Project description:ErfA is a transcription factor of Pseudomonas aeruginosa. We here define the genome-wide binding sites of ErfA by DAP-seq in Pseudomonas aeruginosa PAO1 and IHMA87, Pseudomonas chlororaphis PA23, Pseudomonas protegens CHA0 and Pseudomonas putida KT2440.
Project description:Pseudomonas aeruginosa san ai resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to sub-lethal concentration of cadmium (0.9 mM) adsorbed 75% of added cadmium after 48 hours, while remaining 25% in culture supernatant was adsorbed by exopollysaccaryde, implying a large biosorption potential of leaving biomass and its exopollysaccaryde. Inner cell response on the protein level was investigated by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins in complex protein fractions obtained by size exclusion chromatography used to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 59 proteins were observed as up-regulated in cadmium- amended culture. Almost a third of the total number of up-regulated were metalloproteins. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in cell energy metabolism, amino acid metabolism, cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium- amended culture versus control was found. Our results signify that P. aeruginosa san ai has a great potential for application in bioremediation of cadmium polluted sites.
Project description:Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for high morbity in COPD, burns , implanted medical devices and cystic fibrosis. Pseudomonas aeruginosa is a problematic colonizer of the human lung. P. aeruginosa produces a phospholipase C (PlcH) that degrades choline-containing lipids such as phosphatidylcholine and sphingomylein that are found in lung surfactant and in host membranes. In this study, we analyzed gene expression in mutants defective in PlcH production (delta-plcH and delta-gbdR) and the wild type when growing in medium with lung surfactant.
Project description:Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for high morbity in COPD, burns , implanted medical devices and cystic fibrosis. Pseudomonas aeruginosa is a problematic colonizer of the human lung. P. aeruginosa produces a phospholipase C (PlcH) that degrades choline-containing lipids such as phosphatidylcholine and sphingomylein that are found in lung surfactant and in host membranes. In this study, we analyzed gene expression in mutants defective in PlcH production (delta-plcH and delta-gbdR) and the wild type when growing in medium with lung surfactant. Pseudomonas aeruginosa was cultured in liquid cultures with aeration in a defined medium with Survanta, a lung surfactant replacement. Cultures were harvested during mid-exponential phase, and RNA was isolated for microarray analysis. The P. aeruginosa strain PAO1 wild type gene expression was compared to expression profiles from delta-gbdR and delta-plcHR deletion mutants, two mutants defective in PlcH production.
Project description:The opportunistic pathogen Pseudomonas aeruginosa controls the production of virulence factors via its quorum sensing (QS) systems. The addition of 100 M 4-GB to succinate-containing medium led to a strong reduction of pyocyanin production in strain PAO1gbuA, whereas pyocyanin production by the wildtype was not affected. To investigate possible transcriptional differences between the two strains RNAseq analysis of strains PAO1 and PAO1gbuA in the presence of 4-GB was performed. RNA was extracted in late exponential phase. For the isolation of RNA for RNAseq analysis, pre-cultures of strains PAO1 and PAO1gbuA were grown in medium B containing 20 mM succinate for 12 h at 37C. Cells were washed and used to inoculate main cultures in medium B containing 20 mM succinate and 100 M 4-GB in triplicates with an OD600 of 0.001. After incubation for 11 h at 37C, the respective triplicates were combined, harvested by centrifugation and resuspended in medium B without complementing solutions. From these cell suspensions, RNA was extracted with the innuPREP RNA Minikit from Analytik Jena (Jena, Germany) according to the manufacturers instructions. Residual DNA was removed by digestion with 10 U RNase-free DNase I (Thermo scientific) for 1 h in the presence of RiboLock RNase inhibitor (Thermo scientific). After DNA digestion, the RNA was again purified with the same kit. RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Boblingen, Germany). Ribosomal RNA molecules were removed from total RNA with the Ribo-Zero rRNA Removal Kit (Illumina, San Diega, USA) and removal of rRNA was checked with the Agilent RNA Pico 6000 Kit on an Agilent 2100 Bioanalyzer. cDNA libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, USA), and the resulting cDNA was sequenced paired end on an Illumina MiSeq system using 75 bp read length.
Project description:EV RNA samples from MH-S cells were prepared for small RNA sequencing by TruSeq Small RNA Sample Prep Kits (Illumina, San Diego,USA), using a minimum of 1 μg RNA per sample.
Project description:The ParS/ParR two component regulatory system plays important roles for multidrug resistance in Pseudomonas aeruginosa. In this study we report RNA-seq analyses of the transcriptomes of P. aeruginosa PAO1 wild type and par mutants growing in a minimal medium containing 2% casamino acids. This has allowed the quantification of PAO1 transcriptome, and further defines the regulon that is dependent on the ParS/ParR system for expression. Our RNA-seq analysis produced the first estimates of absolute transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished the expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to affecting drug resistance genes, transcripts of quorum sensing genes (rhlIR and pqsABCDE-phnAB), were significantly up-regulated in both parS and parR mutants. Consistent with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of par genes also lead to overproduction of phenazines and increased swarming motility, consistent with the up-regulation of quorum sensing genes. Our results established a link among ParS/ParR, MexEF-OprN and quorum sensing in Pseudomonas aeruginosa. Based on these results, we propose a model to illustrate the relationship among these regulatory systems in P. aeruginosa. A total of 9 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas aeruginosa PAO1 wild type strain (3 replicates); Pseudomonas aeruginosa parS mutant (3 replicates); Pseudomonas aeruginosa parR mutant (3 replicates).