Halobacterium growth
Ontology highlight
ABSTRACT:
ORGANISM(S): Halobacterium sp. NRC-1
SUBMITTER: Dan Tenenbaum
PROVIDER: E-GEOD-14976 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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 37°C in duplicate flasks. 20µg/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 μg 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 (λ) 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
2010-05-14 | E-GEOD-14835 | biostudies-arrayexpress
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 37°C in duplicate flasks. 20µg/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 μg 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 (λ) 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
2010-05-14 | E-GEOD-14836 | biostudies-arrayexpress
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 37°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 μg 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 (λ) 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.
2010-05-14 | E-GEOD-14832 | biostudies-arrayexpress
Project description:Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways. The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment. Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, AgmR. Like TrmB, its thermophilic archaeal homolog, AgmR regulates glycolytic and gluconeogenic pathways in response to sugar availability. However, using high throughput genome-scale experiments, we find that AgmR directly governs the transcription of nearly 100 additional genes encoding enzymes in diverse metabolic pathways. Genome-scale in vivo binding site location data reveals that >60% of these are direct targets. Integration of these systems-scale datasets with metabolic reconstruction models suggests that AgmR, a sequence-specific bacterial-like regulator, interacts with the general transcription factor machinery to coordinate nitrogen and carbon metabolism with the de novo synthesis of cognate cofactors and reducing equivalents, achieving system-wide redox and energy balance. Halobacterium salinarum NRC-1 (ATCC700922) ura3 parent and VNG1451C strains were grown in rich medium (CM, 250 NaCl, 20 g/L MgSO4?7H2O, 3 g/L sodium citrate, 2 g/L KCl, 10 g/L peptone) with or without varying concentrations of glucose or glycerol at 37ºC under full-spectrum white light. The cells were cross-linked with 1.2% formaldehyde, then lysed and DNA sheared via sonication. Next cleared cell lysate was subject to immunoprecipitation against an anti-myc antibody. Protein-DNA complexes were purified and DNA was isolated after protease and RNAase treatment. DNA was amplified using universal linkers, labeled directly using Kreatech 547 and 647 dyes. Samples were grown in biological duplicate. Each DNA sample was hybridized against 2 microarrays as dyefilps. At least two experiments (including biological duplicates) were carried out.
2010-05-06 | E-GEOD-13498 | biostudies-arrayexpress
Project description:Shotgun proteomics has grown rapidly in recent decades, but a large fraction of tandem mass spectrometry (MS/MS) data in shotgun proteomics are not successfully identified. We have developed a novel database search algorithm, Open-pFind, to efficiently identify peptides even in an ultra-large search space which takes into account unexpected modifications, amino acid mutations, semi- or non-specific digestion and co-eluting peptides. Tested on two metabolically labeled MS/MS datasets, Open-pFind reported 50.5‒117.0% more peptide-spectrum matches (PSMs) than the seven other advanced algorithms. More importantly, the Open-pFind results were more credible judged by the verification experiments using stable isotopic labeling. Tested on four additional large-scale datasets, 70‒85% of the spectra were confidently identified, and high-quality spectra were nearly completely interpreted by Open-pFind. Further, Open-pFind was over 40 times faster than the other three open search algorithms and 2‒3 times faster than three restricted search algorithms. Re-analysis of an entire human proteome dataset consisting of ~25 million spectra using Open-pFind identified a total of 14,064 proteins encoded by 12,723 genes by requiring at least two uniquely identified peptides. In this search results, Open-pFind also excelled in an independent test for false positives based on the presence or absence of olfactory receptors. Thus, a practical use of the open search strategy has been realized by Open-pFind for the truly global-scale proteomics experiments of today and in the future.
2018-07-11 | PXD008783 | Pride
Project description:This SuperSeries is composed of the following subset Series: GSE12923: Halobacterium salinarum NRC-1 growth curve, tiling arrays. GSE12977: Halobacterium salinarum NRC-1 growth curve GSE13108: Halobacterium salinarum NRC-1 conditional ChIP-chip for transcription initiation factor IIB 4 (TFBd) GSE7045: ChIP-Chip of General Transcription factors in Halobacterium NRC-1 GSE15786: Halobacterium sp. NRC-1 ChIP-chip for TFBa, TFBd and TFBf, high resolution array GSE15788: Halobacterium salinarum NRC-1 total RNA hybridization of TFBd overexpression versus Reference sample Despite knowledge of complex prokaryotic transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have played a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ~64% of all genes including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction datasets revealed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes - events usually considered spurious or non-functional. With experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements Refer to individual Series
2010-05-17 | E-GEOD-13150 | biostudies-arrayexpress
Project description:Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways. The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment. Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, VNG1451C. In this experiment we used whole-genome microarray analysis in the VNG1451C deletion mutant vs. H. salinarum NRC-1 ura3 parent strain in rich medium during growth to show that the expression of many metabolic genes is perturbed in the VNG1451C deletion mutant. Halobacterium salinarum NRC-1 (ATCC700922) ura3 parent and VNG1451C strains were grown in complex medium (CM; 250g/L NaCl, 20g/L MgSO4.7H2O, 3g/L sodium citrate, 2g/L KCl, 10g/L peptone) at 37ºC under full-spectrum white light. Biological replicate samples were removed throughout the growth curve at early log, mid log, late log, and stationary phase to measure genome-wide transcription.
2010-05-06 | E-GEOD-13531 | biostudies-arrayexpress
Project description:Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways. The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment. Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, VNG1451C. In this experiment we used whole-genome microarray analysis in the VNG1451C deletion mutant vs. H. salinarum NRC-1 ura3 parent strain in defined medium during growth with and without glucose to show that the expression of many metabolic genes is perturbed in the VNG1451C deletion mutant in response to this sugar. Halobacterium salinarum NRC-1 (ATCC700922) ura3 parent and VNG1451C strains were grown in complete defined medium (CDM; 20 amino acids at concentrations defined by Shand and Perez, 1999. NaCl 250g/L, MgSO4 20 g/L, KCl 1 g/L, NaH2PO4 0.167mM, Biotin 0.02mM, Thiamin 0.015mM, Folic acid 0.0113 mM, MnSO4 0.01mM, FeSO4, 0.01mM, glucose added at 10% w/v where indicated in sample files) at 37ºC under full-spectrum white light. Biological replicate samples were removed throughout the growth curve at early log, mid log, and stationary phase to measure genome-wide transcription.
2010-05-06 | E-GEOD-13529 | biostudies-arrayexpress
Project description:Altering the genetic code for applications in synthetic biology and genetic code expansion involves engineered tRNAs that incorporate amino acids that differ from what is defined by the “standard” genetic code. Since these engineered tRNA variants can be lethal due to proteotoxic stress, regulating their expression is necessary to achieve high levels of the resulting novel proteins. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNA serine variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using mass spectrometry, we determine th frequency of mistranslation in both the induced and repressed conditions of the galactose inducible and tetracycline inducible systems.
2021-11-03 | PXD028496 | Pride
Project description:100mM EMS was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of EMS for 30 minutes, cultures were spun down and pellets were re-suspended in same volume of GN101 media. Cultures were then harvested by centrifugation and pellets were snap frozen on a dry-ice ethanol bath. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes. 16 samples (8 samples from EMS perturbed and 8 non-perturbed controls) were analyzed on replicate arrays (dye-flips) all against the same standard reference sample.
2010-05-27 | E-GEOD-7609 | biostudies-arrayexpress