The ParS/ParR regulon in Pseudomonas aeruginosa PAO1
ABSTRACT: 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:Pseudomonas chlororaphis strain 30-84 is an effective biological control agent against take-all disease of wheat. Phenazines, bacterial secondary metabolites produced by 30-84, are essential for 30-84 to inhibit fungal pathogens, form biofilms, and effectively colonize the rhizosphere. However, how the bacteria themselves respond to phenazines remains unknown. In this study, we conducted an RNA-seq analysis by comparing the wild type strain with a phenazine deficient mutant. RNA-seq analysis identified over 200 genes differentially regulated by phenazines. Consistent with previous findings in Pseudomonas aeruginosa PAO1, phenazines positively contribute to the expression of their own biosynthetic genes. Moreover, phenazine regulatory genes including the phzI/phzR quorum sensing system and the rpeB response regulatory were also expressed at high levels in the presence of phenazines. Besides phenazine biosynthesis and regulatory genes, genes involved in secondary metabolism, exopoysaccharide production and iron uptake as well as amino acid transport were identified as the major components under phenazine control, including many novel genes. We have also demonstrated that mutation of the primary siderophore gene pvdA resulted in up-regulation of phenazine genes when grown in iron-limiting media. These findings implicate phenazines as signaling molecules to regulate gene expression and hence alter metabolism in P. chlororaphis strain 30-84. A total of 4 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (2 replicates); Pseudomonas chlororaphis ZN mutant (2 replicates).
Project description:The purpose of this study was to define the TZD effect in Pseudomonas aeruginosa. Transcriptional profiling of Pseudomonas aeruginosa wild-type strain,reference strain PAO1, as control Vs. PAO1 strain exposed to a final 0.02mM of TZD derivative ((z)-5-octylidenethiazolidine-2,4-dione).
Project description:Oxygenated unsaturated fatty acids, known as oxylipins, are signaling molecules commonly used for cell-to-cell communication in eukaryotes. However, a role for oxylipins in mediating communication in prokaryotes has not previously been described. Bacteria mainly communicate via quorum sensing (QS) , which involves the production and detection of diverse small molecules termed autoinducers. We showed that oleic acid-derived oxylipins 10-HOME and 7,10-DiHOME produced by Pseudomonas aeruginosa function as autoinducers of a novel quorum sensing system termed Oxylipin-Dependent QS Sytem (ODS). This experiment was designed to determine the genes whose expression is altered by these P. aeruginosa oxylipins. We found that the ODS system controls the cell density-dependent expression of a P. aeruginosa gene subset through the mediation of 10-HOME and 7,10-DiHOME oxylipins. Overall design: Pseudomonas aeruginosa mRNA profiles of strain PAO1 ΔPA2076 (ΔodsR ) in the absence or presence of oleic acid or its derived oxylipins 10-HOME and 7,10-DiHOME.
Project description:We examined whether the budding yeast Saccharomyces cerevisiae can sense chemical signals from prokaryotes, specifically a variety of quorum sensing molecules from different bacteria species and from Candida albicans. We found that only N-acyl-3-oxo-dodecanoyl homoserine lactone (C12) from the opportunistic human pathogen Pseudomonas aeruginosa induces a stress response in yeast. Microarray experiments were performed in order to continue investigating the stress response. We treated S. cerevesiae (WT strain W303) with N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing molecule of Pseudomonas aeruginosa, which we found causes a stress response using a GFP reporter for HSP-12. Treatment conditions: 100 uM C12, 100 uM C12-lactam (control: synthetic analogue of C12 that is inactive in P. aeruginosa), DMSO (control: solvent), and 0.3 mM H2O2 (for comparison to oxidative stress).
Project description:ParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in proper folding and initial separation of ori domains by binding to specific parS sites (palindromic centromere-like sequences), mainly localized close to oriC. Bioinformatic analyses identified 10 parS sequences in the Pseudomonas aeruginosa PAO1 genome. One parS from the parS1-parS4 cluster is required for ParB mediated chromosome segregation. To verify the binding of ParB to all known parSs in vivo as well as to identify additional ParB binding sites we performed chromation immunoprecipitation (ChIP) using polyclonal anti-ParB antibodies followed by high throughput sequencing. ChIP was performed with P. aeruginosa PAO1161 (WT) cells, PAO1161 pKB9 (ParB+++) cells with a slight, non-toxic ParB overproduction as well as with 3 strains containing parS modifications impairing ParB binding to these sites. The data confirmed ParB binding to all known parS sequences with the exception of parS5. Moreover, we identified more than a 1000 of new ParB-bound regions, majority of which contained a DNA motif corresponding to inner 7 nt from one arm of the parS palindrome. ParB interactions with these numerous sites could affect chromosome topology, compaction and gene expression classifying P. aeruginosa ParB as a Nucleoid Associated Protein (NAP). Overall design: ChIP was performed with P. aeruginosa PAO1161 (WT) cells, PAO1161 pKB9 (araBADp-parB) cells with a 5-fold ParB overproduction. Additionally, three parS mutants were included: parSnull with all ten parS sites mutated, parS1-4 mutant with the four high affinity ParB sites inactivated, and parS2+ strain with nine parS sequences modified and only parS2 left intact (Jecz et al., 2015). PAO1161 parBnull mutant, with parB gene disrupted was used as a negative control. Each strain was analysed in duplicate. Additionally, input DNA (DNA before incubation with antibodies) was sequenced for WT strain.
Project description:The GacS/GacA two component regulatory system globally activates the production of secondary metabolites including phenazines in Pseudomonas chlororaphis 30-84. To better understand the regulatory role of the Gac system, we conducted RNA-seq analyses to determine the regulon of the response regulator GacA. Transcriptome analyses identified over 700 genes differentially regulated by GacA. Consistent with our previous findings, phenazine biosynthetic genes were significantly down-regulated in a gacA mutant. The expression levels of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS and pip were also decreased. Moreover, the expression of three none-coding RNAs (ncRNAs) including rsmX, rsmY and rsmZ was significantly decreased by gacA mutation consistent with the presence of GacA binding sites in their promoters. Our results also demonstrated that over-expression of rsmZ from a non-gac regulated promoter resulted in the restoration of AHL and phenazine production as well as the expression of other secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production was also investigated. Over-expression of rsmE, but not rsmA, resulted in decreased AHL production and phenazine gene expression in P. chlororaphis. Consistently, a mutation in rsmE bypassed the requirement of GacA in phenazine gene expression. On the contrary, constitutive expression of the phzI/phzR quorum sensing system was not able to rescue phenazine production in the gacA mutant indicating the direct impact of Gac system on the transcript stability of phenazine biosynthetic genes. Together, these results indicate that the Gac system regulates phenazine production at multiple levels and exerts its positive effect on AHL and phenazine biosynthesis via RsmZ and RsmE. A model is proposed to illustrate the GacA regulon in P. chlororaphis 30-84. A total of 6 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (3 replicates); Pseudomonas chlororaphis gacA mutant (3 replicates).
Project description:Oberhardt2008 - Genome-scale metabolic
network of Pseudomonas aeruginosa (iMO1056)
This model is described in the article:
network analysis of the opportunistic pathogen Pseudomonas
Oberhardt MA, Puchałka J, Fryer
KE, Martins dos Santos VA, Papin JA.
J. Bacteriol. 2008 Apr; 190(8):
Pseudomonas aeruginosa is a major life-threatening
opportunistic pathogen that commonly infects immunocompromised
patients. This bacterium owes its success as a pathogen largely
to its metabolic versatility and flexibility. A thorough
understanding of P. aeruginosa's metabolism is thus pivotal for
the design of effective intervention strategies. Here we aim to
provide, through systems analysis, a basis for the
characterization of the genome-scale properties of this
pathogen's versatile metabolic network. To this end, we
reconstructed a genome-scale metabolic network of Pseudomonas
aeruginosa PAO1. This reconstruction accounts for 1,056 genes
(19% of the genome), 1,030 proteins, and 883 reactions. Flux
balance analysis was used to identify key features of P.
aeruginosa metabolism, such as growth yield, under defined
conditions and with defined knowledge gaps within the network.
BIOLOG substrate oxidation data were used in model expansion,
and a genome-scale transposon knockout set was compared against
in silico knockout predictions to validate the model.
Ultimately, this genome-scale model provides a basic modeling
framework with which to explore the metabolism of P. aeruginosa
in the context of its environmental and genetic constraints,
thereby contributing to a more thorough understanding of the
genotype-phenotype relationships in this resourceful and
This model is hosted on
and identified by:
To cite BioModels Database, please use:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
Public Domain Dedication for more information.
Project description:Pseudomonas chlororaphis strain 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we conducted an RNA-seq analysis by comparing the wild type strain with a Hfe deficient mutant. RNA-seq analysis identified over 900 genes differentially regulated by Hfq. A total of 4 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (2 replicates); Pseudomonas chlororaphis ZN mutant (2 replicates).
Project description:Genomic DNA from Pseudomonas aeruginosa strains PAO1 and PA14 Overall design: Pseudomonas aeruginosa genomic DNA was isolated, fragmented and hybridized to Affymetrix Pseudomonas GeneChips.