Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of E.coli MG1655 and a strain with a precise deletion in soxR treated with paraquat - time series

ABSTRACT: SoxR and SoxS constitute an intracellular signal response system that rapidly detects changes in superoxide levels and modulates gene expression in E. coli. A time series microarray design was used to identify co-regulated SoxRS dependent and independent genes affected by superoxide Experiment Overall Design: To determine the immediate transcriptional response of E. coli to superoxide, we conducted a time series assay of mRNA levels immediately following the addition of paraquat. The wild type strain MG1655 and a strain with a precise deletion in soxR was grown in EZ Rich Defined Medium, a modification of Neidhardtâs defined rich medium. When cultures reached an OD600=0.5, 250ÂµM paraquat (PQ) was added, a concentration that triggers the SoxRS transcriptional cascade, but only has a small effect on exponential growth rate in rich media. Samples were taken immediately before exposure to PQ, and every 2 minutes after exposure, for 10 minutes.

ORGANISM(S): Escherichia coli

SUBMITTER: Jeffrey Blanchard

PROVIDER: E-GEOD-6992 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of superoxide, a progenitor reactive oxygen species, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since reactive carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin-coated beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ mass spectrometry-based proteomics approach. While Paraquat exposure and Sod2 knockdown have similar phenotypes, differences in protein carbonylation were anticipated because Paraquat exposure was expected to increase the concentration of superoxide throughout the cell while Sod2 knockdown was only expected to raise the concentration of superoxide in the mitochondrial matrix. Paraquat exposure resulted in widespread increases in carbonylated protein relative abundance: the median Paraquat-exposed to control carbonylated protein relative abundance ratio was 1.53. For Sod2 knockdown, in contrast, the median carbonylated protein relative abundance ratios were 1.13 versus the RNA interference driver control and 1.05 versus the RNA interference transgene control. However, some proteins did show large increases in carbonylated protein relative abundance on Sod2 knockdown, most notably cytochrome c oxidase subunit Vb, possibly providing some indication of the molecular basis of the Sod2-knockdown phenotype.

Dataset Information

Transcription profiling of E.coli MG1655 and a strain with a precise deletion in soxR treated with paraquat - time series

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