Project description:Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed a great genome diversity among P. aeruginosa clinical strains and revealed important regulatory traits during chronic adaptation. While current investigation of epigenetics of P. aeruginosa is still lacking, understanding the epigenetic regulation may provide biomarkers for diagnosis and reveal important regulatory mechanisms. The present study focused on characterization of DNA methyltransferases (MTases) in a chronically adapted P. aeruginosa clinical strain TBCF10839. Single-molecule real-time sequencing (SMRT-seq) was used to characterize the methylome of TBCF. RCCANNNNNNNTGAR and TRGANNNNNNTGC were identified as target motifs of DNA MTases, M.PaeTBCFI and M.PaeTBCFII, respectively.
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:Taxonomic outliers of Pseudomonas aeruginosa recently emerged as infectious for humans. Here we present the first analysis of a hyper-virulent isolate that cause hemorrhagic pneumonia. We demonstrated that, in two sequential clones CLJ1 and CLJ3 recovered from a patient with chronic obstructive pulmonary disease undergoing antibiotic therapy, insertion of a mobile genetic element into the P. aeruginosa chromosome affected major virulence-associated phenotypes and led to increased resistance to antibiotics used to treat the patient. Our work reveals insertion sequences as major players in enhancing the pathogenic potential of a P. aeruginosa taxonomic outlier by modulating both the virulence and resistance to antimicrobials. This also explains the ability of this bacterium to adapt to an infected host and cause a serious disease.
Project description:This study addresses the impact of zinc limitation on the opportunistic human pathogen, Pseudomonas aeruginosa. Zinc limitation was assessed in the P. aeruginosa PAO1 strain using an isogenic deletion mutant lacking the periplasmic, zinc solute-binding protein, znuA (PA5498). ZnuA delivers bound zinc to its cognate ABC transporter, ZnuBC, for import into the cytoplasm. Our transcriptional analyses revealed P. aeruginosa to possess a multitude of zinc acquisition mechanisms, each of which were highly up-regulated in the zinc-deficient znuA mutant strain. P. aeruginosa also utilized zinc-independent paralogues of zinc-dependent genes to maintain cellular function under zinc limitation. Together, these data reveal the complex transcriptional response and versatility of P. aeruginosa to zinc depletion.
Project description:Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). The P. aeruginosa CF isolate PASS4 has reduced ability to catabolise various carbon sources however can grow on DNA as a sole carbon source but, with a higher biomass production than P. aeruginosa burns wound, laboratory strain PAO1. Therefore, proteomic profiling of PASS4 and PAO1 was conducted following growth on DNA as a sole carbon source. To compare the protein expression of P. aeruginosa strains PAO1 and PASS4 following growth in DNA, the amino acid, asparagine was used a control condition, as asparagine was one of the amino acids PASS4 could utilise.
Project description:Pseudomonas aeruginosa is a predominant pathogen in chronic lung infections in individuals with cystic fibrosis (CF). Epidemic strains of P. aeruginosa, such as the Liverpool Epidemic Strain (LES), are capable of transferring between CF patients and have been associated with increased hospital visits and antibiotic treatments. We used label-free quantitative proteomics to compare the laboratory strain PAO1, beta-lactam resistant isolate LESB58, and beta-lactam susceptible isolate LESlike1 and their responses to three beta-lactams (aztreonam, carbenicillin, piperacillin), the aminoglycoside tobramycin, and hydrogen peroxide. Across all samples, we identified 3019 proteins with a minimum of two peptides. We found that LESB58 showed a large response to treatment with the beta-lactam carbenicillin, with 644 proteins significantly increased in abundance and 590 proteins significantly decreased in abundance (Students t-test, p≤0.05, FDR=0.05, S0=1). Proteomic characterization of an additional beta-lactam resistant isolate, LES431, exposed to carbenicillin showed that this response was shared by both isolates. Part of the response to carbenicillin in LESB58 included an increase in abundance in proteins involved in cell wall synthesis and division.
Project description:Analysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in minimal medium with glucose as carbon source (M9G). SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF in Pseudomonas aeruginosa. Deletion of the ECF sigma factor sigX gene provide insights into the SigX role in several virulence and biofilm- related phenotypes in Pseudomonas aeruginosa.