Coordinate regulation of ammonium transporters and components of the mating and pathogenic programs in Ustilago maydis
ABSTRACT: Next generation sequencing was used to assess transcriptome changes in haploid U. maydis wild type and mutant strains grown under nitrogen-replete and low ammonium media. Overall design: Wild type and mutant strains were grown for 48 hours at 26 C, in microarray media ((6.25% Holliday Salt Solution (Holliday, 1974), 1% glucose, and 2% agar) with either 30 mM Glutamine/50 mM Ammonium sulfate (Hi) or 50 uM Ammonium sulfate (Lo)
Project description:The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL disrupted) and wild-type strain, and to what was reported for the wild-type strain under exponential growth related to key processes involved in driving the process of nitrogen fixation in A. vinelandii. These results further illuminate a number of additional genes associated with siderophore synthesis, molybdate transfer and electron transfer that are likely associated with biological nitrogen fixation. Overall design: Azotobacter vinelandii was grown in standard Burk's media and standard Burk's media containing 20 mM Ammonium sulfate. Azotobacter vinelandii mutant strain deregulated for nitrogen fixation was grown in standard Burk's media. All samples were harvested at various time points, total RNA was extracted and mRNA was enriched. Libraries were constructed, and RNA was sequenced using Illumina paired-end reads.
Project description:In this study we show the transcriptional analysis after shifting the Ustilago maydis from media with ammonium as a nitrogen source to media lacking ammonium. From these, 49 genes were up-regulated and 41 were down-regulated. The functional description and gene ontology terms associated to the diferentially expressed genes revealed that various key pathways were represented, including, secondary metabolism, the metabolism of nitrogen, amino acid, fatty acid and amino sugar, among others, suggesting that the interplay of U. maydis with its N2 fixing bacterial endosymbiont is a flexible process that may be active during the adaptation to the fungus to the different nitrogen sources, and possibly during its pathogenic style of life. Overall design: The strain of U. maydis used in this study was FB2. The strain, was maintained at -70 ºC, recovered in complete medium and then grown in liquid minimum medium (MM) at 28 ºC. U. maydis cells were inoculated to MM with (NH4)NO3 as nitrogen source (+N), and in the same basal medium without (NH4)NO3 (-N), and incubated under shaking conditions at 28 ºC for 24 h. To identify the differentially expressed genes of U. maydis cells growing fixation conditions, we prepared two RNA-Seq libraries from three biological replicates pooled of yeast-like cells of U. maydis FB2 strain grown with an added nitrogen source (+N), or without an added nitrogen source (-N) obtained at the end of the log phase (24h). The RNA-seq libraries were sequenced by Illumina HiSeq200 using the paired-end protocol (Illumina Inc. San Diego, CA, USA).
Project description:In the phytopathogenic Basidiomcete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled via the b-mating type locus. The b-mating type locus encodes two homeodomain transcription factors, bE and bW, which form an active heterodimeric complex when they are derived from different b-alleles. Rbf1 encodes a zinc-finger transcription factor that is expressed upon formation of an active bE/bW heterodimer. To identify gene regulated by rbf1 independently from b, the b mating type locus was replaced by a copy of rbf1 under control of the arabinose-inducible crg1 promoter (strain CP27). Samples were taken 5h after induction of rbf1 gene expression. Strain JB2, carrying a deletion of the b-locus, was used as control. Overall design: Strains were grown to an OD600 of 0.4-0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter-sterilized). For induction of the bE and bW genes, cells were inoculated in liquid array medium containing 1% (w/v) arabinose instead of glucose as a carbon source. For induction, cells were washed once with inducing medium. Experiments were performed in two biological replicates for each time point.
Project description:We used the previously designed oligonucleotide-based microarray (Burgmann et al. Environmental Microbiology 2007, 9: 2742-2755) to detect the transcripts of R. pomeroyi DSS-3 genes when the cells were cultured under steady-state carbon (glucose), nitrogen (ammonium), phosphorus (phosphate), or sulfur (sulfate) limitation. Overall design: A total of 14 mRNA samples were hybridized to the arrays (three biological replicates from glucose, ammonium, phosphate, or sulfate limitation and one technical replicate each for ammonium or sulfate limitation)
Project description:We used the previously designed oligonucleotide-based microarray (Burgmann et al. Environmental Microbiology 2007, 9: 2742-2755) to detect the transcripts of R. pomeroyi DSS-3 genes when the cells were cultured under steady-state carbon (glucose), nitrogen (ammonium), phosphorus (phosphate), or sulfur (sulfate) limitation. A total of 14 mRNA samples were hybridized to the arrays (three biological replicates from glucose, ammonium, phosphate, or sulfate limitation and one technical replicate each for ammonium or sulfate limitation)
Project description:J1 mouse embryonic stem cells (mESCs) and NIH-3T3 fibroblasts were grown in standard media conditions. Hybridized cells were fixed with 4% paraformaldehyde; incubated for 12 hours at 30C in an RNA preserving hybridization (RPH) buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C in with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM pH 8.0 Tris, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K). one replicate per sample
Project description:Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc, and grown in standard 2i/serum media conditions. Hybridized iPSCs were fixed with 4% paraformaldehyde; incubated for 12 hours at 30C in an RNA preserving hybridization buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C in with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM pH 8.0 Tris, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K). RNA extracted from live and hybridized iPSCs (1 sample each) was compared by microarray.
Project description:Iron is an essential cofactor for enzymes involved in numerous cellular processes. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Overall design: Saccharomyces cerevisiae DBY7286 strain was grown from very low density to mid-log phase (A600 = 0.5, approximately 18 hrs.) in defined-iron SD minimal medium containing only the supplements necessary to meet auxotrophic requirements. Defined-iron SD minimal media were prepared with yeast nitrogen base lacking iron and copper, supplemented with 1 µM copper sulfate, 25 mM MES pH 6.1, 1 mM Ferrozine (Fluka), and the indicated concentrations of ferrous ammonium sulfate 10 µM (low iron) or 300 µM (high iron). All cells were grown at 30°C with shaking and four independent cultures were prepared for each growth condition
Project description:Plants aquire nitrogen from the soil, most commonly in the form of either nitrate or ammonium. Unlike ammonium, nitrate must be reduced (with NADH and ferredoxin as electron donors) prior to assimilation. Thus, nitrate nutrition imposes a substantially greater energetic cost than ammonium nutrition. Our goal was to compare the transcriptomes of nitrate-supplied and ammonium-supplied plants, with a particular interest in characterizing the differences in redox metabolism elicited by different forms of inorganic nitrogen. We used microarrays to compare the short-term transcriptional response to either nitrogen supply or ammonium supply in Arabidopsis roots. Genes upregulated or downregulated by nitrate only, ammonium only, or both ammonium and nitrate were identified and analyzed. Arabidopsis thaliana (Col-0) plants were grown hydroponically until they reached growth stage 5.10. They were then transferred to a nitrogen-free medium for 26 hr and then supplied with 1 mM nitrate or 1 mM ammonium. RNA isolation (and subsequent microarray analysis) was performed on root tissue isolated just before nitrogen supply (time 0) and at 1.5 hr and 8 hr after nitrogen supply (1.5 hr nitrate, 8 hr nitrate, 1.5 hr ammonium, 8 hr ammonium).
Project description:Transcriptional profiling of Candida parapsilosis in media with a preferred nitrogen source (ammonium sulfate) and a non-preferred source (isoleucine) to identify genes that are subject to Nitrogen Catabolite Expression. Gene expression of wild type and dal81 deletion strains were compared during growth in complex nitrogen sources (YPD), in minimal media with a preferred nitrogen source (YNB+ammonium sulfate) and minimal medium with a non-preferred source (GABA). Overall design: 8 independent samples with three replicates of each, generating 24 samples.