Summary of Study ST000399

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 PR000312. The data can be accessed directly via it's Project DOI: 10.21228/M8KP4K This work is supported by NIH grant, U2C- DK119886.

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Study IDST000399
Study TitleE.coli effects on growth and substrate uptake of green algae (part I - HILIC)
Study SummaryThe purpose of this project was to quantify the exchange of thiamine between bacteria and algae. We previously observed that the model bacteria, Escherichia coli, enhanced the growth and substrate uptake of the green algae, Auxenochlorella protothecoides. We hypothesized that this growth enhancement was due to the secretion of thiamine derivatives or degradation products by E. coli followed by uptake of these compounds by A. protothecoides. Targeted and untargeted LCMS revealed the presence of thiamine dervatives in E. coli cell extracts. These LCMS methods were also used to quantify thiamine derivatives and two degradation products, HMP and THZ, present in E. coli medium after cell removal. The LCMS results along with culture studies were employed to show that thiamine derivatives and degradation products were the primary mechanism of symbiosis between E. coli and A. protothecoides.
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 Date2015-04-20
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/M8KP4K
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000312
Project DOI:doi: 10.21228/M8KP4K
Project Title:E.coli effects on growth and substrate uptake of green algae
Project Summary:The purpose of this project was to quantify the exchange of thiamine between bacteria and algae. We previously observed that the model bacteria, Escherichia coli, enhanced the growth and substrate uptake of the green algae, Auxenochlorella protothecoides. We hypothesized that this growth enhancement was due to the secretion of thiamine derivatives or degradation products by E. coli followed by uptake of these compounds by A. protothecoides. Targeted and untargeted LCMS revealed the presence of thiamine dervatives in E. coli cell extracts. These LCMS methods were also used to quantify thiamine derivatives and two degradation products, HMP and THZ, present in E. coli medium after cell removal. The LCMS results along with culture studies were employed to show that thiamine derivatives and degradation products were the primary mechanism of symbiosis between E. coli and A. protothecoides.
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

Subject:

Subject ID:SU000420
Subject Type:Cells
Subject Species:Escherichia coli
Taxonomy ID:562
Species Group:Microorganism

Factors:

Subject type: Cells; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id local_sample_id Type of media samples
SA019197BH28_HILIC.dBase Degraded Thiamine
SA019198BH33_HILIC.dFractionated using RP column
SA019199BH34_HILIC.dFractionated using RP column
SA019200BH32_HILIC.dFractionated using RP column
SA019201BH31_HILIC.dFractionated using RP column
SA019202BH30_HILIC.dFractionated using RP column
SA019203BH29_HILIC.dFractionated using RP column
SA019204BH35_HILIC.dMethanol Extracted
SA019205BH36_HILIC.dMethanol Extracted
Showing results 1 to 9 of 9

Collection:

Collection ID:CO000414
Collection Summary:Cultures were harvested after 5 days of growth, washed with dH2O, and freeze dried 5 ml of trichloroacetic acid was added to entire freeze dried cell pellet in 15 ml tube, vortexed, and incubated on shaker at 150 rpm for 3 hours TCA was extracted twice w/ 5ml diethyl ether, then sample was washed with 5 ml chloroform, and aqueous phase was recovered Freeze-dried aqueous phase, resuspended in 1 ml MeOH and incubated on shaker for 1 hour at 150 rpm, centrifuged Supernatant was trasnferred to 2 ml tube, sample washed with additional 0.5 ml MeOH and added to 2 ml tube Freeze dried. Note that a method blank and 2 recovery standards containing thiamine were included in this processing
Collection Protocol Filename:Thiamine_BH_IG032715QTrap.xlsx
Sample Type:Cell

Treatment:

Treatment ID:TR000434
Treatment Summary:BH28 was base degraded thiamine. It did not come from an organism. It was suspended thiamine in strong base for an extended period of time. This was done to see what types of degraded products would form. BH29-34 were E. coli medium samples that were fractionated using a reverse phase column. BH35-36 were media samples after E. coli growth where methanol was used for extraction.

Sample Preparation:

Sampleprep ID:SP000427
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 AN000636 AN000637
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Agilent 6530 Agilent 6550
Column Waters Acquity BEH Amide (150 x 2.1mm,1.7um) Waters Acquity BEH Amide (150 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:CH000461
Methods Filename:Data_Dictionary_Fiehn_laboratory_HILIC_QTOF_MS.pdf
Instrument Name:Agilent 6530
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Pressure:200-700 bar
Column Temperature:45 C
Flow Gradient:100% B to 30% B
Flow Rate:0.4 mL/min
Injection Temperature:4 C
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:3uL
Solvent A:100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
Solvent B:100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
Analytical Time:14 min
Capillary Voltage:4500 V
Time Program:16.75 min
Weak Wash Solvent Name:1:1 ACN:H2O
Strong Wash Solvent Name:1:1 ACN:H2O
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel generated
Chromatography Type:HILIC
  
Chromatography ID:CH000462
Methods Filename:Data_Dictionary_Fiehn_laboratory_HILIC_QTOF_MS.pdf
Instrument Name:Agilent 6550
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Pressure:200-700 bar
Column Temperature:45 C
Flow Gradient:100% B to 30% B
Flow Rate:0.4 mL/min
Injection Temperature:4 C
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:3uL
Solvent A:100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
Solvent B:95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
Analytical Time:14 min
Capillary Voltage:4500 V
Time Program:16.75 min
Weak Wash Solvent Name:1:1 ACN:H2O
Strong Wash Solvent Name:1:1 ACN:H2O
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel generated
Chromatography Type:HILIC

MS:

MS ID:MS000568
Analysis ID:AN000636
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
Ion Spray Voltage:1000
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:1850 V
  
MS ID:MS000569
Analysis ID:AN000637
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:1850 V
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