Comparison of Aspergillus fumigatus transcriptome grown on selected nitrogen sources
ABSTRACT: Transcriptome of A. fumigatus shifted from ammoniumtartrate to different nitrogen sources and incubated for a defined time was compared. After 16h preculture, the fungus was transferred into fresh medium containing ammonium tartrate, sodium nitrate, proline or bsa as nitrogen source. After 1h, fungus was reisolated, RNA was prepared from fungus, transcriptome was assessed and used for further analysis. Ammoniumtartrate, sodiumnitrate, BSA and proline as nitrogen sources, 2 biological replicates for each source. A. fumigatus liquid media shifts were performed according to ref. (Narendja et al.,2002) with minor modifications: 200 ml minimal medium base with 5 mM ammonium tartrate was inoculated with 10^8 conidia freshly harvested. A. fumigatus ATCC 46645 conidia were grown at 37°C and 150 rpm for 16 hours. This pre-culture was then harvested, washed liberally with sterile saline and divided into mycelial masses of equal size on a sterile surface. The portions were then added to 100 ml of minimal medium base without nitrogen source. When a defined carbon source was needed, 1% glucose was added. Flasks were incubated for 20 min at 37°C. Thereafter, one of the following sterile nitrogen sources was added: ammonium tartrate to a final concentration of 5 mM, Sodiumnitrate to a final concentration of 10 mM, 1 g proline suspended in 2 ml minimal medium base, or 0.5 g BSA (Albumin Fraktion V, Roth), suspended in 15 ml minimal medium base. The cultures were incubated for another 60 minutes, harvested by filtering through Miracloth, snap frozen in liquid nitrogen, and ground using a cooled mortar to obtain a fine powder.
Project description:Aspergillus fumigatus is an opportunistic, airborne pathogen causing invasive aspergillosis in immunocompromised patients. During the infection process A. fumigatus is challenged by hypoxic microenvironments occurring in inflammatory, necrotic tissue. To gain further insights into the adaptation mechanism, A. fumigatus was cultivated in an oxygen-controlled chemostat under hypoxic and normoxic conditions. Transcriptome analysis revealed significant increases in transcripts associated with cell wall polysaccharide metabolism, amino acid and metal ion transport, nitrogen metabolism and glycolysis. A concomitant reduction in transcript levels was observed with cellular trafficking and G-protein coupled signaling. To learn more about the functional roles of hypoxia-induced transcripts we deleted A. fumigatus genes putatively involved in reactive nitrogen species detoxification (fhpA), NAD+ regeneration (frdA, osmA) nitrogen metabolism (niaD, niiA) and respiration (rcfB). We show that the NO-detoxifying flavohemoprotein fhpA is strongly induced by hypoxia independent of the nitrogen source, but is dispensable for hypoxic survival. By deleting the nitrate reductase gene niaD, the nitrite reductase gene niiA and the two fumarate reductases genes frdA and osmA, we found that alternative electron acceptors such as nitrate and fumarate do not have a significant impact on growth of A. fumigatus during hypoxia, but that functional mitochondrial respiratory chain complexes are essential under these conditions. Inhibition studies indicated that primarily complex III and IV play a crucial role in the hypoxic growth of A. fumigatus.
Project description:The response of P. putida KT2440 (PB2440) to preferential carbon source and varying nitrogen levels was studied in this experiment. The culture was grown in minimal medium with 2mM glucose as carbon source and different concentration of ammonium chloride as nitrogen source. For nitrogen limiting condition 2mM ammonium chloride was used whereas, for nitrogen rich condition 20mM ammonium chloride was added in the medium. Overall it was seen that the strain could grow efficiently in varying nitrogen conditions by adapting or regulating their metabolism without compromising on the growth rate. To ascertain how the cells were coping with the nitrogen stress, gene expression by microarray was performed. RNA extraction was done using Qiagen RNeasy minikit (Germany). Standard Affymetrix protocol was followed for hybridization with GeneChip P. aeruginosa Genome Array supplied by Affymetrix as GeneChip for P. putida were not supplied by any company.
Project description:The response of P. putida CF600 to preferential carbon source and varying nitrogen levels was studied in this experiment. The culture was grown in minimal medium with 2mM phenol as carbon source and different concentration of ammonium chloride as nitrogen source. For nitrogen limiting condition 2mM ammonium chloride was used whereas, for nitrogen rich condition 20mM ammonium chloride was added in the medium. Overall it was seen that the strain could grow efficiently in varying nitrogen conditions by adapting or regulating their metabolism without compromising on the growth rate. To ascertain how the cells were coping with the nitrogen stress, gene expression by microarray was performed. RNA extraction was done using Qiagen RNeasy minikit (Germany). Standard Affymetrix protocol was followed for hybridization with GeneChip P. aeruginosa Genome Array supplied by Affymetrix as GeneChip for P. putida were not supplied by any company.
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.
Project description:Corynebacterium glutamicum, a gram-positive soil bacterium used for the industrial production of amino acids such as L-glutamate and L-lysine, is able to use a number of different nitrogen sources, such as ammonium, urea, or creatinine. In this communication, we show that L-glutamine serves as an excellent nitrogen source for C. glutamicum and allows similar growth rates in glucose minimal medium as ammonium. A transcriptome comparison revealed a strong induction of the nitrogen starvation response when glutamine was used as nitrogen source. Subsequent growth experiments with a variety of mutants defective in nitrogen metabolism showed that glutamate synthase is crucial for glutamine utilization, while a putative glutaminase is dispensable under the experimental conditions used. The fact that the glutamate synthase encoding gltBD operon is under strict nitrogen control explains the necessity for induction of the nitrogen starvation response. The paradox situation that the nitrogen starvation response is induced although intracellular L-glutamine levels are high has implications on nitrogen sensing. In contrast to other gram-positive and gram-negative bacteria such as Bacillus subtilis, Salmonella typhimurium, and Klebsiella pneumoniae, a drop in glutamine concentration obviously does not serve as a nitrogen starvation signal in C. glutamicum. Three biological replicates were performed. To analyse how L-glutamine influences global gene expression when used as sole nitrogen source instead of ammonium, DNA microarray analyses were performed. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either L-glutamine or ammonium sulphate as nitrogen source.
Project description:The basidiomycete Ustilago maydis is the causal agent of corn smut disease and induces tumor formation during biotrophic growth in its host plant maize. The Usilago maydis genome harbors a homolog to the GATA transcription factors Nit2 and AreA that act as global regulators of nitrogen catabolite repression in filamentous model fungi Neurospora crassa and Aspergillus nidulans. We aimed at resolving the role of the Ustilago maydis homolog Ncr1 for the utilization of complex nitrogen sources and pathogenicity. Sporidia of the indicated Ustilago maydis strains were grown overnight in ammonium minimal medium (Holliday, 1976) and samples for total RNA extraction were taken 2h after transfer to minimal medium lacking any nitrogen source (-N) during the exponential growth phase to assess those genes that are regulated in response to nitrogen starvation. The solopathogenic strain SG200 (control) and deletion mutants of (Nitrogen catabolite repression1) Ncr1 and (Target of Ncr1) Ton1, both being in the SG200 background, were studied in two independent experiments (one experiment for Ton1). Per strain and experiment, three biological replicate samples were analyzed (except for only biological replicates for Ncr1 in the second experiment).
Project description:This study is measuring the steady-state levels of mRNA in wild-type Caulobacter crescentus grown in M2 defined medium containing either ammonium or nitrate as the sole nitrogen source. Four independent cultures of Caulobacter crecentus were grown in each of two medium conditions: M2(nitrate)glucose and M2(ammonium)glucose. Cultures in each medium type were grown to OD660=0.3 and RNA was isolated from each.
Project description:Objective: We analyzed changes in A. fumigatus gene expression profile at various stages of an in vitro model of aspergillosis to study the adaptation of A. fumigatus to the blood environment. Results: Most of virulence factors described to be involved in aspergillosis were not activated during the blood phase. We found three active processes to be activated in the later phase that may help to the adaptation: Iron homeostasis, a partial secondary metabolite cluster and the formation of detoxification enzymes. Conclusions: We propose that A. fumigatus is unable to grow in blood and it requires a metabolic change that allows the organism to shut down all uptake and energy-consume mechanisms, resulting in a resting mycelial stage. We performed gene expression profile by sequencing mRNA of A. fumigatus that were growm under two conditions, Minimal Medium (M) and human blood (B), and at different times: before placing the fungus in the final medium (pre), at 30' and at 180', with 2 biological replicates per condition.
Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored. RNA from cells grown on nitrogen-free synthetic medium under nitrogen fixation (experiment) and combined nitrogen source as ammonium chloride (control) was used for two-color whole-genome microarray approach.
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