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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000399 |
| Study Title | E.coli effects on growth and substrate uptake of green algae (part I - HILIC) |
| Study 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 |
| ofiehn@ucdavis.edu | |
| Phone | (530) 754-8258 |
| Submit Date | 2015-04-20 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | d |
| Analysis Type Detail | LC-MS |
| Release Date | 2016-06-18 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | Bacteria |
Factors:
Subject type: Cells; Subject species: Escherichia coli (Factor headings shown in green)
| mb_sample_id | local_sample_id | Type of media samples |
|---|---|---|
| SA019197 | BH28_HILIC.d | Base Degraded Thiamine |
| SA019198 | BH33_HILIC.d | Fractionated using RP column |
| SA019199 | BH34_HILIC.d | Fractionated using RP column |
| SA019200 | BH32_HILIC.d | Fractionated using RP column |
| SA019201 | BH31_HILIC.d | Fractionated using RP column |
| SA019202 | BH30_HILIC.d | Fractionated using RP column |
| SA019203 | BH29_HILIC.d | Fractionated using RP column |
| SA019204 | BH35_HILIC.d | Methanol Extracted |
| SA019205 | BH36_HILIC.d | Methanol 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 |
