Unknown,Transcriptomics,Genomics,Proteomics

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Coordination of frontline defense mechanisms under severe oxidative stress.

ABSTRACT: Oxidative stress (OS) results from genetic defects or stressful environmental challenges that cause unchecked production of reactive oxygen species (ROS). OS has been implicated in many diseases due to its wide ranging damage to nucleic acids, proteins and lipids. Using Halobacterium salinarum NRC-1, an extremophile that thrives under conditions of excessive OS, we have constructed a systems model for oxidative stress response (OSR) by integrating transcriptional changes induced by treatments with H2O2 and paraquat, functional associations inferred through comparative genomics, and de novo discovered cis-regulatory motifs. Together with phenotypic analysis of mutant strains this has revealed a multi-tiered OS management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids, and gas vesicles, metal trafficking, and various other aspects of metabolism. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was previously constructed from cellular responses to diverse environmental perturbations –this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of g rays. Mid-log phase cultures of H. salinarum NRC-1 (2 biological replicates) grown in flasks were treated with either sub-lethal concentrations of 25mM H2O2 or 4mM PQ and incubated with shaking for up to 240 min. For each treatment, two time courses were run to determine the transcriptional responses to (1) constant stress and (2) recovery of H. salinarum NRC-1 to H2O2 and PQ. During constant stress, culture aliquots (~4ml) were collected over a time course (-1, 5, 10, 20, 40, 80 and 160 minutes), centrifuged (16000g, 90 seconds) and flash frozen. For recovery, cells were first treated for 2-hours with either 25 mM H2O2 or 4mM PQ, recovered by centrifugation, washed and re-suspended in CM. Cultures were returned to the incubator with shaking and samples were taken at 0, 10, 20, 30, 40, 50, 60, 120, and 240 minutes and processed as described previously. Analysis of temporal transcriptional changes (-1, 0, 5, 10, 20, 40, 80, 160 and 320m) to sub-inhibitory concentrations of PQ (0.25mM) was also characterized. Total RNA was prepared and labeled with Alexa547 and Alexa647 dyes. Intensities were normalized between the Alexa 546 and Alexa 647 channels by scaling all intensities in one channel such that the 75th percentile in each channel was made equal.

ORGANISM(S): Halobacterium salinarum

SUBMITTER: Dan Tenenbaum

PROVIDER: E-GEOD-17515 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Coordination of frontline defense mechanisms under severe oxidative stress.

Kaur Amardeep A Van Phu T PT Busch Courtney R CR Robinson Courtney K CK Pan Min M Pang Wyming Lee WL Reiss David J DJ DiRuggiero Jocelyne J Baliga Nitin S NS

Molecular systems biology 20100701

Complexity of cellular response to oxidative stress (OS) stems from its wide-ranging damage to nucleic acids, proteins, carbohydrates, and lipids. We have constructed a systems model of OS response (OSR) for Halobacterium salinarum NRC-1 in an attempt to understand the architecture of its regulatory network that coordinates this complex response. This has revealed a multi-tiered OS-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, pr ...[more]

PMID: 20664639

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Project description:We report detailed characterization of physiological changes encoded by 63% of all genes within the archaeon Halobacterium salinarum subsp. NRC-1 during routine laboratory growth. While the majority of these changes occur during the transition from rapid exponential growth to the stationary phase, we were also able to detect transient changes in gene expression. This demonstrated the presence of several additional albeit subtle physiological states that exist beyond what might be anticipated from a direct interpretation of the growth profile. Changes in the abundance of several key intracellular metabolites over the growth curve corroborated observations of changes in gene expression of enzymes that catalyze their synthesis. Next, we investigated the roles of general transcription factors (GTFs) in mediating these global growth-associated gene expression changes. This revealed numerous phenotypic perturbations including slowed growth, gas vesicle biogenesis deficiency and morphologic abnormalities upon the overexpression of a single GTF - TBPd. These phenotypes were quantified and were attributed to a perturbation in the regulation of aconitase. Importantly, our results demonstrate why an experiment design as simple as a simple batch culture can be enormously informative of activities and interrelationships of a large fraction of all genes in a microbe. Halobacterium NRC-1 was grown in shaken (220RPM) complete medium liquid culture at 37Â°C in duplicate flasks. Samples were selected at 7 timepoints during the growth of this organism representing different phases of growth (e.g. early exponential, logrithmic, stationary etc.). RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 Î¼g of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (Î») values determined by a maximum likelihood method.Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84.

Project description:We report detailed characterization of physiological changes encoded by 63% of all genes within the archaeon Halobacterium salinarum subsp. NRC-1 during routine laboratory growth. While the majority of these changes occur during the transition from rapid exponential growth to the stationary phase, we were also able to detect transient changes in gene expression. This demonstrated the presence of several additional albeit subtle physiological states that exist beyond what might be anticipated from a direct interpretation of the growth profile. Changes in the abundance of several key intracellular metabolites over the growth curve corroborated observations of changes in gene expression of enzymes that catalyze their synthesis. Next, we investigated the roles of general transcription factors (GTFs) in mediating these global growth-associated gene expression changes. This revealed numerous phenotypic perturbations including slowed growth, gas vesicle biogenesis deficiency and morphologic abnormalities upon the overexpression of a single GTF - TBPd. These phenotypes were quantified and were attributed to a perturbation in the regulation of aconitase. Importantly, our results demonstrate why an experiment design as simple as a simple batch culture can be enormously informative of activities and interrelationships of a large fraction of all genes in a microbe. Halobacterium NRC-1 delta-ura3 strain was grown in shaken (220RPM) complete medium liquid culture at 37Â°C in duplicate flasks. 20Âµg/ml uracil was added to the media. Samples were selected at 6 timepoints during the growth of this organism representing different phases of growth (e.g. early exponential, logrithmic, stationary etc.). RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 Î¼g of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (Î») values determined by a maximum likelihood method.Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84. Keywords: growth curve, archaea, halobacterium

Project description:We report detailed characterization of physiological changes encoded by 63% of all genes within the archaeon Halobacterium salinarum subsp. NRC-1 during routine laboratory growth. While the majority of these changes occur during the transition from rapid exponential growth to the stationary phase, we were also able to detect transient changes in gene expression. This demonstrated the presence of several additional albeit subtle physiological states that exist beyond what might be anticipated from a direct interpretation of the growth profile. Changes in the abundance of several key intracellular metabolites over the growth curve corroborated observations of changes in gene expression of enzymes that catalyze their synthesis. Next, we investigated the roles of general transcription factors (GTFs) in mediating these global growth-associated gene expression changes. This revealed numerous phenotypic perturbations including slowed growth, gas vesicle biogenesis deficiency and morphologic abnormalities upon the overexpression of a single GTF - TBPd. These phenotypes were quantified and were attributed to a perturbation in the regulation of aconitase. Importantly, our results demonstrate why an experiment design as simple as a simple batch culture can be enormously informative of activities and interrelationships of a large fraction of all genes in a microbe. Halobacterium NRC-1 was grown in shaken (220RPM) complete medium liquid culture at 37Â°C in duplicate flasks. 20Âµg/ml uracil was added to the media. Samples were selected at 7 timepoints during the growth of this organism representing different phases of growth (e.g. early exponential, logrithmic, stationary etc.). RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 Î¼g of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (Î») values determined by a maximum likelihood method.Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84. Keywords: growth curve, archaea, halobacterium

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Coordination of frontline defense mechanisms under severe oxidative stress.

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Coordination of frontline defense mechanisms under severe oxidative stress.

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