Circadian clock regulation of the glycogen synthase (gsn) gene by WCC is critical for rhythmic glycogen metabolism in Neurospora crassa.

Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in Neurospora crassa We find that glycogen synthase (gsn) mRNA, glycogen phosphorylase (gpn) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while gsn was necessary for glycogen production, constitutive gsn expression resulted in high and arrhythmic glycogen levels, and deletion of gpn abolished gsn mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that gsn promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic gsn mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated gsn transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis.

The copper-inducible copAB operon in Xanthomonas citri subsp. citri is regulated at transcriptional and translational levels.

Upstream open reading frames (ORFs) are frequently found in the 5'-flanking regions of genes and may have a regulatory role in gene expression. A small ORF (named cohL here) was identified upstream from the copAB copper operon in Xanthomonascitri subsp. citri (Xac). We previously demonstrated that copAB expression was induced by copper and that gene inactivation produced a mutant strain that was unable to grow in the presence of copper. Here, we address the role of cohL in copAB expression control. We demonstrate that cohL expression is induced by copper in a copAB-independent manner. Although cohL is transcribed, the CohL protein is either not expressed in vivo or is synthesized at undetectable levels. Inactivation of cohL (X. citri cohL polar mutant strain) leads to an inability to synthesize cohL and copAB transcripts and consequently the inability to grow in the presence of copper. Bioinformatic tools predicted a stem-loop structure for the cohL-copAB intergenic region and revealed that this region may arrange itself in a secondary structure. Using in vitro gene expression, we found out that the structured 5'-UTR mRNA of copAB is responsible for sequestering the ribosome-binding site that drives the translation of copA. However, copper alone was not able to release the sequence. Based on the results, we speculate that cohL plays a role as a regulatory RNA rather than as a protein-coding gene.

Functional diversity in the pH signaling pathway: an overview of the pathway regulation in Neurospora crassa.

Microorganisms have the ability to adapt and respond to different environmental conditions, whether they are stressful or not. Although the detection and/or responding mechanisms are often unknown, a large number of proteins may participate in signal transduction pathways involved in environmental stimulus to induce physiological and cellular events. Here, we examine the important role in cell homeostasis that extracellular pH plays in different fungi, and summarize the recent data reported in distinct organisms, by comparing them to the well-characterized mechanisms firstly described in Aspergillus and yeast. While most of the knowledge regarding the cellular processes triggered by the pH signaling pathway is based on the work in these two organisms, new data have been emerging in a diverse group of filamentous fungi, namely the involvement of this signaling pathway in metabolism and fungal pathogenicity. In this review, we present the major aspects of the pH signaling pathway in different model organisms, focusing on the protein components and the biological processes influenced by this pathway. In particular, we discuss novel cellular processes regulated by this pathway in the fungus Neurospora crassa. The diversity of functional processes that are affected under pH stress highlights how broadly this condition impacts on basic cellular processes in fungi and reveals how divergent fungal species are.

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α.

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways.

BACKGROUND: Glycogen and trehalose are storage carbohydrates and their levels in microorganisms vary according to environmental conditions. In Neurospora crassa, alkaline pH stress highly influences glycogen levels, and in Saccharomyces cerevisiae, the response to pH stress also involves the calcineurin signaling pathway mediated by the Crz1 transcription factor. Recently, in yeast, pH stress response genes were identified as targets of Crz1 including genes involved in glycogen and trehalose metabolism. In this work, we present evidence that in N. crassa the glycogen and trehalose metabolism is modulated by alkaline pH and calcium stresses. RESULTS: We demonstrated that the pH signaling pathway in N. crassa controls the accumulation of the reserve carbohydrates glycogen and trehalose via the PAC-3 transcription factor, which is the central regulator of the signaling pathway. The protein binds to the promoters of most of the genes encoding enzymes of glycogen and trehalose metabolism and regulates their expression. We also demonstrated that the reserve carbohydrate levels and gene expression are both modulated under calcium stress and that the response to calcium stress may involve the concerted action of PAC-3. Calcium activates growth of the Δpac-3 strain and influences its glycogen and trehalose accumulation. In addition, calcium stress differently regulates glycogen and trehalose metabolism in the mutant strain compared to the wild-type strain. While glycogen levels are decreased in both strains, the trehalose levels are significantly increased in the wild-type strain and not affected by calcium in the mutant strain when compared to mycelium not exposed to calcium. CONCLUSIONS: We previously reported the role of PAC-3 as a transcription factor involved in glycogen metabolism regulation by controlling the expression of the gsn gene, which encodes an enzyme of glycogen synthesis. In this work, we extended the investigation by studying in greater detail the effects of pH on the metabolism of the reserve carbohydrate glycogen and trehalose. We also demonstrated that calcium stress affects the reserve carbohydrate levels and the response to calcium stress may require PAC-3. Considering that the reserve carbohydrate metabolism may be subjected to different signaling pathways control, our data contribute to the understanding of the N. crassa responses under pH and calcium stresses.

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator.

The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative carbon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identification of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1(KO), rcm-1(RIP) and cre-1(KO) strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also confirmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1(KO) strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (5'-SYGGRG-3') in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

A genome-wide screen for Neurospora crassa transcription factors regulating glycogen metabolism.

Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5'-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression.

Extracellular lipids of Candida albicans biofilm induce lipid droplet formation and decreased response to a topoisomerase I inhibitor in dysplastic and neoplastic oral cells.

OBJECTIVE: Some microorganisms, i.e., Candida albicans, have been associated with cancer onset and development, although whether the fungus promotes cancer or whether cancer facilitates the growth of C. albicans is unclear. In this context, microbial-derived molecules can modulate the growth and resistance of cancer cells. This study isolated extracellular lipids (ECL) from a 36-h Candida albicans biofilm incubated with oral dysplastic (DOK) and neoplastic (SCC 25) cells, which were further challenged with the topoisomerase I inhibitor camptothecin (CPT), a lipophilic anti-tumoral molecule. METHODOLOGY: ECL were extracted from a 36-h Candida albicans biofilm with the methanol/chloroform precipitation method and identified with Nuclear Magnetic Resonance (1H-NMR). The MTT tetrazolium assay measured ECL cytotoxicity in DOK and SCC 25 cells, alamarBlue™ assessed cell metabolism, flow cytometry measured cell cycle, and confocal microscopy determined intracellular features. RESULTS: Three major classes of ECL of C. albicans biofilm were found: phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The ECL of C. albicans biofilm had no cytotoxic effect on neither cell after 24 hours, with a tendency to disturb the SCC 25 cell cycle profile (without statistical significance). The ECL-induced intracellular lipid droplet (LD) formation on both cell lines after 72 hours. In this context, ECL enhanced cell metabolism, decreased the response to CPT, and modified intracellular drug distribution. CONCLUSION: The ECL (PI, PC, and PG) of 36-h Candida albicans biofilm directly interacts with dysplastic and neoplastic oral cells, highlighting the relevance of better understanding C. albicans biofilm signaling in the microenvironment of tumor cells.

The Neurospora crassa PCL-1 cyclin is a PHO85-1 (PGOV) kinase partner that directs the complex to glycogen metabolism and is involved in calcium metabolism regulation.

Cyclins are a family of proteins characterized by possessing a cyclin box domain that mediates binding to cyclin dependent kinases (CDKs) partners. In this study, the search for a partner cyclin of the PHO85-1 CDK retrieved PCL-1 an ortholog of yeast Pcls (for Pho85 cyclins) that performs functions common to Pcls belonging to different cyclin families. We show here that PCL-1, as a typical cyclin, is involved in cell cycle control and cell progression. In addition, PCL-1 regulates glycogen metabolism; Δpcl-1 cells accumulate higher glycogen levels than wild-type cells and the glycogen synthase (GSN) enzyme is less phosphorylated and, therefore, more active in the mutant cells. Together with PHO85-1, PCL-1 phosphorylates in vitro GSN at the Ser636 amino acid residue. Modeling studies identified PHO85-1 and PCL-1 as a CDK/cyclin complex, with a conserved intermolecular region stabilized by hydrophobic and polar interactions. PCL-1 is also involved in calcium and NaCl stress response. Δpcl-1 cells are sensitive to high NaCl concentration; on the contrary, they grow better and overexpress calcium responsive genes under high calcium chloride concentration compared to the wild-type strain. The expression of the calcium-responsive CRZ-1 transcription factor is modulated by PCL-1, and this transcription factor seems to be less phosphorylated in Δpcl-1 cells since exhibits nuclear location in these cells in the absence of calcium. Our results show that PCL-1 locates at different cell regions suggesting that it may determine its activity by controlling its intracellular location and reveal an interesting functional divergence between yeast and filamentous fungus cyclins.

Transcriptional Control of the Production of Aspergillus fumigatus Conidia-Borne Secondary Metabolite Fumiquinazoline C Important for Phagocytosis Protection.

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.

Biochemical and biophysical characterization of the RVB-1/RVB-2 protein complex, the RuvBL/RVB homologues in Neurospora crassa.

The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation.

The cAMP-PKA signaling pathway plays an important role in many biological processes including glycogen metabolism. In this work we investigated its role in the Neurospora crassa glycogen metabolism control using mutant strains affected in components of the pathway, the cr-1 strain deficient in adenylyl cyclase activity therefore has the PKA pathway not active, and the mcb strain a temperature-sensitive mutant defective in the regulatory subunit of PKA therefore is a strain with constitutively active PKA. We analyzed the expression of the gene encoding glycogen synthase (gsn), the regulatory enzyme in glycogen synthesis as a potential target of the regulation. The cr-1 strain accumulated, during vegetative growth, glycogen levels much higher than the wild type strain indicating a role of the PKA pathway in the glycogen accumulation. The gsn transcript was not increased in this strain but the GSN protein was less phosphorylated "in vitro", and therefore more active, suggesting that the post-translational modification of GSN is likely the main mechanism controlling glycogen accumulation during vegetative growth. Heat shock down-regulates gsn gene transcription in the two mutant strains, as well as in the wild type strain, suggesting that the PKA pathway may not be the only pathway having a direct role in gsn transcription under heat shock. DNA-protein complexes were formed between the STRE motif in the gsn promoter and nuclear proteins from heat-shocked mycelium. However STRE was not able to induce transcription of a reporter gene in Saccharomyces cerevisiae, suggesting that the motif might be involved in a different way of regulation in the N. crassa gene expression under heat shock. The CRE-like DNA elements present in the gsn promoter were shown to be bound by different proteins from the PKA mutant strains. The DNA-protein complexes were observed with proteins from the strains grown under normal condition and under heat shock indicating the functionality of this DNA element. In this work we presented some evidences that the PKA signaling pathway regulates glycogen metabolism in N. crassa in a different way when compared to the well-characterized model of regulation existent in S. cerevisiae.

Comparative study of the interactions between fungal transcription factor nuclear localization sequences with mammalian and fungal importin-alpha.

Importin-α (Impα) is an adaptor protein that binds to cargo proteins (containing Nuclear Localization Sequences - NLSs), for their translocation to the nucleus. The specificities of the Impα/NLS interactions have been studied, since these features could be used as important tools to find potential NLSs in nuclear proteins or even for the development of targets to inhibit nuclear import or to design peptides for drug delivery. Few structural studies have compared different Impα variants from the same organism or Impα of different organisms. Previously, we investigated nuclear transport of transcription factors with Neurospora crassa Impα (NcImpα). Herein, NIT-2 and PAC-3 transcription factors NLSs were studied in complex with Mus musculus Impα (MmImpα). Calorimetric assays demonstrated that the PAC-3 NLS peptide interacts with both Impα proteins with approximately the same affinity. The NIT-2 NLS sequence binds with high affinity to the Impα major binding site from both organisms, but its binding to minor binding sites reveals interesting differences due to the presence of additional interactions of NIT-2-NLS with MmImpα. These findings, together with previous results with Impα from other organisms, indicate that the differential affinity of NLSs to minor binding sites may be also responsible for the selectivity of some cargo proteins recognition and transport.

The pH Signaling Transcription Factor PAC-3 Regulates Metabolic and Developmental Processes in Pathogenic Fungi.

The zinc finger transcription factor PAC-3/RIM101/PacC has a defined role in the secretion of enzymes and proteins in response to ambient pH, and also contributes to the virulence of species. Herein we evaluated the role of PAC-3 in the regulation of Neurospora crassa genes, in a model that examined the plant-fungi interactions. N. crassa is a model fungal species capable of exhibiting dynamic responses to its environment by employing endophytic or phytopathogenic behavior according to a given circumstance. Since plant growth and productivity are highly affected by pH and phosphorus (P) acquisition, we sought to verify the impact that induction of a Δpac-3 mutation would have under limited and sufficient Pi availability, while ensuring that the targeted physiological adjustments mimicked ambient pH and nutritional conditions required for efficient fungal growth and development. Our results suggest direct regulatory functions for PAC-3 in cell wall biosynthesis, homeostasis, oxidation-reduction processes, hydrolase activity, transmembrane transport, and modulation of genes associated with fungal virulence. Pi-dependent modulation was observed mainly in genes encoding for transporter proteins or related to cell wall development, thereby advancing the current understanding regarding colonization and adaptation processes in response to challenging environments. We have also provided comprehensive evidence that suggests a role for PAC-3 as a global regulator in plant pathogenic fungi, thus presenting results that have the potential to be applied to various types of microbes, with diverse survival mechanisms.

Characterization of an OrtT-like toxin of Salmonella enterica serovar Houten.

The Escherichia coli GhoT/GhoS system is a type V toxin-antitoxin system in which the antitoxin GhoS cleaves the GhoT mRNA, controlling its translation. GhoT is a small hydrophobic protein that damages bacterial membranes. OrtT is a GhoT-like toxin, but it apparently lacks a corresponding antitoxin and serves a different physiologic role. Using a profile hidden Markov model approach, a Salmonella enterica serovar Houten genome was screened to obtain homologs of GhoT/OrtT. We only found one protein (referred to here as OrtT-Sal) that shared more sequence identity with OrtT than GhoT. The chromosomal region around the coding sequence of OrtT-Sal suggests that it is an orphan toxin and can be under RpoH activation. To study OrtT-Sal, we chemically synthesized and expressed in E. coli the whole toxin and its N- and C-terminal regions (OrtT-Sal1-29 and OrtT-Sal29-57, respectively). Our findings have shown that the overproduction of the polypeptides resulted in severe growth inhibition and cell lysis. Using circular dichroism, we found that OrtT-Sal, OrtT-Sal1-29, and OrtT-Sal29-57 form an alpha-helical structure in the presence of SDS micelles or TFE. Finally, using carboxyfluorescein-loaded lipid vesicles, we determined that the polypeptides damage lipid membrane directly.

Correction to: Characterization of an OrtT-like toxin of Salmonella enterica serovar Houten.

In the article mentioned above an author's name was misspelled.

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa.

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif. DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Neurospora crassa developmental control mediated by the FLB-3 transcription factor.

Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species.

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction.

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.