Project description:Protein complexome analysis of Pseudomonas aeruginosa PAO1 at OD 0.3 in LB media through 10-40% glycerol gradient fractionation in 21 samples.
Project description:b-Oxidative enzymes for fatty acid degradation (Fad) of long-chain fatty acid (LCFA), a component of lung surfactant phosphatidylcholine, are induced in vivo during lung infection in cystic fibrosis patients, which could contribute to nutrient acquisition and pathogenesis of Pseudomonas aeruginosa. In addition, fatty acid biosynthesis (Fab) is essential for the syntheses of two virulence controlling acylated-homoserine-lactone molecules in this organism. We mapped the promoter regions of the fadBA5-operon (PA3014 and PA3013) and a fadE homologue (PA2815) involved in Fad and the fabAB-operon involved in Fab. Focusing on the transposon mutagenesis of strain PAO1 carrying the PfadBA5-lacZ fusion, we identified a regulator for the fadBA5-operon to be PsrA (PA3006). Transcriptome analysis of the DpsrA mutant indicates its importance in regulating b-oxidative enzymes, which confirms a previous proteomic study. We further showed that induction of the fadBA-operon responds to LCFA signals, and this induction requires the presence of PsrA, suggesting that PsrA binds to LCFA to derepress fadBA5. Electrophoresis mobility shift assay indicate specific binding of PsrA to the fadBA5-promoter region. This binding is disrupted by specific LCFA (C18:1D9, C16:0, and to a lesser extent C14:0), but not by the first intermediate of b-oxidation, acyl-CoA. We proposed that PsrA is a Fad-regulator that binds and responds to LCFA signals in Pseudomonas aeruginosa. Experiment Overall Design: PAO1 and PAO1-psrA::Tn cultures grown in LB and cells were harvested at mid-log phase. Total RNA was isolated from both samples, and used for cDNA synthesis. And then, the cDNA for both samples were fragmented and labeled. The cDNA of PAO1 was used for 2 GeneChips, and PAO1-psrA::Tn cDNA was used for three GeneChips.
Project description:P. aeruginosa PAO1 PA2663-UW expression in biofilm cells relative to P. aeruginosa PAO1 WT-UW expression in biofilm cells. All samples cultured in LB with glass wool. Keywords: Mutation
Project description:P. aeruginosa PAO1 wild type and PA2663 mutant strains expression in biofilm cells relative to P. aeruginosa PAO1 wild type strain expression in biofilm cells. All samples cultured in LB with glass wool Keywords: Biofilm
Project description:To analyze the impact of elevated c-di-GMP concentrations in P. aeruginosa, we expressed pleD* on an inducible vector (pHERD20T) in the PAO1 wild-type strain. PleD is a DGC from Caulobacter cresentusand the pleD* construct variant encodes for a constitutively active enzyme due to four amino acid exchanges (T120N, T214A, P234H, N357Y).We aimed to analyze the cellular consequences of increased c-di-GMP levels in the opportunistic pathogen P. aeruginosa on a global scale. We therefore grew the pleD* harboring PAO1 as well as the empty vector control PAO1 in LB medium, added arabinose (0.2%) to the medium to induce pleD* expression and harvested the cells in exponential growth phase, when the cells exhibited elevated c-di-GMP levels of about two-fold (see manuscript). We are discribing the characteristics of elevated c-di-GMP with a Multi-Omics-Dataset.
Project description:Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model.
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.
Project description:To better understand the effects of Dkstatin treatment, we compared transcriptome landscapes of PAO1 grown without or with either Dkstatin. A global view of the entire transcriptome uncovered the substantial differences between cells treated with Dkstatin-1 or Dkstatin-2.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling of control of P. aeruginosa PAO1 (RNA-seq) to transcriptome profiling of farnesol-treated P. aeruginosa PAO1 and to evaluate protocols for optimal high-throughput data analysis. Methods:LB medium (50 mL) was inoculated with exponential growth phase P. aeruginosa PAO1 at a concentration of 108 CFU/mL. Farnesol was then added at a concentration of either 0 (control) or 0.56 mg/mL, in triplicate. All six experiment groups were incubated in a water bath shaker at 37 ºC with a shaking rate of 180 rpm for 5 h. Cells were then sampled and centrifuged from the three control groups and three farnesol treatment groups, respectively. The cell precipitates were separately snap-frozen at -80ºC. Total RNA was isolated from cells using Trizol (Life Technologies, USA) according to the manufacturer’s protocol. Results: Our RNA-seq results showed that less than 100 genes of P. aeruginosa PAO1 were differentially expressed following farnesol treatment. We found that about 1.7% of all detected genes (96 of 5554 genes) were more than two-fold differentially expressed following farnesol treatment. Conclusions: