Transcriptome analysis of Aspergillus fumigatus delta crzA mutant upon 10 and 30 minutes treatment with CaCl2 (200 mM)
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ABSTRACT:
ORGANISM(S): Aspergillus fumigatus
SUBMITTER: Frederico Soriani
PROVIDER: E-GEOD-15432 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory Ca2+/Calmodulin binding subunit. A mutant of A. fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and delta calA mutant strains. Our results showed that although the mitochondrial function is reduced in the delta calA mutant strain, its respiratory chain is functional and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified several genes that encode transcription factors that have increased mRNA expression in the delta calA mutant and that could be involved in the Cal-CrzA pathway. Deletion mutants for these transcription factors had also reduced susceptibility to itraconazole, caspofungin, and sodium dodcyl sulfate. For the time course microarray experiments, 1.0 x 109 conidia of both A. fumigatus wild type [CEA17 delta akuB(KU80)] and mutant strain (delta calA) were used to inoculate 400 ml of pre-warmed liquid cultures in complete medium (YG) in 1 L erlenmeyer flasks. Cultures were allowed to grow for 6, 8, 12, 18 and 24 hours in a reciprocal shaker (250 rpm) at 37°C. At each time point, germilings were harvested by centrifugation or filtration and total RNA was extracted. Hybridization experiments were competitive using Cy3 or Cy5-labeled probes derived from both wild type and delta calA strain grown for 6, 8, 12, 18 and 24 hours. Hybridizations were performed using the wild type RNA as reference in comparison to the mutant strain (test RNA) for the same time point. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene in-slide replicates were condensed (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects (see Supplementary files linked below).
2008-12-22 | E-GEOD-13939 | biostudies-arrayexpress
Project description:Marine cyanobacteria are thought to be the most sensitive of the phytoplankton groups to copper toxicity, yet little is known of the transcriptional response of marine Synechococcus to copper shock. Global transcriptional response to two levels of copper shock was assayed in both a coastal and an open ocean strain of marine Synechococcus using whole genome expression microarrays. Both strains showed an osmoregulatory-like response, perhaps as a result of increasing membrane permeability. This could have implications for marine carbon cycling if copper shock leads to dissolved organic carbon leakage in Synechococcus. The two strains additionally showed a reduction in photosynthetic gene transcripts. Contrastingly, the open ocean strain showed a typical stress response whereas the coastal strain exhibited a more specific oxidative or heavy metal type response. In addition, the coastal strain activated more regulatory elements and transporters, many of which are not conserved in other marine Synechococcus strains and may have been acquired by horizontal gene transfer. Thus, tolerance to copper shock in some marine Synechococcus may in part be a result of an increased ability to sense and respond in a more specialized manner. In this series four conditions have been analyzed. These are moderate copper shock for Synechococcus sp. WH8102 and CC9311 (pCu 11.1 and pCu 10.1, respectively), and high copper shock for WH8102 and CC9311 (pCu 10.1 and pCu 9.1, respectively). For each slide, an experimental RNA sample was labeled with Cy3 or Cy5 and was hybridized with a reference RNA from a non-copper-shocked sample labeled with the other Cy dye. There are six or eight slides per condition, each with two biological replicates. There are three or four technical replicates for each biological replicate including at least one flip-dye comparison. Each slide contains six replicate spots per gene.
2010-05-17 | E-GEOD-13910 | biostudies-arrayexpress
Project description:Primary productivity of open ocean environments, such as those inhabited by marine picocyanobacteria Synechococcus sp.WH8102, are often limited by low inorganic phosphate (P). To observe how this organism copes with P starvation, we constructed a full genome microarray and examined differences in gene expression under P-limited and P-replete growth conditions. To determine the temporal nature of the responses, comparisons were made for cells newly entered into P-stress (at a time point corresponding to the induction of extracellular alkaline phosphatase activity) and a later time point (late log phase). In almost all instances the P starvation response was transitory, with 36 genes showing significant upregulation (>log2 fold) while 23 genes were highly downregulated at the early time point; however, these changes in expression were maintained for only five of the upregulated genes. Knockout mutants were constructed for genes SYNW0947 or SYNW0948, comprising a two component regulator hypothesized to play a key role in regulating the response to P-limitation. A high degree of overlap in the sets of genes affected by P-limited conditions and in the knockout mutants supports this hypothesis; however there is some indication that other regulators may play a role in this response in Synechococcus sp. WH8102. Consistent with what has been observed in many other cyanobacteria, the Pho regulon of this strain is comprised largely of genes for alkaline phosphatases, P transport or P metabolism. Interestingly, however, the exact composition and arrangement of the Pho regulon appears highly variable in marine cyanobacteria. In this series four conditions have been analyzed. These are low phosphate stress during early log phase, low phosphate stress during late log phase, SYNW0947 mutation, and SYNW0948 mutation. There are six slides per condition, each with two biological replicates. There are three technical replicates for each biological replicate including one flip-dye comparison. The exception is the low phosphate stress during late log phase experiment which has a total of five slides, two biological replicates with three and two technical replicates, respectively, and one flip-dye comparison for each biological replicate. Each slide contains six replicate spots per gene.
2010-05-18 | E-GEOD-13170 | biostudies-arrayexpress
Project description:The GacS/GacA signal transduction system is a central regulator in Pseudomonas spp., including the biological control strain P. fluorescens Pf-5, in which GacS/GacA controls the production of secondary metabolites and exoenzymes that suppress plant pathogens. A whole genome oligonucleotide microarray was developed for Pf-5 and used to assess the global transcriptomic consequences of a gacA mutation in P. fluorescens Pf-5. In cultures at the transition from exponential to stationary growth phase, GacA significantly influenced transcript levels of 632 genes, representing more than 10% of the 6147 annotated genes in the Pf-5 genome. Transcripts of genes involved in the production of hydrogen cyanide, the antibiotic pyoluteorin, and the extracellular protease AprA were at a low level in the gacA mutant, whereas those functioning in siderophore production and other aspects of iron homeostasis were significantly higher in the gacA mutant than in wild-type Pf-5. Notable effects of gacA inactivation were also observed in the transcription of genes encoding components of a type VI secretion system and cytochrome C oxidase subunits. Two novel gene clusters expressed under the control of gacA were identified from transcriptome analysis, and we propose global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp. In this series two conditions have been analyzed. A gacA mutant of Pseudomonas fluorescens Pf-5 was harvested at early (OD 0.5) and late (OD 2.4) time points and compared to wild-type Pf-5 harvested in parallel. For each slide, an experimental RNA sample from a gacA mutant was labeled with Cy3 or Cy5 and was hybridized with a reference RNA sample from wild-type Pf-5 labeled with the other Cy dye. There are six slides per condition. Each condition is represented by three biological replicates. There are two flip-dye replicates for each biological replicate. Each slide contains three replicate spots per gene.
2010-03-03 | E-GEOD-16898 | biostudies-arrayexpress
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 µM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (anaerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), K2SO4 (15 mM), sodium fumarate (50 mM), Luria Broth (5%) and can amino acids (0.1%). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in mini fermenter vessels in 175 ml of the above defined minimal media (anaerobic). The vessel was continually sparged with N2 to ensure anaerobiosis. CORM-3 was added at a final concentration of 100 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (OD600 of 0.4) before addition of CO-RM. For RNA extraction, 30 ml was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5à First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2à SSC/0.1 % SDS, 1à SSC, 0.2à SSC and 0.01à SSC. Microarray slides were dried by centrifugation at 1500 à g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-24 | E-GEOD-12878 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE12877: Transcriptional profiling of Escherichia coli after addition of CO-RMs to aerobically growing cells GSE12878: Transcriptional profiling of Escherichia coli after addition of CO-RMs to anaerobically growing cells Refer to individual Series
2010-06-24 | E-GEOD-13048 | biostudies-arrayexpress
Project description:Escherichia coli strain MG1655 was grown to mid-log phase in defined aerobic media. One sample was treated with 30 µM CORM-3, the other sample was a control. The volume (30 ml), temperature (37oC) and shaking (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Cells were grown in defined minimal medium (aerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), and K2SO4 (15 mM). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher. E. coli strain MG1655 was grown in 250 ml side arm flasks in 30 ml of the above defined minimal media (aerobic). CORM-3 was added at a final concentration of 30 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (Klett values of 30-40) before addition of CO-RM. The total 30 ml volume was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagenâs RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min. This was supplemented with 6 microlitres of 5à First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 3 hours. The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2à SSC/0.1 % SDS, 1à SSC, 0.2à SSC and 0.01à SSC. Microarray slides were dried by centrifugation at 1500 à g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ⥠twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.
2010-06-24 | E-GEOD-12877 | biostudies-arrayexpress
Project description:Host-pathogen interactions are of great importance in understanding the pathogenesis of infectious microorganisms. We developed in vitro models to study porcine respiratory tract pathogens using an immortalized epithelial cell line, the St. Jude Porcine Lung (SJPL) cell line. In conditions where cytotoxicity was absent or low, the transcriptomic profile of A. pleuropneumoniae after contact with the porcine lung epithelial cells was determined using DNA microarrays. Genes such as tadB, rcpA, members of a putative adhesion locus, and a gene with high homology to the Hsf autotransporter adhesin of Haemophilus influenzae were upregulated as well as genes pgaBC involved in biofilm biosynthesis, while capsular polysaccharide-associated genes were downregulated. Samples from A. pleuropneumoniae grown by either adhesion to SJPL cells or planktonic growth over SJPK cells were combined with samples from A. pleuropneumoniae grown in DMEM medium and co-hybridized on the microarray. Four hybridizations were conducted for each sample combination type. Microarray analysis was carried out using the TM4 Suite of softwares (TIGR) and the SAM algorithm, using a false discovery rate (FDR) value of 0%. For the planktonic and adhesion experiments, Cy5 signal (test) was compared to Cy3 signal (control) in order to obtain a list of significant differentially expressed genes. In order to obtain the list of differentially expressed genes between planktonic growth and adhesion, log2 ratios were compared in TM4 also using the SAM algorithm. Functional classification was performed using TIGR’s Comprehensive Microbial Resource.
2008-07-09 | E-GEOD-12009 | biostudies-arrayexpress
Project description:Aflatoxins are carcinogenic fungal secondary metabolites. Levels of aflatoxins in agricultural commodities are stringently regulated by many countries. A cluster of genes is responsible for aflatoxin biosynthesis by Aspergillus flavus and other closely related species. Expression of the clustered aflatoxin genes is governed by a complex network of regulatory mechanisms. To better understand the molecular events that are associated with aflatoxin production, transcription profiling by microarray analyses which compared three independent aflatoxigenic A. flavus strains to individual isogenic progenies that no longer produced aflatoxins after serial transfers was carried out. Twenty-two significantly differentially expressed features were identified. After physical mapping using the A. oryzae genome sequence as the reference, the number of unique genes was reduced to 16. Compared to the parental strains, changes in the aflatoxin gene expression levels in the progenies were not significant, which suggests that the inability to produce aflatoxins is not caused by decreased expression. The only gene showing higher expression levels in the progenies is homologous to glutathione S-transferease genes. Overexpression of this gene, named hcc, at six- to nine-fold in an aflatoxigenic A. flavus did not cause discernible changes in colony morphology or aflatoxin production. Loss of aflatoxin production after serial transfers may not result from a single event but caused by multiple factors. Keywords: Compartiave hybridization toxigenic and atoxigenic lines of Aspergillus Aspergillus flavus NRRL 29459, NRRL 29474, and NRRL 29490 are aflatoxigenic strains originated from soil collection in a peanut field (Terrell Co., Georgia, USA). Strains 459B-20-2, 474A-20, and 499A-20 were nonaflatoxigenic isolates obtained after 20 serial transfers of the parental strains on potato dextrose agar slants (Horn and Dorner 2002). Comparsions in each experiment consisted of one aflatoxigenic parental strain and one nonaflatoxigenic progeny, compared after 48- or 72-hr growth. Each comparison was repeated with duplicate dye-flip.
2010-05-26 | E-GEOD-8185 | biostudies-arrayexpress
Project description:Microarray analysis was used to identify the calcium-responsive genes dependent on CrzA in the filamentous fungus Aspergillus nidulans. In order to identify such genes, we conducted the two types of experiment. One was a comparison between wild type with calcium treatment and wild type without calcium treatment. Another was a comparison between wild type with calcium treatment and crzA mutant with calcium treatment. From a comparison between the results of these experiments, we could identify the genes whose expression was induced or repressed in response to calcium in a manner dependent on CrzA. KEY WORD; Aspergillus nidulans, calcium response, crzA Conidia of wild type or crzA mutant strains were incubated at 37C in CD medium for 18h and treated with or without calcium (final concentration; 50 mM) for 15 min. The mycelia were harvested and frozen in liquid nitrogen, ground to a powder, and used for RNA preparation. mRNA was purified and used for hybridization experiments. A total of 2 hybridizations were performed for each microarray experiment described in the summary. The following replicates were carried out: 1. In-slide replicates were carried out for each analyses. 2. Dye swap replicates were carried out for each analyses. The slides were scanned with an Axon GenePix 4000B scanner (Molecular Devices). The resulting TIFF images were imported into GenePix Pro and fluorescent intensity of spots were calculated for each of the Cy3 and Cy5 channels. Global normalization was applied to all analyses. Following normalization, spots whose Cy3 or Cy5 intensity was less than 0 were removed from the data set (the exceptional case was that intensity of the other channel was more than 100). The dye-swap replicates and in-slide replicates were subjected to all analyses. Finally, gene expression ratios (channel 2/channel 1) were calculated for each replicates. Gene expression was considered to be significantly higher or lower whenever the spot intensity changed by at least 2-fold in all four replicates.
2008-10-02 | E-GEOD-12953 | biostudies-arrayexpress