Unknown,Transcriptomics,Genomics,Proteomics

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Mechanisms of adaptation to stress and control of virulence in Pseudomonas aeruginosa: the OxyR regulons

ABSTRACT: Oxidative stress caused by exposure to reactive oxygen species (ROS), is a major challenge for aerobic and especially anaerobic organisms. Bacteria coordinate the response to oxidative stress through the LysR-type transcriptional regulator (LTTR) OxyR. Extensive studies have focused on the classical Escherichia coli system to shed light on the mode of action of defensive weapons against oxidative stress. The underlying mechanism is mediated via the formation of redox-dependent disulfide bond between two conserved cysteines of OxyR, thus activating transcription of members of the OxyR regulon. However, only fragmentary information on the regulation and function of OxyR has been gleaned through genetic and biochemical analyses in the important opportunistic pathogen P. aeruginosa. In this report, we used a comprehensive transcriptional profiling analysis to delineate the OxyR regulon under three conditions (KingM-bM-^@M-^Ys A medium [Pseudomonas medium or PM], Luria Broth (LB), and LB when oxyR is overexpressed), to investigate its roles in different cellular aspects that are independent of the classical oxidative stress response. Interestingly, when grown in LB, OxyR was found to regulating many genes involved in the process of inter-cellular communication known as quorum sensing (QS). In contrast, when grown in PM, OxyR regulate the expression of a newly identified CSS (cell-surface signaling) system in an OxyR-dependent fashion. In addition, the results from oxyR overexpression further confirmed that OxyR was linked to regulation of QS and Type 3 Secretion (T3SS) in addition to the regulation of antioxidative genes. Taken together, our results show that, apart from its dominant role in defense against oxidative stress in P. aeruginosa, OxyR acts as a global regulator that provides a link between the regulation of oxidative stress response, QS and virulence. 15 samples, representing 5 different biological conditions, including 3 biological replicates for each condition

ORGANISM(S): Pseudomonas aeruginosa PAO1

SUBMITTER: Pieter Monsieurs

PROVIDER: E-GEOD-33871 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organised quorum sensing (QS) systems via small acyl-homoserine lactone (AHL) signal molecules. Recently, a third bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the QS systems. To further dissect the various independent regulation levels for many QS induced genes and to evaluate the impact of PQS on the QS circuitry, we performed a transcriptome analysis of PAO1 cultures supplemented with PQS. The global transcriptional profile in response to PQS revealed a marked up-regulation of genes belonging to the tightly interdependent functional groups of iron acquisition and oxidative stress response. Remarkably, not only most of the differentially regulated genes but also the induction of a lacZ transcriptional fusion of rhlR could be traced back to a iron chelating effect of PQS. Nevertheless, although iron deficiency per se induced rhlR, there seems to be PQS specific effects that are independent of the PQS effect on P. aeruginosa iron homeostasis Experiment Overall Design: For RNA extraction bacteria were harvested at early logarithmic, late logarithmic and stationary phase of growth. Three independent cultures of PQS-treated and untreated PAO1 each were pooled and the RNA was immediately stabilized with RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). RNA was isolated using the RNeasy kit (Qiagen), treated with DNaseI (Roche) for 30 min at 37M-BM-0C and re-purified with the RNeasy spin column. The subsequent steps of cDNA generation and Biotin-ddUTP terminal labelling were performed as described in the manufacturerM-bM-^@M-^Ys instructions for the P. aeruginosa GeneChipM-CM-^R. Fragmented and labelled cDNA was hybridised to a GeneChip at 50 M-BM-0C for 16 h. The washing steps, staining and scanning of the microarrays were performed using the Affymetrix GeneChip system. Data analysis was performed using the Affymetrix Microarray Suite Software 5.0 with Affymetrix default parameters. As expression analysis was performed in duplicate, a total of two GeneChips per culture condition was scanned at 570 nm, 3 M-BM-5m resolution in an Affymetrix GeneChip scanner. The signals were multiplied by a scaling factor to make the average signal for all the arrays equivalent. The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. Data were combined with the latest annotation from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at www.pseudomonas.com.

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Mechanisms of adaptation to stress and control of virulence in Pseudomonas aeruginosa: the OxyR regulons

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