Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Pseudomonas aeruginosa PAO1-psrA::Tn vs. PAO1 in LB

ABSTRACT: 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.

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Tung Hoang

PROVIDER: E-GEOD-8083 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:PAO1 and FRD1 were cultured planktonically and in biofilms with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined for PAO1 cultured planktonically, for FRD1 cultured planktonically, for PAO1 cutured in biofilms, and for FRD1 cultured in biofilms. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.

Project description:PAO1 was cultured planktonically to stationary phase with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.

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Transcription profiling of Pseudomonas aeruginosa PAO1-psrA::Tn vs. PAO1 in LB

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