Unknown,Transcriptomics,Genomics,Proteomics

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Microarray and transcript expression analysis of P. aeruginosa grown with or without bacterial (Delftia tsuruhatensis SJ01) extracts

ABSTRACT: Differential expression of regulatory genes of reference strain P. aeruginosa PA01, involved in the quorum sensing was analyzed using Microarray. Total RNA was isolated from reference strain P. aeruginosa PA01, grown with or without bacterial extracts (0.1 mg/ml) using TRI reagent. Total RNA was quantified, and 10 Âµg RNA was converted to cDNA, fragmented and labelled by following GeneChipÂ® P. aeruginosa PA01 genome array user manual . Labelled cDNAs were hybridized with P. aeruginosa genome array gene chip , washed and stained. Hybridized chips were scanned, processed and analyzed using expression console and transcriptome analysis console.

ORGANISM(S): Pseudomonas aeruginosa PAO1

SUBMITTER: Avinash Mishra

PROVIDER: E-MTAB-5693 | 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.

Project description:Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of P. aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into GABA and succinate before entering the TCA cycle in support of cell growth as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were identified to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown indispensable for polyamine utilization. The newly identified dadRAX locus, encoding the regulator, alanine transaminase and racemase respectively, coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintain alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, the alternative gamma-glutamylation pathway for the conversion of putrescine into GABA was also discussed. Subsequently, GabD, GabT and PA5313 were identified for GABA utilization. Growth defect of PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA was also demonstrated in vitro. Polyamine utilization in general was proven independent of the PhoPQ two-component system even the expression of which was induced by polyamines. Multiple potent catabolic pathways as depicted in this study could serve pivotal roles in control of intracellular polyamine levels. Experiment Overall Design: P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of L-glutamate alone or with the addition of putrescine, agmatine or GABA at 20 mM. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Only genes showing consistent expression profiles in duplicates were selected for further analysis.

Dataset Information

Microarray and transcript expression analysis of P. aeruginosa grown with or without bacterial (Delftia tsuruhatensis SJ01) extracts

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