Transcription profiling of Pseudomonas aeruginosa roxSR and anr mutant strains under aerobic conditions
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ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa
SUBMITTER: Hiroyuki Arai
PROVIDER: E-GEOD-17296 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To investigate the gene expression profile of pellicle cells of Pseudomonas aeruginosa, microarray analysis was performed. Transcriptome profiles of pellicle cells and planktonic cells grown in LB medium were determined by Affymetrix GeneChip. Gene expression pattern that is specific to pellicle cells was evaluated by comparing the data set with that of planktonic cells. Pseudomonas aeruginosa wild type (PAO1ut) strain was cultivated aerobically in LB in Erlenmeyer flasks under static or shaking conditions, and total RNAs were extracted at 24 hours (static culture) and early stationary phase (OD600 = 1.4, shaking culture). The experiment was performed in duplicate independent cultures.
2011-03-31 | E-GEOD-24784 | biostudies-arrayexpress
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.
2009-07-31 | GSE17296 | GEO
Project description:DNA microarray analysis was employed to investigate the transcriptome response to nitric oxide in Pseudomonas aeruginosa. We focused on the role played by the nitric oxide-response regulators DNR and FhpR and an oxygen-response regulator ANR in the response. The transcriptome profiles of the P. aeruginosa strains before and after exposure to nitric oxide under the microaerobic conditions were analyzed. Wild type, its anr, dnr, and fhpR mutants, and the anr mutant that express dnr were used for the analyses. Pseudomonas aeruginosa wild type (PAO1ut), anr mutant (RManr), dnr mutant (RMdnr), anr mutant that constitutively expresses DNR (RManrEXdnr), and fhpR mutant (PDM2665) were cultivated microaerobically in LB in 1-liter jar fermenter. When optical density at 600 nm reached 0.3, nitric oxide-saturated water was added to the medium (final nitric oxide concentration was 20 micro-M). RNA was isolated from a 10 ml aliquot of the culture prior to the addition of nitric oxide and at 5 min after the addition. The experiment was performed in duplicate independent cultures.
2014-12-08 | E-GEOD-58862 | biostudies-arrayexpress
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.
2010-07-07 | E-GEOD-17179 | biostudies-arrayexpress
Project description:Transcriptional profiles of Escherichia coli MG1655 in mixed culture with Pseudomonas aeruginosa PAO1 showed a number of E. coli genes to be upregulated including purA-F and other genes associated with purine synthesis. In contrast, genes associated with pyrimidine synthesis were unaffected. Competition experiments in both planktonic and biofilm cultures, using three purine synthesis mutants, purD, purH, and purT showed little difference in E. coli survival from the parent strain. As purines are components of the cell signals, cAMP and c-di-GMP, we conducted competition experiments with E. coli mutants lacking adenylate cyclase (cyaA), cAMP phosphodiesterase (cpdA), and the catabolite receptor protein (crp), as well as ydeH, an uncharacterized gene that has been associated with c-di-GMP synthesis. Survival of the cyaA and crp mutants during co-culture were significantly less than the parent strain. Supplementation of the media with 1mM cAMP could restore survival of the cyaA mutant but not the crp mutant. In contrast, survival of the cpdA mutant was similar to the parent strain. Survival of the ydeH mutant was moderately less than the parent, suggesting that cAMP has more impact on E. coli mixed culture growth than c-di-GMP. Addition of 1 mM indole restored the survival of both the cyaA and crp mutations. Mutants in genes for tryptophan synthesis (trpE) and indole production (tnaA) showed a loss of competition and recovery through indole supplementation, comparable to the cyaA and crp mutants. Overall, these results suggest indole and cAMP as major contributing factors to E. coli growth in mixed culture. Two replicates of LB grown E. coli ZK126 and P. aeruginosa PAO1 were grown in pure culture at 37C to an OD of 0.3 (log phase) and then mixed 1:1 and incubated for an additional 45 min. RNA was extracted, purified, reverse transcribed to cDNA and then analyzed on E. coli and P. aeruginosa chips from Affymetrix. Expression profiles were compared to pure culture E. coli and P. aeruginosa grown on LB.
2011-11-09 | E-GEOD-26931 | biostudies-arrayexpress
Project description:Pseudomonas aeruginosa is one of the most frequent pathogen dominant in complicated urinary tract infections (UTI). To unravel the adaptation strategies of P. aeruginosa to the conditions in the urinary tract and to define the underlying regulatory network an artificial growth system mimicking the conditions in the urinary tract was established. Transcriptome analyses were used to investigate the physiological status of P. aeruginosa under this conditions. We performed comparisons to identify genes induced under artificial urinary tract conditions to unravel the adaptive strategies and the underlying regulatory network used by Pseudomonas aeruginosa during urinary tract infections using Affimetrix GeneChips. Pseudomonas aeruginosa wild type strain PAO1 was grown in an artificial in vitro growth system mimicking the conditions in the urinary tract. Therefore, biofilms were grown on the surface of membrane filters placed on agar plates at 37 °C up to the late logarithmic state under aerobic and anaerobic conditions (incubated in an anaerobic beanch). An artificial urine medium (AUM) simulating the averaged urine of an human adult was used as nutrient souce. 10-fold diluted Luria Bertani (LB)-medium was used as reference medium. For growth under oxygen depletion the media were supplemented with 50 mM KNO3 to sustain anaerobic respiration. The biofilms were harveted at this time points and resuspsended in 0.9% (w/v) NaCl. The OD578 of biofilm suspension was 0.8 for all tested conditions. First comparison: Identification of genes induced or repressed under aerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown aerobically for 18 h to the late logarithmic phase in biofilms on AUM with the transcriptome profile of the PAO1 strain, which was grown aerobically for 18 h to the late logarithmic phase in biofilms on 10-fold diluted LB. Second 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 anaerobically for 2 days up to the late logarithmic phase in biofilms on AUM supplemented with 50 mM nitrate with the transcriptome profile of the PAO1 strain, which was grown anaerobically for 2 days up to the late logarithmic phase in biofilms on 10-fold diluted LB supplemented with 50 mM nitrate.
2013-06-30 | E-GEOD-33160 | biostudies-arrayexpress
Project description:Among multiple interconnected pathways for L-Lysine (L-Lys) catabolism in pseudomonads, Pseudomonas aeruginosa PAO1 employed the decarboxylase and the transaminase pathways. However, up till now several genes involved in the operation and regulation of these pathways were still missing. Transcriptome analyses coupled with promoter activity measurements and mutant growth phenotype analysis lead us to identify several new members of the L-Lys and D-Lys catabolic pathways and their regulatory elements, including argR to trigger lysine decarboxylation into cadaverine, PauR for the γ-glutamylation pathway of polyamine catabolism into 5-aminovalerate, gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for L-Lys efflux, and lysP for L-Lys uptake. Gene expression microarray, including probe preparation, hybridization, fluidics run and chip scan, was performed by Georgia State University DNA/Protein Core Facility. P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of 10mM L-glutamate supplemented with 10 mM L-lysine, cadaverine, 5-amino valerate, glutaric acid, D-lysine or 5mM L-pipecolate. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Data collection was carried out using GCOS 1.4 software (Affymetrix). Probe intensity values were normalized to a target value of 500 with normalization factor equal to 1. Data analysis was performed using GeneSpring GX 11 Software (Aglient, Palo Alto,CA). Related Research Papers: 1. Indurthi SM, Chou HT, Lu CD. Molecular Characterization of lysR-lysXE, gcdR-gcdHG, and amaR-amaAB Operons for Lysine Export and Catabolism: A Comprehensive Lysine Catabolic Network in Pseudomonas aeruginosa PAO1. Microbiology. 2015 Submitted [EMID:04803de65c782] 2. Chou HT, Li J, and Lu CD. Functional Characterization of the agtABCD and agtSR Operons for γ-Aminobutyrate and δ-Aminovalerate Uptake and Regulation in Pseudomonas aeruginosa PAO1. Curr. Microbiology. 2014 Jan;68(1):59-63. 3. Chou HT, Li JY, Peng YC, Lu CD. Molecular characterization of PauR and its role in control of putrescine and cadaverine catabolism through the γ-glutamylation pathway in Pseudomonas aeruginosa PAO1. J Bacteriol. 2013 Sep; 195(17):3906-13. 4. Chou HT, Hegazy M, Lu CD. L-lysine Catabolism is Controlled by Arginine/ArgR in Pseudomonas aeruginosa PAO1. J Bacteriol. 2010 Nov;192(22):5874-80
2015-12-09 | E-GEOD-75502 | biostudies-arrayexpress
Project description:The transcriptome of two different Pseudomonas aeruginosa mutant strains were compared to the Pseudomonas aeruginosa wild type strain in the stationary growth phase
2010-04-24 | E-MEXP-2606 | biostudies-arrayexpress
Project description:Phagocytosis and killing of the opportunistic pathogen Pseudomonas aeruginosa by polymorphonuclear neutrophils (PMNs) is the major antipseudomonal host defense of vertebrates. By screening a transposon library of the clinical P. aeruginosa isolate TBCF10839 that can grow and replicate in PMNs, a mutant was identified that was most strongly sensitized to killing by PMNs. The inactivated gene PA1572 termed nadK1 was found to encode an ATP:NAD kinase. The transcriptomes of the TBCF10839 wild type and nadK1 mutant were investigated in the presence of PMNs or H2O2. Exposure to H2O2 led to diametrical mRNA expression profiles. H2O2-degrading enzymes were upregulated by wild type, but not by nadK1 mutant. This data demonstrates that NadK1 is crucial for the response of P. aeruginosa to reactive oxygen species. The transcriptomes of Pseudomonas aeruginosa TBCF10839 wild type and nadK1 mutant were comparatively investigated in the presence of polymorphonuclear neutrophils or H2O2 in order to investigate the role of NadK1 in oxidative stress response. These samples represent test samples. The expression profile of Pseudomonas aeruginosa strain TBCF10839 wild type cells, isolated from a Cystic Fibrosis patient, was investigated under standard growth conditions in batch culture in Luria broth (LB) with no treatment. These samples represent reference samples. All samples were analyzed in duplicate.
2010-05-27 | E-GEOD-21704 | biostudies-arrayexpress
Project description:We have tested the effect of deletion of Topoisomerase I on genes transcription of the bacteria Pseudomonas. aeruginosa. Topoisomerase I is a conserved protein that modulate the topology state of the DNA from super-coiled to relaxed. To this end we have compared the transcription profile of a topA::GentR mutant to that of the wild-type strain. The topA mutant contains in topA gene a Mariner transposon with a gentamicin resistance cassette. It is part of the non-redundant mutant library of P. aeruginosa PA14 strains. WT and mutant cells were grown to OD 2.0 in LB 200rpm shaking at 37C.
2011-12-21 | E-GEOD-24038 | biostudies-arrayexpress