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Transcriptional profiling of E. coli after addition of peroxynitrite or hydrogen peroxide to aerobically growing cells

ABSTRACT: Peroxynitrite is formed in macrophages by the diffusion-limited reaction of superoxide and nitric oxide. This highly reactive species is capable of causing both oxidative and nitrosative stress in Escherichia coli. Previous studies have focused on the reactions of peroxynitrite with specific proteins or the effects of peroxynitrite on the growth and viability of whole cells. This work shows for the first time the transcriptomic response of E. coli to peroxynitrite, highlighting specific areas targeted by the stress. Upregulation of the cysteine biosynthesis pathway and subsequent identification of an increase in S-nitrosothiol levels suggests S-nitrosylation as a consequence of peroxynitrite exposure. Genes involved in the assembly / repair of iron-sulfur clusters also show enhanced transcription identifying another target of this reactive species. Unexpectedly arginine biosynthesis gene transcription levels were also elevated after treatment with peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed that the post-translational nitration of tyrosine residues within this protein is responsible for its degradation in vitro. Further upregulation is seen in oxidative stress response genes including katG and ahpCF. Probabilistic modelling of this data identified 5 altered transcription factors in response to peroxynitrite exposure including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in commercially available peroxynitrite preparations. Transcriptomic analysis of cells treated with hydrogen peroxide also showed an upregulation of oxidative stress response genes; however it did not show increased transcription of many other genes which are upregulated by peroxynitrite suggesting that cellular responses to peroxynitrite and hydrogen peroxide are distinct.

ORGANISM(S): Escherichia coli Escherichia coli K-12

PROVIDER: GSE19370 | GEO | 2010/05/24

SECONDARY ACCESSION(S): PRJNA120413

REPOSITORIES: GEO

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Project description:Peroxynitrite is formed in macrophages by the diffusion-limited reaction of superoxide and nitric oxide. This highly reactive species is capable of causing both oxidative and nitrosative stress in Escherichia coli. Previous studies have focused on the reactions of peroxynitrite with specific proteins or the effects of peroxynitrite on the growth and viability of whole cells. This work shows for the first time the transcriptomic response of E. coli to peroxynitrite, highlighting specific areas targeted by the stress. Upregulation of the cysteine biosynthesis pathway and subsequent identification of an increase in S-nitrosothiol levels suggests S-nitrosylation as a consequence of peroxynitrite exposure. Genes involved in the assembly / repair of iron-sulfur clusters also show enhanced transcription identifying another target of this reactive species. Unexpectedly arginine biosynthesis gene transcription levels were also elevated after treatment with peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed that the post-translational nitration of tyrosine residues within this protein is responsible for its degradation in vitro. Further upregulation is seen in oxidative stress response genes including katG and ahpCF. Probabilistic modelling of this data identified 5 altered transcription factors in response to peroxynitrite exposure including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in commercially available peroxynitrite preparations. Transcriptomic analysis of cells treated with hydrogen peroxide also showed an upregulation of oxidative stress response genes; however it did not show increased transcription of many other genes which are upregulated by peroxynitrite suggesting that cellular responses to peroxynitrite and hydrogen peroxide are distinct. Biological experiments (i.e. a comparison of control and plus peroxynitrite cells) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed. Additional biological experiments (i.e. a comparison of control and plus hydrogen peroxide cells) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

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Transcriptional profiling of E. coli after addition of peroxynitrite or hydrogen peroxide to aerobically growing cells

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