Summary of Study ST000394

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000308. The data can be accessed directly via it's Project DOI: 10.21228/M82P59 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000394
Study TitleThe circadian oscillator in Synechococcus elongatus controls metabolite partitioning during diurnal growth (part I)
Study SummaryCyanobacteria are increasingly being considered for use in large-scale outdoor production of fuels and industrial chemicals. Cyanobacteria can anticipate daily changes in light availability using an internal circadian clock and rapidly alter their metabolic processes in response to changes light availability. Understanding how signals from the internal circadian clock and external light availability are integrated to control metabolic shifts will be important for engineering cyanobacteria for production in natural outdoor environments. This study has assessed how “knowing” the correct time of day, via the circadian clock, affects metabolic changes when a cyanobacterium goes through a dark-to-light transition. Our data show that the circadian clock plays an important role in inhibiting activation of the oxidative pentose phosphate pathway in the morning. Synechococcus elongatus PCC 7942 is a genetically tractable model cyanobacterium that has been engineered to produce industrially relevant biomolecules and is the best-studied model for a prokaryotic circadian clock. However, the organism is commonly grown in continuous light in the laboratory, and data on metabolic processes under diurnal conditions are lacking. Moreover, the influence of the circadian clock on diurnal metabolism has been investigated only briefly. Here, we demonstrate that the circadian oscillator influences rhythms of metabolism during diurnal growth, even though light–dark cycles can drive metabolic rhythms independently. Moreover, the phenotype associated with loss of the core oscillator protein, KaiC, is distinct from that caused by absence of the circadian output transcriptional regulator, RpaA (regulator of phycobilisome-associated A). Although RpaA activity is important for carbon degradation at night, KaiC is dispensable for those processes. Untargeted metabolomics analysis and glycogen kinetics suggest that functional KaiC is important for metabolite partitioning in the morning. Additionally, output from the oscillator functions to inhibit RpaA activity in the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active output state, phenocopies a ΔrpaA strain. Inhibition of RpaA by the oscillator in the morning suppresses metabolic processes that normally are active at night, and kaiC-null strains show indications of oxidative pentose phosphate pathway activation as well as increased abundance of primary metabolites. Inhibitory clock output may serve to allow secondary metabolite biosynthesis in the morning, and some metabolites resulting from these processes may feed back to reinforce clock timing.
Institute
University of California, Davis
DepartmentGenome and Biomedical Sciences Facility
LaboratoryWCMC Metabolomics Core
Last NameFiehn
First NameOliver
Address1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Emailofiehn@ucdavis.edu
Phone(530) 754-8258
Submit Date2016-05-04
Study CommentsThe first 4 samples were a test run to see how efficient the analysis was and were run on a lipidomics platform. The next 12 samples were the used in the paper and were the same as the original 4 samples, but they were split into 3 biological replicates and run on the GC platform.
Publicationsdoi: 10.1073/pnas.1504576112
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2016-06-18
Release Version1
Oliver Fiehn Oliver Fiehn
https://dx.doi.org/10.21228/M82P59
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000308
Project DOI:doi: 10.21228/M82P59
Project Title:The circadian oscillator in Synechococcus elongatus controls metabolite partitioning during diurnal growth
Project Summary:Cyanobacteria are increasingly being considered for use in large-scale outdoor production of fuels and industrial chemicals. Cyanobacteria can anticipate daily changes in light availability using an internal circadian clock and rapidly alter their metabolic processes in response to changes light availability. Understanding how signals from the internal circadian clock and external light availability are integrated to control metabolic shifts will be important for engineering cyanobacteria for production in natural outdoor environments. This study has assessed how “knowing” the correct time of day, via the circadian clock, affects metabolic changes when a cyanobacterium goes through a dark-to-light transition. Our data show that the circadian clock plays an important role in inhibiting activation of the oxidative pentose phosphate pathway in the morning. Synechococcus elongatus PCC 7942 is a genetically tractable model cyanobacterium that has been engineered to produce industrially relevant biomolecules and is the best-studied model for a prokaryotic circadian clock. However, the organism is commonly grown in continuous light in the laboratory, and data on metabolic processes under diurnal conditions are lacking. Moreover, the influence of the circadian clock on diurnal metabolism has been investigated only briefly. Here, we demonstrate that the circadian oscillator influences rhythms of metabolism during diurnal growth, even though light–dark cycles can drive metabolic rhythms independently. Moreover, the phenotype associated with loss of the core oscillator protein, KaiC, is distinct from that caused by absence of the circadian output transcriptional regulator, RpaA (regulator of phycobilisome-associated A). Although RpaA activity is important for carbon degradation at night, KaiC is dispensable for those processes. Untargeted metabolomics analysis and glycogen kinetics suggest that functional KaiC is important for metabolite partitioning in the morning. Additionally, output from the oscillator functions to inhibit RpaA activity in the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active output state, phenocopies a ΔrpaA strain. Inhibition of RpaA by the oscillator in the morning suppresses metabolic processes that normally are active at night, and kaiC-null strains show indications of oxidative pentose phosphate pathway activation as well as increased abundance of primary metabolites. Inhibitory clock output may serve to allow secondary metabolite biosynthesis in the morning, and some metabolites resulting from these processes may feed back to reinforce clock timing.
Institute:University of California, Davis
Department:Genome and Biomedical Sciences Facility
Laboratory:WCMC Metabolomics Core
Last Name:Fiehn
First Name:Oliver
Address:1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Email:ofiehn@ucdavis.edu
Phone:(530) 754-8258
Funding Source:NIH U24DK097154
Publications:doi: 10.1073/pnas.1504576112

Subject:

Subject ID:SU000415
Subject Type:Cells
Subject Species:Synechococcus elongatus PCC 7942
Taxonomy ID:1140
Species Group:Microorganism

Factors:

Subject type: Cells; Subject species: Synechococcus elongatus PCC 7942 (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Time Point
SA018744SDiamInj03_KaiC T0_CSH.dKaiC mutant -
SA018745SDiamInj02_KaiC T4_CSH.dKaiC mutant 4
SA018746SDiamInj04_WT T0_CSH.dWT -
SA018747SDiamInj05_WT T4_CSH.dWT 4
Showing results 1 to 4 of 4

Collection:

Collection ID:CO000409
Collection Summary:Bacteria were grown in a turbidostat/bioreactor at equal cell density (measured by optical density at 750nm), under a 12:12h Light/Dark cycle. After collection samples were immediately placed on ice and then centrifuged at 5000RPM for 10min at ­4 degrees Celsius. After centrifugation supernatant was decanted and cell pellets were immediately frozen in liquid N2.
Collection Protocol Filename:StudyDesign-SpencerDiamond-10814.pdf
Sample Type:Cell
Collection Time:Samples were collected at T0 (beginning of day) and T4 (4h into day).
Volumeoramount Collected:40ml of sample was collected at each time point
Storage Conditions:Samples were put into a 50mL conical tube containing ice up to the 30ml mark.

Treatment:

Treatment ID:TR000429
Treatment Summary:2: WT bacteria and KaiC mutant The phenotype associated with loss of the core oscillator protein, KaiC, is distinct from that caused by absence of the circadian output transcriptional regulator, RpaA (regulator of phycobilisome-associated A). Untargeted metabolomics analysis and glycogen kinetics suggest that functional KaiC is important for metabolite partitioning in the morning. Additionally, output from the oscillator functions to inhibit RpaA activity in the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active output state, phenocopies a ΔrpaA strain. KaiC-null strains show indications of oxidative pentose phosphate pathway activation as well as increased abundance of primary metabolites. Inhibitory clock output may serve to allow secondary metabolite biosynthesis in the morning, and some metabolites resulting from these processes may feed back to reinforce clock timing.
Treatment Protocol Filename:StudyDesign-SpencerDiamond-10814.pdf

Sample Preparation:

Sampleprep ID:SP000422
Sampleprep Summary:1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells, transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding beads to eppendorf tube 3. Use the GenoGrinder to grind for 3 minutes at 1,250 rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine with supernatant collected in step 5. Total volume of extracted sample will be approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube for GC-TOF analysis. 9. Store backups in -20 or -80C.
Sampleprep Protocol Filename:SOP_Extraction_of_Yeast_Cells.pdf

Combined analysis:

Analysis ID AN000630 AN000631
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Agilent 6530 Agilent 6550
Column Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Agilent 6530 QTOF Agilent 6550 QTOF
Ion Mode POSITIVE NEGATIVE
Units counts counts

Chromatography:

Chromatography ID:CH000455
Methods Filename:Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics_05-29-2014.pdf
Instrument Name:Agilent 6530
Column Name:Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
Column Pressure:450-850 bar
Column Temperature:65 C
Flow Gradient:15% B to 99%B
Flow Rate:0.6 mL/min
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:1.67 uL
Solvent A:60:40 Acetonitrile:Water +10mM Ammonium Formate +10mM Formic Acid
Solvent B:9:1 Isopropanol:Acetonitrile +10mM Ammonium Formate +10mM Formic Acid
Analytical Time:13 min
Capillary Voltage:3500 V
Time Program:15 min
Weak Wash Solvent Name:Isopropanol
Strong Wash Solvent Name:Isopropanol
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel generated
Chromatography Type:Reversed phase
  
Chromatography ID:CH000456
Methods Filename:Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics_05-29-2014.pdf
Instrument Name:Agilent 6550
Column Name:Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
Column Pressure:450-850 bar
Column Temperature:65 C
Flow Gradient:15% B to 99%B
Flow Rate:0.6 mL/min
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:5 uL
Solvent A:60:40 Acetonitrile:Water +10mM Ammonium Acetate +10mM Acetic Acid
Solvent B:9:1 Isopropanol:Acetonitrile +10mM Ammonium Acetate +10mM Acetic Acid
Analytical Time:13 min
Capillary Voltage:3500 V
Time Program:15 min
Weak Wash Solvent Name:Isopropanol
Strong Wash Solvent Name:Isopropanol
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel generated
Chromatography Type:Reversed phase

MS:

MS ID:MS000563
Analysis ID:AN000630
Instrument Name:Agilent 6530 QTOF
Instrument Type:QTOF
MS Type:ESI
Ion Mode:POSITIVE
Capillary Voltage:3500 V
Collision Gas:Nitrogen
Dry Gas Flow:8 L/min
Dry Gas Temp:325 C
Fragment Voltage:120 V
Fragmentation Method:Auto MS/MS
Ion Source Temperature:325 C
Ion Spray Voltage:1000 V
Ionization:Pos
Precursor Type:Intact Molecule
Reagent Gas:Nitrogen
Source Temperature:325 C
Dataformat:.d
Desolvation Gas Flow:11 L/min
Desolvation Temperature:350 C
Nebulizer:35 psig
Octpole Voltage:750 V
Resolution Setting:extended dynamic range
Scan Range Moverz:60-1700 Da
Scanning Cycle:2 Hz
Scanning Range:60-1700 Da
Skimmer Voltage:65 V
  
MS ID:MS000564
Analysis ID:AN000631
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Voltage:3500 V
Collision Gas:Nitrogen
Dry Gas Flow:13 L/min
Dry Gas Temp:200 C
Fragment Voltage:175 V
Fragmentation Method:Auto MS/MS
Ion Source Temperature:325 C
Ion Spray Voltage:1000 V
Ionization:Neg
Precursor Type:Intact Molecule
Reagent Gas:Nitrogen
Source Temperature:325 C
Dataformat:.d
Desolvation Gas Flow:11 L/min
Desolvation Temperature:350 C
Nebulizer:35 psig
Octpole Voltage:750 V
Resolution Setting:extended dynamic range
Scan Range Moverz:60-1700 Da
Scanning Cycle:2 Hz
Scanning Range:60-1700 Da
Skimmer Voltage:65 V
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