Project description:To further determine the origin of the increased virulence of Pseudomonas aeruginosa PA14 compared to Pseudomonas aeruginosa PAO1, we report a transcriptomic approach through RNA sequencing. Next-generation sequencing (NGS) has revolutioned sistems-based analsis of transcriptomic pathways. The goals of this study are to compare the transcriptomic profile of all 5263 orthologous genes of these nearly two strains of Pseudomonas aeruginosa.
Project description:Analysis of Pseudomonas aeruginosa PAO1 treated with 200 µM sphingomyelin. Results provide insight into the response to sphingomyelin in P. aeruginosa.
Project description:Pseudomonas aeruginosa is a major cause of infection in hospitalised patients, with a large genome which makes it highly versatile and resistant to most antimicrobial agents. Ceftazidime-avibactam (CZA) offers an alternative treatment, but resistance is quickly evolving. There is limited knowledge on the exact resistance mechanisms to this drug, or on cross-resistance to meropenem (MEM). This laboratory experiment aimed to decipher these mechanisms in order to provide guidance for the best treatment choice in meropenem pre-treated P. aeruginosa infections. Six clinical isolates of P. aeruginosa were subjected to multistep resistance selection in sub inhibitory concentrations of CZA and MEM. MICs were also determined in the presence of the efflux pump inhibitor phenyl-Arginine-β-Naphthylamide (PAβN). Molecular analyses were performed by whole genome sequencing, whole -gene- and -protein expression profiles. CRISPR/Cas9 genome editing was performed using a two-plasmid method for selected mutations
Project description:We identified PhaF as an RNA binding protein in Pseudomonas aeruginosa. In order to identify the different target transcripts of PhaF, we carried out the CLIP (Crosslinking and immunoprecipitation) and CLAP-Seq (Covalent linkage affinity purification) approaches, which utilizes UV irradiation to crosslink PhaF with the transcripts that are in close vicinity to it. For CLIP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal VSVG tag on PhaF. In order to determine the importance of the C-terminal domain (CTD) of PhaF on RNA binding, we used PAO1ΔphaF strains carrying plasmids that express a C-terminal VSVG tagged version of PhaF-CTD (pPhaF-CTD-V), along with a control strain that carries the same plasmid (pPhaF-CTD) but without the VSVG tag. For CLAP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal Halo tag on PhaF. All the strains were subjected to UV irradiation, lysed, immunoprecipitated using either anti-VSVG-antibody coated beads or Magne Halo tagged beads. Following immunoprecipitation, the RNAs were purified. We also purified Total (Tot) RNA (samples collected before immunoprecipitation) from the PAO1 strains harboring the C-terminal VSVG tag on PhaF. The purified RNA samples were converted to cDNA libraries, which were subsequently sequenced using the Illumina NextSeq. The sequences were mapped to the genome to identify the target transcripts. In a separate series of experiments we also carried out RNA-Seq to identify the genes that are differentially regulated using Pseudomonas aeruginosa PAO1 and PAO1ΔphaF strains. The isolated RNA was sent to SeqCenter (Pittsburgh, PA) and the sequences obtained were mapped to the genome and DESeq analysis was performed.
Project description:We identified PhaF as an RNA binding protein in Pseudomonas aeruginosa. In order to identify the different target transcripts of PhaF, we carried out the CLIP (Crosslinking and immunoprecipitation) and CLAP-Seq (Covalent linkage affinity purification) approaches, which utilizes UV irradiation to crosslink PhaF with the transcripts that are in close vicinity to it. For CLIP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal VSVG tag on PhaF. In order to determine the importance of the C-terminal domain (CTD) of PhaF on RNA binding, we used PAO1ΔphaF strains carrying plasmids that express a C-terminal VSVG tagged version of PhaF-CTD (pPhaF-CTD-V), along with a control strain that carries the same plasmid (pPhaF-CTD) but without the VSVG tag. For CLAP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal Halo tag on PhaF. All the strains were subjected to UV irradiation, lysed, immunoprecipitated using either anti-VSVG-antibody coated beads or Magne Halo tagged beads. Following immunoprecipitation, the RNAs were purified. We also purified Total (Tot) RNA (samples collected before immunoprecipitation) from the PAO1 strains harboring the C-terminal VSVG tag on PhaF. The purified RNA samples were converted to cDNA libraries, which were subsequently sequenced using the Illumina NextSeq. The sequences were mapped to the genome to identify the target transcripts. In a separate series of experiments we also carried out RNA-Seq to identify the genes that are differentially regulated using Pseudomonas aeruginosa PAO1 and PAO1ΔphaF strains. The isolated RNA was sent to SeqCenter (Pittsburgh, PA) and the sequences obtained were mapped to the genome and DESeq analysis was performed.
Project description:We report RNA sequencing data for mRNA transcripts obtained from tobramycin exposed phoenix colonies, VBNCs, and various controls (untreated lawn, edge of the zone of clearance of tobramycin, treated outer background lawn). Extracted mRNA was sequenced using an Illumina HiSeq 4000, mapped to a Pseudomonas aeruginosa PAO1 reference genome, and processed to obtain counts for all gene transcripts for each sample. This is the first sequencing data generated for Pseudomonas aeruginosa phoenix colonies and VBNCs.