Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators.
Project description:rs12-04_rnanonpolya - nad9 knockdown 1 - Mitochondrial transcriptome regulation and coordination with the nucleus - Following a previously established strategy (Val et al., 2011, Nucleic Acids Res. 39, 9262–9274), we express, from an estradiol-inducible nuclear transgene, a trans-cleaving ribozyme directed against the nad9 mitochondrial mRNA and associated as a trailor sequence to a tRNA mimic. The latter serves as a shuttle and ensures mitochondrial uptake of the chimeric RNA through the natural tRNA import pathway. In mitochondria, the ribozyme triggers cleavage and degradation of the target mRNA. The impact of the nad9 mRNA knockdown on the overall plant transcriptome. Control samples correspond to plants not induced with estradiol.
Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 µM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.
Project description:rs12-06_a9-mrnonpolya - matr knockdown 1 - Mitochondrial transcriptome regulation and coordination with the nucleus - Following a previously established strategy (Val et al., 2011, Nucleic Acids Res. 39, 9262–9274), we express, from an estradiol-inducible nuclear transgene, a trans-cleaving ribozyme directed against the matR mitochondrial mRNA and associated as a trailor sequence to a tRNA mimic. The latter serves as a shuttle and ensures mitochondrial uptake of the chimeric RNA through the natural tRNA import pathway. In mitochondria, the ribozyme triggers cleavage and degradation of the target mRNA. The impact of the matR mRNA knockdown on the overall plant transcriptome. Control plants express either no ribozyme (C0a and C0b) or the shuttle RNA combined with a ribozyme that has no specific target in A. thaliana (SD).
Project description:To assess the role of two redox-sensitive transcriptional regulators, RoxSR and ANR, in Pseudomonas aeruginosa under aerobic conditions, microarray analysis was performed. Transcriptome profiles of roxSR mutant and anr mutant aerobically grown in LB medium were determined by Affymetrix GeneChip at both the exponential phase and early stationary phase and compared to that of the wild type strain. Experiment Overall Design: Pseudomonas aeruginosa wild type (PAO1ut), roxSR mutant (ROX1), and anr mutant (PAO6261) strains were cultivated aerobically in LB in Erlenmeyer flasks, and total RNAs were extracted at both the exponential phase (OD600 = 0.3) and early stationary phase (OD600 = 1.4). The experiment was performed in duplicate independent cultures.
Project description:To assess the role of two redox-sensitive transcriptional regulators, RoxSR and ANR, in Pseudomonas aeruginosa under aerobic conditions, microarray analysis was performed. Transcriptome profiles of roxSR mutant and anr mutant aerobically grown in LB medium were determined by Affymetrix GeneChip at both the exponential phase and early stationary phase and compared to that of the wild type strain.
Project description:The transcription factor Anr regulates the response to low oxygen in P. aeruginosa and is inhibited by oxygen. We used microarrys to compare gene expression in P. aeruginosa PAO1 wild-type with an isogenic anr mutant in order to determine which transcripts are affected by Anr. We grew P. aeruginosa cells as biofilms on CFBE cells in order to model cystic fibrosis airways infections.
Project description:Purpose : The goal of this study was to use RNA Seq to define the regulon of the transciption factor Anr by comparing global transcriptional profiles of Pseudomonas aeruginosa strain PAO1 and a clinical isolate with their isogenic ?anr mutants, grown in colony biofilms at 1% oxygen. Methods : mRNA profiles were generated for laboratory strain PAO1 and for a clinical isolate J215, as well as for ?anr derivatives of each strain, in duplicate, by deep sequencing. Strains were grown for 12 hours in colony biofilms at 1% O2, 5% CO2 prior to RNA harvest. Ribosomal and transfer RNAs were removed using the MICROBExpress kit (Life Technologies). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters. mRNA profiles of 12 hour colony biofilms were generated for P. aeruginosa strains PAO1 WT, PAO1 ?anr, clinical isolate J215, and J215 ?anr, each in duplicate, by deep sequencing using Illumina HiSeq.