Expression data from PAO1 and its creBC derivative mutant (PAOΔcreBC)
ABSTRACT: We used microarrays to determine the expression profile of the P. aeruginosa creBC mutant regarding its parental wild type strain PAO1. Expression analysis from three independent replicates of PAO1 and its creBC derivative mutant (PAOΔcreBC) were performed under basal conditions and with cefoxitin and ceftazidime. cDNA synthesis, fragmentation, labeling and hybridization were performed according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol.
Project description:The transcriptional regulator AmpR controls expression of the AmpC ß-lactamase in P. aeruginosa and other bacteria. Studies have demonstrated that in addition to regulating ampC expression, AmpR also regulates the expression of the sigma factor AlgT/U and the production of some quorum-sensing regulated virulence factors. In order to understand the ampR regulon, we compared the expression profiles of PAO1 and its isogenic ampR mutant, PAO∆ampR in the presence and absence of sub-MIC ß-lactam stress. The analysis demonstrates that the ampR regulon is much more extensive than previously thought, with the deletion of ampR affecting the expression of over 300 genes. Expression of an additional 207 genes are affected by AmpR when the cells are exposed to sub-MIC ß-lactam stress, indicating that the ampR regulon in P. aeruginosa is much more extensive than previously thought. An inframe deletion of ampR was generated in P. aeruginosa PAO1. The wild type and ampR mutant strains were grown to mid-log phase and subjected to sub-MIC ß-lactam exposure. Total RNA was isolated from 2-hour ß-lactam exposed and unexposed cells to monitor changes in gene expression arising due to loss of ampR in the presence and absence of ß-lactam exposure.
Project description:P. aeruginosa PAO1 grown as lawns on Nematode Growth Medium prepared without supplementation (NGM Pi<0.1 mM) has high killing ability against C. elegans, however, no mortality in worms has been observed during 48 hrs when feeding on PAO1 lawns grown on phosphate supplemented full NGM Pi 25 mM, pH 6.0 medium. We used a microarray to define the virulence-related genes in P. aeruginosa grown as lawns in NGM Pi<0.1 mM vs NGM Pi25 mM pH 6.0 All samples for gene expression analysis were prepared in biological triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or NGM/Pi<0.1 mM were used for RNA isolation. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility
Project description:In this experiment the transcriptional response of the opportunistic human pathogen Pseudomonas aeruginosa to sublethal concentrations of NaClO was investigated. To this aim, four independent cultures of P. aeruginosa PAO1 grown in minimal medium BM2 were treated with NaClO (2 ug/ml) for 1 h at 37 C followed by RNA extraction and microarray analysis. Untreated cultures served as controls.
Project description:The bacterial transcription factor RpoN regulates an extensive network of genes whose products are involved in diverse biological functions. We constructed a small peptide termed the RpoN molecular roadblock, which binds to and blocks transcription from RpoN promoters. This RpoN molecular roadblock can be used in any bacterium to obtain information on the RpoN regulon. We expressed the RpoN molecular roadblock in P. aeruginosa PAO1 and used microarrays to identify genes that were differentially transcribed due to the RpoN molecular roadblock. The RpoN molecular roadblock was expressed in P. aeruginosa PAO1 in mid-exponential phase in rich media. Total RNA was isolated and prepped for Affymetrix GeneChips.
Project description:In this experiment the transcriptional response of the opportunistic human pathogen Pseudomonas aeruginosa towards physiological concentrations of the major human host defense peptide LL-37 was investigated using microarrays. To this aim, three independent cultures of P. aeruginosa PAO1 were grown until mid-log phase in Mueller-Hinton broth and subsequently incubated with either sublethal LL-37 concencentrations (20 µg/ml) or without peptide for 2 h at 37 °C following RNA extraction and microarray analysis.
Project description:The purpose of this study was to define the TZD effect in Pseudomonas aeruginosa. Transcriptional profiling of Pseudomonas aeruginosa wild-type strain,reference strain PAO1, as control Vs. PAO1 strain exposed to a final 0.02mM of TZD derivative ((z)-5-octylidenethiazolidine-2,4-dione).
Project description:D-Glutamate (D-Glu), an essential component of peptidoglycans, can be utilized as carbon and nitrogen source by Pseudomonas aeruginosa. DNA microarrays were employed to identify genes involved in D-Glu catabolism. Through gene knockout and growth phenotype analysis, the divergent dguR-dguABC (D-glutamate utilization) gene cluster was shown to participate in D-Glu catabolism and regulation. The dguA gene encodes a FAD-dependent D-amino acid dehydrogenase with D-Glu as its preferred substrate, and its promoter was specifically induced by exogenous D-Glu and D-Gln. The function of DguR as transcriptional activator of the dguABC operon was demonstrated by promoter activity measurements in vivo and by mobility shift assays with purified His-tagged DguR in vitro. Although the DNA-binding activity of DguR did not require D-Glu, the presence of D-Glu in the binding reaction was found to stabilize a preferred nucleoprotein complex. The dguB gene encodes a putative enamine/imine deaminase of the RidA family, but its role in D-Glu catabolism remained to be determined. While a lesion in dguC encoding a periplasmic solute binding protein did not affect growth on D-Glu, the AatJMQP transporter for acidic amino acid uptake was found essential for D-Glu and L-Glu utilization. Expression of this uptake system was subjected to induction by exogenous DL-Glu, most likely via the AauSR two-component system. In summary, DguA was identified in this study as a new member of the FAD-dependent amino acid dehydrogenase family for D-amino acid catabolism. DguR serves as a D-Glu sensor and transcriptional activator of the dguA promoter. Pseudomonas aeruginosa PAO1 was chosen as model to study gene response with different D-amino acids.
Project description:To see the effect of sub-lethal concentration of tobramcyin on PAO1 under anaerobic conditions. RNA was isolated from 5 biological repeats of PAO1 grown to mid-log phase in cationic adjusted mueller hinton broth containing 15mM KNO3 and 5 biological repeats of PAO1 grown to mid-log phase in cationic adjusted mueller hinton broth containing 15mM KNO3 and then treated for 30 minutes with 2 ug/ml tobramycin. All flasks were sealed to create anaerobic conditions. null
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:The transcriptome of P. aeruginosa PAO1 in the presence of extracelluar 2-oxoglutarate at a concentration of 20 mM. We determined the transcriptional response of P. aeruignosa PAO1 to extracellular 2-oxoglutarate. P. aeruginosa PAO1 was grown in nutrient broth (Oxoid number 2) and induced with 20 mM 2-oxoglutarate. At 30 min post induction, total RNA was isolated and prepped for Affymetrix GeneChips.