Project description:More than 7% of the Pseudomonas aeruginosa genes are encoding transcriptional regulators, many of which with unknown functions. Among them, those belonging to the LysR family are the most represented. The PA4203 gene lies upstream of the previously characterized ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, which detoxifies gluconolactone by converting it to gluconate. Upstream of PA4203 and in the opposite orientation are the PA4202 gene coding for a nitronate monooxygenase and ddlA (PA4201) encoding a D-alanine alanine ligase. This genetic organization is conserved in all P. aeruginosa genomes, but not in other pseudomonads. The intergenic regions between PA4203 and ppgL, and PA4202 are very short (79 and 107 nucleotides, respectively). PA4203 is a repressor of PA4202 and of its own transcription. A chromatin immunoprecipation analysis confirmed the presence of a single PA4203 binding site between PA4202 and PA4203. Electrophoretic mobility shift assays (EMSAs) with the purified PA4203 protein and in41 gel footprinting with the 1, 10-phenanthroline-copper ion, combined with primer extension analysis to determine transcriptional startpoints allowed the identification of a LysR binding motive in the PA4202 and PA4203 intergenic region. Despite this, a transcriptome analysis revealed more genes to be affected in a PA4203 mutant, likely due to the overexpression of the nitronate monooxygenase (PA4202). Deletion of the PA4202 gene resulted in an increased sensitivity of the cells to 3- nitropropionic acid (3-NPA).
Project description:Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. The LTTR family (LysR-Type Transcriptional Regulators) consists of proteins involved in regulation of various processes including stress response, motility, virulence or amino acid metabolism. The aim of this study was characterization of the LysR-type regulator BsrA (PA2121), identified previously as a negative regulator of biofilm formation in P. aeruginosa. To identify the BsrA binding sites in P. aeruginosa the ChIP-seq analysis was performed. It revealed 765 BsrA binding sites in P. aeruginosa PAO1161 genome, among them 367 was localized in the intergenic regions. Parallel transcriptomic analysis identified altered expression of 157 genes in response to BsrA excess, among them 35 had a BsrA binding site in the corresponding promoter regions, indicating direct influence of BsrA on expression of these genes. BsrA-repressed loci encompass genes encoding proteins engaged in key metabolic pathways including the tricarboxylic acid cycle. A group of directly activated genes by BsrA, consists of several loci encoding proteins involved in pili/fimbriae assembly as well as secretion and transport systems. Results also confirmed that BsrA acts as an autorepressor. Presented data uncover the regulon of BsrA protein with its role as transcriptional regulator of genes engaged in vital cellular processes in P. aeruginosa.
Project description:Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. The LTTR family (LysR-Type Transcriptional Regulators) consists of proteins involved in regulation of various processes including stress response, motility, virulence or amino acid metabolism. The aim of this study was characterization of the LysR-type regulator BsrA (PA2121), identified previously as a negative regulator of biofilm formation in P. aeruginosa. To identify the BsrA binding sites in P. aeruginosa the ChIP-seq analysis was performed. It revealed 765 BsrA binding sites in P. aeruginosa PAO1161 genome, among them 367 was localized in the intergenic regions. Parallel transcriptomic analysis identified altered expression of 157 genes in response to BsrA excess, among them 35 had a BsrA binding site in the corresponding promoter regions, indicating direct influence of BsrA on expression of these genes. BsrA-repressed loci encompass genes encoding proteins engaged in key metabolic pathways including the tricarboxylic acid cycle. A group of directly activated genes by BsrA, consists of several loci encoding proteins involved in pili/fimbriae assembly as well as secretion and transport systems. Results also confirmed that BsrA acts as an autorepressor. Presented data uncover the regulon of BsrA protein with its role as transcriptional regulator of genes engaged in vital cellular processes in P. 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:The Pseudomonas aeruginosa PAO1 gene phaF (PA5060) is a transcriptional regulator in the closely related pseudomonad P. putida. phaF is expressed at higher levels in P. aeruginosa clinical isolates from the cystic fibrosis respiratory tract. To determine the role of phaF in regulating P. aeruginosa gene expression, we cloned it under control of the pBAD promoter in expression vector pJN105 and compared expression in this strain relative to an empty vector control strain. We used microarrays to study overall gene expression in a P. aeruginosa PAO1 phaF overexpression strain.
Project description:The Pseudomonas aeruginosa PAO1 gene phaF (PA5060) is a transcriptional regulator in the closely related pseudomonad P. putida. phaF is expressed at higher levels in P. aeruginosa clinical isolates from the cystic fibrosis respiratory tract. To determine the role of phaF in regulating P. aeruginosa gene expression, we cloned it under control of the pBAD promoter in expression vector pJN105 and compared expression in this strain relative to an empty vector control strain. We used microarrays to study overall gene expression in a P. aeruginosa PAO1 phaF overexpression strain. A P. aeruginosa PAO1 strain over-expressing the transcriptional regulator phaF, PAO1 pJN105-phaF, and the corresponding empty control strain, P. aeruginosa PAO1 pJN105, were grown to exponential phase, RNA was isolated, and gene expression was analyzed via microarray analysis and compared.
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: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).
Project description:Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety, health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions, two spaceflight-analogue culture systems, i.e. the rotating wall vessel (RWV) and the random position machine (RPM), were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG), revealed a regulatory role for AlgU (RpoE). Specifically, P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts, including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore, cultivation in LSMMG increased heat- and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly, the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed, with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public.