Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Evolution of Context Dependent Regulation by Expansion of Feast/Famine Regulatory Proteins

ABSTRACT: We validated context dependent regulation by two FFRPs AsnC and VNG1237C by deleting the FFRP and testing the effect on downstream target genes. For validtion of AsnC condition specific regulation we grew wild-type and delta-AsnC in the presence of 4mM paraquat (PQ) and samples at 1 and 160 minutes post addition of this sub-lethal dose of PQ. For validation of VNG1237C we grew wild-type and delta-VNG1237C in a standard growth curve sampling at OD 0.18 and 1.15. The asnC deletion strain (M-bM-^HM-^FasnC) and the control M-bM-^HM-^Fura3 strain were grown in 4mM paraquat (PQ) and were sampled at 1 and 160 minutes after PQ addition. Sampling timing for M-bM-^HM-^FVNG1237C was designed such that the starting ODs were as similar as possible and once an OD of 1.17 was reached for M-bM-^HM-^Fura3 the M-bM-^HM-^FVNG1237C was sampled at this same time point.

ORGANISM(S): Halobacterium sp. NRC-1

SUBMITTER: Christopher Plaisier

PROVIDER: E-GEOD-61975 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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.

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Evolution of Context Dependent Regulation by Expansion of Feast/Famine Regulatory Proteins

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