strain. GC/MS analysis of the wine revealed that the ure2 mutant produced less ethyl carbamate but more ethanol than the wild type strain when arginine, di-ammoniumphosphate, asparagine or glutamine were added as nitrogen sources, in combinations and separately. There was no significant difference between the wild type fermentation and the ure2 mutant fermentation when no nitrogen was added. It was found that a combination between the deletion of URE2 and the addition of a good nitrogen source resulted in lower levels of ethyl carbamate. High density micro array analysis done on an industrial strain wine yeast in Chardonnay grape must revealed that the GAP1, CAN1, CAR1 and DUR1,2 genes, responsible for transport and metabolism of arginine and degradation of urea, are NCR sensitive. These data strongly suggest that NCR functions in industrial yeast strains.
AFRIKAANSE OPSOMMING: Die wyngis Saccharomyces cerevisiae kan verskillende stikstofbronne gebruik om in sy stikstofbehoeftes te voldoen. Stikstofbronne word as goeie of swak stikstofbronne geklassifiseer op grond van die groeitempo van die gis op die betrokke stikstofbron. 'n Goeie stikstofbron laat die gis vinniger groei as wat dit op 'n swak stikstofbron sou groei. In omgewings soos druiwemos waar daar 'n verskeidenheid van stikstofbronne teenwoordig is, sal die gis eers die goeie bronne en daarna die swak bronne benut. Stikstofbronne soos ammonium, asparagien en glutamien word geklassifiseer as goeie bronne. Allantoïen, y-amino-butaraat (GABA), prolien en arginien word as swak stikstofbronne geklassifiseer. Die meganisme waarmee S. cerevisiae tussen die stikstofbronne onderskei, staan as stikstof kataboliet onderdrukking (NCR) bekend. Die proteïene wat vir verantwoordelik is NCR naamlik Ure2p, Gln3p, Gat1 p, Dal80p en Deh1 p, bind met die uitsondering van Ure2p, almal aan cis-werkende elemente in die promoters van NCR-sensitiewe gene. Die trans-werkende faktore reguleer die transkripsie van NCR-sensitiewe gene. NCR werk hoofsaaklik deur die inhibering van Gln3p deur Ure2p in die teenwoordigheid van 'n goeie stikstofbron. Die oorgrote meerderheid NCR-sensitiewe gene word deur Gln3p in die afwesigheid van 'n goeie stikstofbron geaktiveer. Heelwat navorsing is op die prionvormings vermoë van Ure2p gedoen. Ure2p het twee domeine: 'n N-terminale domein wat vir prionvorming verantwoordelik is en die C-terminale domein waar die NCR funksie van Ure2p gesetel is. Die doel van die studie was (i) om te bepaal waar in die C-terminale domein van Ure2p die NCR regulering geleë is, (ii) of ure2 delesie mutante minder etielkarbamaat tydens wynfermentasies produseer en (iii) of NCR in industriële gisrasse funksioneel is. Delesie analises van URE2 het getoon dat die NCR regulerings domein in die laaste tien aminosure gesetel is. Dit is vas gestel m.b.v. noordlike klad tegniek analises op die OALS, CAN1 en GAP1 gene.Etielkarbamaat in wyn word deur die spontane chemiese reaksie tussen ureum en alkohol geproduseer. Ureum word gedurende die metabolisme van arginien in S. cerevisiae geproduseer. Arginien word deur arginase, produk van die CAR1 geen, na ornitien en ureum afgebreek. Die bi-funksionele ureum amidoliase, gekodeer deur die DUR1,2 geen, breek ureum na CO2 en NH/ af. As gevolg van die NCRsensitiwiteit van dié gene is ondersoek ingestel na In ure2 mutant se vermoë om minder etielkarbamaat tydens wynfermentasies te produseer. Chardonnay druiwemos is met In diploiede laboratorium ras en die isogeniese ure2 mutant gefermenteer. GC/MS analise op die wyn het getoon dat die ure2 mutant minder etielkarbamaat, maar meer alkohol in vergelyking met die wilde tipe gis produseer, as arginien, di-ammoniumfosfaat, asparagien en glutamien, afsonderlik of gesamentlik byvoeg is. Daar was egter nie In merkwaardige verskil tussen die fermentasies waar geen stikstof bygevoeg is nie. Dit dui daarop dat In kombinasie van In URE2 delesie en die byvoeging van stikstof etielkarbamaat vlakke verlaag. Mikro-skyfie analise van In industriële gis in Chardonnay mos het getoon dat die GAP1, CAN1, CAR1 en DUR1,2 gene wat verantwoordelik is vir die transport en metabolisme van arginien en degradasie van ureum, wel NCR-sensitief is. Dit dui daarop dat NCRwel in industriële gisrasse funksioneel is.

A characteristic feature of species of Trichoderma is the production of concentric rings of conidia in response to alternating light-dark conditions. In response to a single burst of light, a single ring of conidia forms at what was the colony perimeter. On the basis of these observations, competency to photoconidiate has been proposed to be due to the age and metabolic rate of the hyphal cell. In this study, conidiation was investigated in five biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens and T. harzianum) using both a morphological and molecular approach. All five isolates produced concentric conidial rings under alternating light-dark conditions on potato-dextrose agar (PDA), however, in response to a 15 min burst of blue light, only T. asperellum and T. virens produced a clearly, defined conidial ring which correlated with the colony margin at the time of light exposure. Both T. harzianum and T. hamatum photoconidiated in a disk-like fashion and T. atroviride produced a broken ring with a partially filled in appearance. On the basis of these results, it was postulated that competency to photoconidiate is a factor of the metabolic state of the hyphal cell rather than chronological age or metabolic rate. The influence of the source of nitrogen on photoconidiation was assessed on pH-buffered (pH 5.4) minimal medium (MM) amended with glutamine, urea or KNO₃. In the presence of glutamine or urea, T. asperellum and T. harzianum conidiated in a disk, whereas, when KNO₃ was the sole nitrogen source, a ring of conidia was produced. Further, in the presence of increasing amounts of glutamine, the clearly defined photoconidial ring produced on PDA by T. asperellum became disk-like. These results clearly demonstrated that primary nitrogen promotes photoconidiation in these isolates and strongly suggests that competency of a hyphal cell to conidiate in response to light is dependent on the nitrogen catabolite repression state of the cell. The experiments were repeated for all five isolates on unbuffered MM. Differences were apparent between the buffered and unbuffered experiments for T. atroviride. No photoconidiation was observed in T. atroviride on buffered medium whereas on unbuffered medium, rings of conidia were produced on both primary and secondary nitrogen. These results show that photoconidiation in T. atroviride is influenced by the buffering capacity of the medium. Conidiation in response to light by T. hamatum and T. virens was absent in all nitrogen experiments, regardless of the nitrogen source and buffering capacity, whereas both isolates conidiated in response to light on PDA. These results imply that either both sources of nitrogen are required for photoconidiation, or a factor essential for conidiation in these two isolates was absent in the minimal medium. Mycelial injury was also investigated in five biocontrol isolates of Trichoderma. On PDA, all isolates except T. hamatum conidiated in response to injury. On nitrogen amended MM, conidiation in response to injury was again observed in all isolates except for T. hamatum. In T. atroviride, injury-induced conidiation was observed on all medium combinations except the pH-buffered MM amended with glutamine or urea and T. virens conidiated in response to injury on primary nitrogen only, regardless of the buffering capacity. These results have revealed conidiation in response to injury to be differentially regulated between isolates/species of Trichoderma. On unbuffered MM amended with glutamine or urea, conidiation in response to injury occurred at the colony perimeter only in T. atroviride. It was hypothesised that the restriction of conidiation to the perimeter may be due to changes in the pH of the agar. The experiment was repeated and the pH values of the agar under the growing colony measured at the time of light induction (48 h) or injury (72 h). The areas under the hyphal fronts were acidified to below the starting value of the medium (pH 5.4) and the centres of the plates were alkalinised. The region of acidification at the time of stimuli correlated with the production of conidia, which implicates a role for crossregulation of conidiation by the ambient pH. The influence of the ambient pH on injury-induced conidiation was investigated in T. hamatum and T. atroviride on MM amended with glutamine and PDA, pH-buffered from pH 2.8 to 5.6. Thickening of the hyphae around the injury site was observed at the lowest pH values on MM in both T. atroviride and T. hamatum, however no conidia were produced, whereas both Trichoderma species conidiated on pH-buffered PDA in a strictly low pH-dependent fashion. This is the first observation of injury-induced conidiation in T. hamatum. The influence of the ambient pH on photoconidiation was assessed in T. hamatum, T. atroviride and T. harzianum using both buffered and unbuffered PDA from pH 2.8 to 5.2. On buffered PDA, no conidiation in response to light was observed above pH 3.2 in T. hamatum, above 4.0 in T. atroviride and above 4.4 in T. harzianum, whereas on unbuffered PDA it occurred at all pH values tested. It was postulated that conidiation at pH values above 4.4 on unbuffered PDA was due to acidification of the agar. The pH values of the agar under the growing colony were measured at the time of light exposure and in contrast to the MM with glutamine experiments, alkalisation of the agar had occurred in both T. atroviride and T. hamatum. No change in medium pH was recorded under the growing T. harzianum colony. These results indicate that low pH-dependence of photoconidiation is directly related to the buffering capacity of the medium. Recent studies have linked regulation of conidiation in T. harzianum to Pac1, the PacC orthologue. In fungi, PacC regulates gene expression in response to the ambient pH. In these studies pH-dependent photoconidiation occurred only on buffered PDA and on unbuffered PDA conidiation occurred at significantly higher ambient pH levels. It is proposed that the influence of ambient pH on conidiation in the isolates used in this study is not due to direct Pac1 regulation. The T. harzianum isolate used in this study produced profuse amounts of the yellow anthraquinone pachybasin. Production of this secondary metabolite was strictly pH-dependent, irrespective of the buffering capacity of the medium. Studies in T. harzianum have linked Pac1 regulation to production of an antifungal α-pyrone. pH-dependence on both buffered and unbuffered media strongly suggests that pachybasin production may also be under the control of Pac1. Photoconidiation studies on broth-soaked filter paper, revealed rhythmic conidiation in the pachybasin producing T. harzianum isolate. Diffuse rings of conidia were produced in dark-grown cultures and, in cultures exposed to light for 15 min at 48 h, the rings were clearly defined. These results show that conidiation is under the control of an endogenous rhythm in T. harzianum and represent the first report of circadian conidiation in a wild-type Trichoderma. A Free-Running Rhythm (FRR) assay was used to investigate rhythmic gene expression in T. atroviride IMI206040 and a mutant derivative, in which the wc-2 orthologue, blr-2, was disrupted. Over a 3 d period, expression of gpd, which encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, oscillated with a period of about 48 h. In the Δblr-2 mutant, the gpd rhythm was absent. These results revealed that in T. atroviride, gpd expression is under the control of an endogenous clock and that clock-regulated expression of gpd is associated with a functional BLR complex. Using degenerate primers, a portion of frq, which encodes the N. crassa clock oscillator FREQUENCY, was isolated from T. atroviride and used to probe the FRR assay northern blots. No frq expression was detected at any time point, which suggests that the circadian clock in Trichoderma does not involve FREQUENCY. In a concurrent study, orthologues of rco-1 (rcoT) were isolated and sequenced from T. atroviride and T. hamatum using a combination of degenerate, inverse and specific PCR. RcoT is an orthologue of the yeast global co-repressor Tup1 and in the filamentous fungi, RcoT orthologues have been demonstrated to negatively regulate conidiation. Genomic analysis of all available rcoT orthologues revealed the conservation of erg3, a major ergosterol biosynthesis gene, upstream from rcoT in ascomycetous filamentous fungi, but not in the ascomycetous yeast or in the basidiomycetes. These studies have significantly contributed to our understanding of the regulatory factors controlling conidiation in Trichoderma and have multiple implications for Trichoderma biocontrol; most notable the promotion of conidiation by primary nitrogen and low pH. Incubation conditions can be altered to suit the nitrogen and pH preferences of a biocontrol strain in order to promote cost effective conidial production, however this is not easily achieved in the soil, where the biocontrol strain must perform in a highly buffered environment optimised for plant growth. Successful use of Trichoderma biocontrol strains may involve the screening and targeting of strains to the appropriate pH conditions or the selection of new strains on the basis of capacity to perform under a given range of conditions.

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As fontes de nitrogênio do meio são classificadas como preferenciais e nãopreferenciais, de maneira que as primeiras inibem a expressão dos genes responsáveis pela metabolização das segundas por um mecanismo chamado de Repressão Catabólica do Nitrogênio (Nitrogen Catabolic Repression - NCR). No presente estudo avaliamos o padrão de regulação dos genes do metabolismo central do nitrogênio na levedura Dekkera bruxellensis. Foram definidos quatro grupos de fontes de nitrogênio baseados no crescimento celular. Em seguida, o padrão de expressão dos genes do metabolismo central do nitrogênio mostrou que fenilalanina, embora do grupo quatro, é o maior indutor das permeases Gap1p e Put4p, resultando em sua elevada taxa de consumo. Já a histidina, indutora da permease Put4p, promove maior indução dos genes que codificam as enzimas de assimilação de amônia. Quando o mecanismo NCR é inibido pela presença de metionina sulfoximina no meio, ocorre a desrrepressão dos genes que codificam as permeases. E finalmente, os resultados mostram que nitrato, definido no grupo dois induz o mecanismo de sinalização intracelular de regulação gênica semelhante ao que se observa quando as células estão no estado de privação de nitrogênio no meio. Isto complementa os estudos anteriores nos quais mostramos que a assimilação de nitrato altera o estado fisiológico da célula para respiração mesmo na presença de alta concentração de glicose no meio.
The nitrogen sources in the medium are classified as preferential or non-preferrential, so that the first inhibit expression of genes responsible for metabolism of the latter by a mechanism called Nitrogen Catabolic Repression (NCR). In the present study we evaluated the pattern of gene regulation of the central nitrogen metabolism in yeast Dekkera bruxellensis. It was defined four groups of nitrogen sources based on cell growth. Then, the expression pattern of the central nitrogen metabolism genes showed that phenylalanine, though belonging to group four, is the biggest inducer of genes of permeases Gap1p Put4p, resulting in its high consumption rate. Moreover, histidine induces the gene encoding permease Put4p and promoted the highest induction of the genes encoding the enzymes of ammonia assimilation. When the NCR mechanism was inhibited by the presence of methionine sulfoximine in the medium there was derepression of the genes encoding for permeases. Finally, the results showed that nitrate, defined in the group two, induced the intracellular signaling pathway gene regulation similar to that seen when cells are in a state of nitrogen deprivation in the middle. This complements our previous studies that showed that the nitrate assimilation alter the physiological state of the cell to respiration even in presence of high glucose concentration in medium.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The Paracoccidioides genus is composed of thermodimorphic fungus that causes paracoccidioidomycosis (PCM), an endemic human systemic mycosis in Latin America. These organisms grow as mycelium in temperatures below 28 °C and as yeast form in temperatures above 37 °C. Nitrogen is an important element in this microorganism’s nutrition that participates in the synthesis of proteins, nucleic acids and others biomolecules. In this regard, nitrogen uptake and metabolism are essential to growth and fungal establishment. When nitrogen levels and sources such as glutamine and ammonia concentration are limited, pathogenic fungus use a regulation system called Nitrogen Catabolic Repression that induces the expression of genes encoding permeases and enzymes required for the catabolism of secondary nitrogen sources, such as formamidase, gamma-glutamiltranspeptidase and urease. Gamma-glutamiltranspeptidase is an enzyme that catalyzes the first reaction of glutationa degradation and it has been the target of several studies about nitrogen starvation in various fungi. It has been observed that the expression of the gene encoding this enzyme was induced in limiting conditions of nitrogen and was repressed when the availability of nitrogen was high. Urease is an enzyme that catalyzes the degradation of urea in ammonia and carbonic acid. This enzyme is already known as a virulence factor in fungi such as Cryptococcus. neoformans, and also has been the target of studies about nitrogen starvation. In this study we expressed gamma-GT and urease proteins from Paracoccidioides brasiliensis, isolate Pb18, in Escherichia coli. The gene coding for Ggt and Ure were cloned in pET32a expression vector, and used for E. coli pLysS transformation. The recombinant proteins produced were shown to be catalytically active. Together, data obtained in this work could add knowledge about the role of gamma-GT and urease and can be used as a foundation for complementary experiments regarding nitrogen metabolism regulation, as well as in Paracoccidioides spp pathogenesis.
Resumo: O gênero Paracoccidioides é composto por fungos termodimórficos que causam a paracoccidioidomicose (PCM), uma micose sistêmica humana endêmica na América Latina. Quando cultivados em temperaturas menores que 28 °C o fungo cresce como micélio e em temperaturas em torno de 37 °C, como levedura. O nitrogênio é um importante nutriente para os micro-organismos, pois participa da síntese de proteínas, ácidos nucléicos e outras biomoléculas. Nesse sentido, a captação e o metabolismo de nitrogênio são essenciais para o crescimento e o estabelecimento do fungo no hospedeiro. Quando os níveis de nitrogênio e fontes como glutamina e amônia estão em concentrações limitantes, os fungos patogênicos utilizam um sistema de regulação chamado Repressão Catabólica de Nitrogênio que induz a expressão de genes que codificam permeases e enzimas necessárias para o catabolismo de fontes secundárias de nitrogênio como a formamidase, a gama-glutamil transpeptidase e a urease. A gama-glutamil transpeptidase é uma enzima que catalisa a primeira reação da degradação da glutationa. Ela tem sido alvo de estudos de privação de nitrogênio em diversos fungos, nos quais foi observada uma alta expressão do gene codificador dessa enzima em condições limitantes de nitrogênio, enquanto que, em alta disponibilidade de nitrogênio a sua expressão era reprimida. A urease é uma enzima que degrada uréia em amônia e ácido carbônico. Ela já é conhecida por ser um fator de virulência em alguns fungos, como Cryptococcus neoformans, e também tem sido alvo de estudos de privação de nitrogênio. Neste estudo nós expressamos as proteínas gama-GT e urease de Paracoccidioides brasiliensis, isolado Pb18, em sistema heterólogo bacteriano de Escherichia coli. O fragmento dos genes codificadores de Ggt e Ure foram clonados em vetor de expressão pET32a e os respectivos clones foram utilizados na transformação de células de E. coli pLySs. As proteínas recombinantes produzidas mostraram estar cataliticamente ativas. Os dados obtidos neste trabalho puderam acrescentar conhecimentos sobre as enzimas gama-GT e urease e podem ser usados como base para experimentos complementares em relação à regulação do metabolismo de nitrogênio bem como na patogênese de Paracoccidioides spp.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.