Genome-wide mapping of the RNA targets of the Pseudomonas aeruginosa riboregulatory protein RsmN
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ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa
PROVIDER: GSE94113 | GEO | 2018/05/01
REPOSITORIES: GEO
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Project description:Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences that are bound in vivo by Rsm proteins, in order to identify potential regulatory targets for each of them. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to identify 709 targets, 75 of them common to the three proteins. Relevant targets include genes encoding proteins involved in metabolism, transport and secretion, stress responses, signal transduction and regulation, and the turnover of the second messenger c-di-GMP. The role of Rsm proteins in the utilization of basic amino acids and bacterial fitness in the plant rhizosphere are also exposed.
2020-09-03 | GSE154204 | GEO
Project description:Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes.
2019-02-05 | GSE110803 | GEO
Project description:In the opportunistic pathogen Pseudomonas aeruginosa RsmA is an RNA-binding protein that plays critical roles in the control of virulence, interbacterial interactions and biofilm formation. Although RsmA is thought to exert its regulatory effects by binding full-length transcripts, the extent to which RsmA binds nascent transcripts has not been addressed. Moreover, which transcripts are direct targets of this key post-transcriptional regulator is largely unknown. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, with cells grown in the presence and absence of the RNA polymerase inhibitor rifampicin, we identify hundreds of nascent transcripts that RsmA associates with in P. aeruginosa. We also find that the RNA chaperone Hfq targets a subset of the RsmA-associated nascent transcripts and that the two RNA-binding proteins can exert regulatory effects on common targets. Our findings establish that RsmA associates with many transcripts as they are being synthesized in P. aeruginosa, identify the direct targets of RsmA, and suggest that RsmA and Hfq may act in a combinatorial fashion on certain target transcripts. More broadly, our data suggest that the binding of post-transcriptional regulators to nascent transcripts may be commonplace in bacteria where distinct regulators can function alone or in concert to achieve control over the translation of transcripts as soon as they emerge from RNA polymerase.
2020-04-20 | GSE138338 | GEO
Project description:Global expression profiling of airway epithelial cells infected with Pseudomonas aeruginosa and the rsmA mutant. Recent work in our laboratory investigated the impact of RsmA on a number of airway epithelial cell phenotypes including actin depolymerization, cytotoxicity and invasion. These cellular phenotypes were influenced by the positive effect of RsmA on the expression of the TTSS in P. aeruginosa (Mulcahy et al. 2006. Infect. Immun.). Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death. Keywords: Expression profiling, Pseudomonas aeruginosa, microbial infection, airway epithelial cells, cystic fibrosis, exoenzymes, ExoS, ExoT, ExoU, ExoY, Kruppel-like factors, KLF2, KLF6
2006-09-15 | GSE4485 | GEO
Project description:Global expression profiling of airway epithelial cells infected with Pseudomonas aeruginosa and the rsmA mutant. Recent work in our laboratory investigated the impact of RsmA on a number of airway epithelial cell phenotypes including actin depolymerization, cytotoxicity and invasion. These cellular phenotypes were influenced by the positive effect of RsmA on the expression of the TTSS in P. aeruginosa (Mulcahy et al. 2006. Infect. Immun.). Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding ; protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in ; the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors ; KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death. Experiment Overall Design: RNA isolation and microarray sample preparation Experiment Overall Design: Biological samples from two independent infection experiments were used in two independent microarray experiments as outlined below. Total RNA was isolated from epithelial cells using a RNeasy Kit (Qiagen) according to manufacturer's instructions. Genomic DNA was removed using DNA-free (Ambion) and confirmed by PCR to be free of DNA, prior to cDNA synthesis. Isolated RNA was used in the preparation of fragmented cRNA for microarray hybridization according to the manufacturer's protocol (Affymetrix). Briefly, 8.5ug of RNA was converted to double stranded cDNA by reverse transcription, using a Superscript cDNA synthesis kit (Invitrogen), with an oligo dT primer containing a T7 RNA polymerase site added 3' of the poly (T) (Affymetrix). After second strand synthesis, cDNA was converted to biotin-labelled cRNA by in vitro transcription (Enzo). The labelled cRNA was purified using Genechip clean up module (Qiagen). Total cRNA quality and yield was measured using a GeneQuant spectrophotometer and 20ug of cRNA was fragmented at 94ºC for 35 mins in fragmentation buffer (40 mM tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). Full length and fragmented cRNA sample integrity was assessed by electrophoresis on a 1% agarose-TBE gel visualized by staining with SyBr green. Fragmented cRNA was sent to MRC geneservice (Cambridge, UK) for assessment of RNA quality using a Bioanalyzer (Agilent) before hybridization and scanning. The arrays were washed and stained in a fluidics station and scanned according to the manufacturer's protocols (Affymetrix). Experiment Overall Design: Microarray data analysis Experiment Overall Design: Raw image analysis was performed using Microarray Suite v5.0 software (Affymetrix). Downstream data analysis was performed using GeneSight software version 2.7 (Biodiscovery). For all analyses, the signal intensities from two independent infection and microarray experiments were used. Statistically significant changes in gene expression were identified by firstly performing analysis of variance (ANOVA), for comparison between multiple groups, with a P value of < 0.05. The list of transcripts generated using ANOVA was then subjected to a Student's t-test, for comparison between two groups, with a P value of < 0.05. To identify different levels of gene expression compared to baseline, a fold change cut-off of greater than or equal to 1.5 was applied to this data.
2007-10-27 | E-GEOD-4485 | biostudies-arrayexpress
Project description:The opportunistic human pathogen Pseudomonas aeruginosa can utilize several carbon and nitrogen compounds as energy sources, which allows the bacterium to grow on a variety of different environments. Nevertheless, the uptake and utilization of these compounds is organized in a hierarchical manner, which is guaranteed by a mechanism named catabolite repression. In P. aeruginosa catabolite repression is a post-transcriptional process with the translational repressor protein, Crc, as the main component. Crc recognizes CA-motifs (acronym for catabolite activity) present in the vicinity of the ribosome binfing site of corresponding target mRNAs and therefore compete with ribosome binding. Certain conditions, which are mainly related to changes in the carbon to nitrogen ratio, induce the two component system CbrAB, which activates the transcription of the sRNA CrcZ. The sRNA sequesters Crc and allows the translation of the target mRNAs. The main focus of this study was to identify novel direct targets of the CbrAB/Crc system with the use of a transcriptome analysis in combination with the search for CA-motifs. We were able to identify five novel targets (estA, acsA, dctP, bkdR and aroP2), which were involved in the uptake and utilization of less preferred carbon sources and amino acids. Direct interaction of Crc with these genes and the resulting regulation by CbrB and CrcZ were verified using mutational analysis and in vitro and in vivo experiments. Moreover, these targets were discussed in the light of growth and biofilm development in synthetic CF sputum medium which emphasised the importance of the CbrAB/Crc system as a regulator of chronic infection. keyword(s): genetic modification Comparative transcriptome analysis with the PAO1 wild type strain and PAO6673 (delta crc), PAO66711 (delta cbrB), PAO6679 (delta crcZ) strains grown in BSM-Succinate medium.
2012-03-31 | E-GEOD-33241 | biostudies-arrayexpress
Project description:The opportunistic human pathogen Pseudomonas aeruginosa can utilize several carbon and nitrogen compounds as energy sources, which allows the bacterium to grow on a variety of different environments. Nevertheless, the uptake and utilization of these compounds is organized in a hierarchical manner, which is guaranteed by a mechanism named catabolite repression. In P. aeruginosa catabolite repression is a post-transcriptional process with the translational repressor protein, Crc, as the main component. Crc recognizes CA-motifs (acronym for catabolite activity) present in the vicinity of the ribosome binfing site of corresponding target mRNAs and therefore compete with ribosome binding. Certain conditions, which are mainly related to changes in the carbon to nitrogen ratio, induce the two component system CbrAB, which activates the transcription of the sRNA CrcZ. The sRNA sequesters Crc and allows the translation of the target mRNAs. The main focus of this study was to identify novel direct targets of the CbrAB/Crc system with the use of a transcriptome analysis in combination with the search for CA-motifs. We were able to identify five novel targets (estA, acsA, dctP, bkdR and aroP2), which were involved in the uptake and utilization of less preferred carbon sources and amino acids. Direct interaction of Crc with these genes and the resulting regulation by CbrB and CrcZ were verified using mutational analysis and in vitro and in vivo experiments. Moreover, these targets were discussed in the light of growth and biofilm development in synthetic CF sputum medium which emphasised the importance of the CbrAB/Crc system as a regulator of chronic infection. Comparative transcriptome analysis with the PAO1 wild type strain and PAO6673 (delta crc), PAO66711 (delta cbrB), PAO6679 (delta crcZ) strains grown in LB medium.
2012-03-31 | E-GEOD-33244 | biostudies-arrayexpress
Project description:Among multiple interconnected pathways for L-Lysine (L-Lys) catabolism in pseudomonads, Pseudomonas aeruginosa PAO1 employed the decarboxylase and the transaminase pathways. However, up till now several genes involved in the operation and regulation of these pathways were still missing. Transcriptome analyses coupled with promoter activity measurements and mutant growth phenotype analysis lead us to identify several new members of the L-Lys and D-Lys catabolic pathways and their regulatory elements, including argR to trigger lysine decarboxylation into cadaverine, PauR for the γ-glutamylation pathway of polyamine catabolism into 5-aminovalerate, gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for L-Lys efflux, and lysP for L-Lys uptake.
2015-12-09 | GSE75502 | GEO
Project description:P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. We identified bacterial metabolite - phenylacetic acid (PAA) which acts as an inhibitory molecule counteracting its pathogenic infection. Microarray and genetic analyses were conducted to investigate the inhibitory mechanism of the identified inhibitor PAA on bacterial virulence. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of type 3 secretion systems (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Our findings present a new insight to the puzzle of high-cell-density-modulated virulence attenuation in P. aeruginosa and the regulatory mechanisms of T3SS which is associated with bacterial acute infection.
2013-02-21 | GSE43641 | GEO