Project description:Purpose: Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). We provide an insight to the DNA auxotrophy of P. aeruginosa PASS4 isolate. Better understanding of P. aeruginosa adaptations in the CF lung environment can have a great impact in the development of specialised treatment regimes aimed at the eradications of P. aeruginosa infections. Methods: P. aeruginosa strains PAO1 and PASS4 were grown in minimal medium with either L-Asparagine or DNA as a carbon source, in biological triplicates. RNA was extracted and sequenced on Illumina HiSeq 1000 platform. The sequence reads that passed quality filters were analyzed using EdgePro and DESeq packages, as well as the Rockhopper tool. Results: We mapped > 10 million paired sequence reads per sample to the genome of P. aeruginosa PAO1 and identified a total of 576 genes differentially expressed by PASS4 when grown in DNA (P value < 0.01, log2 fold-change 1< to < -1), with 322 genes upregulated and 254 genes downregulated. There were a total of 423 genes differentially expressed by PAO1 when grown in DNA (P value < 0.01, log2 fold-change 1< to <-1), with 359 genes upregulated and 64 genes downregulated . A total of 129 transcripts displayed similar expression patterns in both organisms, with 112 being upregulated and 17 down-regulated. Conclusions: Our study identified that P. aeruginosa PASS4 was a purine auxotroph. Purine auxotropy may represent a viable microbial strategy for adaptation to DNA rich environments such as the CF lung.
Project description:Purpose : The goal of this study was to use RNA Seq to define the regulon of the transciption factor Anr by comparing global transcriptional profiles of Pseudomonas aeruginosa strain PAO1 and a clinical isolate with their isogenic ∆anr mutants, grown in colony biofilms at 1% oxygen. Methods : mRNA profiles were generated for laboratory strain PAO1 and for a clinical isolate J215, as well as for ∆anr derivatives of each strain, in duplicate, by deep sequencing. Strains were grown for 12 hours in colony biofilms at 1% O2, 5% CO2 prior to RNA harvest. Ribosomal and transfer RNAs were removed using the MICROBExpress kit (Life Technologies). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters.