ABSTRACT: Proteomics analysis of envelope proteins was undertaken to understand the mechanisms and changes within the cell once Mfs1 and Mfs2 were over-expressed.
Project description:Five biological repeats of P.aeruginosa PAO1 and five biological repeats of P.aeruginosa PAO1 phoQ::xylE were grown in mineral medium BM2 using glucose as the carbon source and with 2mM MgSO4. Microarray experiments were done using microarray slides and protocols from TIGR on cells from mid-log phase.
Project description:Microarray experiments were preformed comparing the effect of sub-inhibitory concentrations of ciprofloxacin on five independent cultures of PAO1 and the PAO1 Lon mutant
Project description:Micoarrays were used to obtain gene expression in mid-log phase PAO1::cprR(PA3077) with the treatment of 12ug/ml CP28 vs mid-log phase PAO1 with the treatment of 12ug/ml CP28
Project description:Recent studies have shown that the concentrations of proteins expressed from orthologous genes are often conserved across organisms, and to a greater extent than the abundances of the corresponding mRNAs. However, such studies have not distinguished between evolutionary (e.g., sequence divergence) and environmental (e.g., growth condition) effects on the regulation of steady-state protein and mRNA abundances. Here we systematically investigated the transcriptome and proteome of two closely related Pseudomonas aeruginosa strains, PAO1 and PA14, under identical experimental conditions, thus controlling for environmental effects. For 703 genes observed by both shotgun proteomics and microarray experiments, we find that the protein-to-mRNA ratios are highly correlated between orthologous genes in the two strains, to an extent comparable to protein and mRNA abundances. In spite of this high molecular similarity between PAO1 and PA14, we found that several metabolic, virulence, and antibiotic resistance genes are differentially expressed between the two strains, mostly at the protein but not at the mRNA level. Our data demonstrate that post-transcriptional regulation is important for understanding the discordance between mRNA and protein abundance.
Project description:The TyrR-like enhancer-binding protein GcsR (or PA2449) was shown to regulate the expression of genes required for glycine metabolism. In order to define the regulon of GcsR we compared the transcriptome of a gcsR deletion mutant of P. aeruginosa PAO1 with that of the wild-type using RNA-Seq. Strains were grown under glycine rich conditions (peptone broth) and their transcriptomes were compared using RNA-Seq.
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: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. Comparative genomics and phenotypic assays have shown that antibiotic resistance profiles differ among LES isolates and that genotype–phenotype associations are difficult to establish for resistance phenotypes in clinical isolates of P. aeruginosa based on these comparisons alone. We compared two LES isolates, LESlike1 and LESB58, and the common laboratory strain P. aeruginosa PAO1 using label-free quantitative proteomics to more accurately predict functional differences between strains. The proteomes of the LES isolates were found to be more similar to each other than to PAO1. However, we also observed a number of differences in the abundance of proteins involved in quorum sensing, virulence, and antibiotic resistance, including in the comparison of LESlike1 and LESB58. Specifically, the proteomic data revealed a higher abundance of proteins involved in polymyxin and aminoglycoside resistance in LESlike1. Minimum inhibitory concentration assays confirmed that LESlike1 has higher resistance to antibiotics from these classes. These findings provide an example of the ability of proteomic data to complement genotypic and phenotypic studies to understand resistance in clinical isolates.
Project description:Protein complexome analysis of Pseudomonas aeruginosa PAO1 at OD 0.3 in LB media through 10-40% glycerol gradient fractionation in 21 samples.
Project description:We carried out an experimental evolution in human serum as an ex-vivo model and screened evolved lines for the evolution of resistance phenotypes towards two anti-virulence treatments, gallium and flucytosine, which both target the iron scavenging pyoverdine of Pseudomonas aeruginosa (each at 2 different doses). We performed whole-genome sequencing of 16 evolved clones from the different treatment regimes .