Regulon of transcriptional regulator PA2449 in Pseduomonas aeruginosa PAO1
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ABSTRACT: The putative trancriptional regulator PA2449 was found to be essential for both glycine/serine metabolism and the production of phenazines in P. aeruignosa PAO1. We examined the regulon controlled by PA2449 via microarray analysis between wild-type P. aeruignosa PAO1 and the PA2449-null mutant P. aeruginosa PW5126. Both strains were obtained from the PA-two allele library (Univ. of Washington, Ref. Jacobs et al. 2003. PNAS 100, 14339). Strains were grown under conditions known to induce phenazine biosynthesis (peptone broth), and their resulting transcriptomes were compared. Total RNA was isolated and prepped for Affymetrix GeneChips.
Project description:The putative trancriptional regulator PA2449 was found to be essential for both glycine/serine metabolism and the production of phenazines in P. aeruignosa PAO1. We examined the regulon controlled by PA2449 via microarray analysis between wild-type P. aeruignosa PAO1 and the PA2449-null mutant P. aeruginosa PW5126. Both strains were obtained from the PA-two allele library (Univ. of Washington, Ref. Jacobs et al. 2003. PNAS 100, 14339).
Project description:The transcriptome of P. aeruginosa PAO1 in the presence of extracelluar 2-oxoglutarate at a concentration of 20 mM. We determined the transcriptional response of P. aeruignosa PAO1 to extracellular 2-oxoglutarate. P. aeruginosa PAO1 was grown in nutrient broth (Oxoid number 2) and induced with 20 mM 2-oxoglutarate. At 30 min post induction, total RNA was isolated and prepped for Affymetrix GeneChips.
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: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.
Project description:The bacterial transcription factor RpoN regulates an extensive network of genes whose products are involved in diverse biological functions. We constructed a small peptide termed the RpoN molecular roadblock, which binds to and blocks transcription from RpoN promoters. This RpoN molecular roadblock can be used in any bacterium to obtain information on the RpoN regulon. We expressed the RpoN molecular roadblock in P. aeruginosa PAO1 and used microarrays to identify genes that were differentially transcribed due to the RpoN molecular roadblock. The RpoN molecular roadblock was expressed in P. aeruginosa PAO1 in mid-exponential phase in rich media. Total RNA was isolated and prepped for Affymetrix GeneChips.
Project description:Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection within the cystic fibrosis (CF) lungs. The effects of sub-inhibitory colistin on gene expression in P. aeruginosa were investigated by transcriptome microarray and functional analysis. Analysis revealed an alteration in the expression of 60 genes in total from a variety of pathways. Genes associated with bacterial chronic colonisation and virulence such as response to osmotic stress, motility, and biofilm formation, as well as those associated with LPS modification and quorum sensing are the most highly represented. Most striking among these is the upregulation of the PQS biosynthesis operon including pqsH, pqsE, and the anthranilate biosynthetic genes phnAB. Early activation of this central component of the QS-network may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF-lung. Experiment Overall Design: Three independent cultures of the P. aeruginosa strain PAO1 were exposed to 0.15 µg colistin mlâ1. The untreated and treated samples were grown from OD600 0.05 to 0.8 and subsequently total RNA was extracted using the Ambion RiboPureTm- Bacteria kit according to the manufacturerâs instructions.
Project description:Pseudomonas aeruginosa is an opportunistic human pathogen, infecting immuno-compromised patients and causing persistent respiratory infections in people affected from cystic fibrosis. Pseudomonas strain Pseudomonas aeruginosa PA14 shows higher virulence than Pseudomonas aeruginosa PAO1 in a wide range of hosts including insects, nematodes and plants but the precise cause of this difference is not fully understood. Little is known about the host response upon infection with Pseudomonas and whether or not transcription is being affected as a host defense mechanism or altered in the benefit of the pathogen. In this context the social amoeba Dictyostelium discoideum has been described as a suitable host to study virulence of Pseudomonas and other opportunistic pathogens.
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:20 P. aeruginosa strains were genotyped using a custom spotted oligo array (Ausubel P. aruginosa genotyping 4.8K v1), which included primarily oligos for genes present in strain PA14 but absent in PAO1, and oligos for genes present in PAO1 but absent in PA14 (as well as controls).
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, PCA and O star with a phenazine deficient mutant. RNA-seq analysis identified over 800 genes differentially regulated by phenazines.