Summary of Study ST001308
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 PR000889. The data can be accessed directly via it's Project DOI: 10.21228/M8S39G This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001308 |
| Study Title | 1H NMR metabolomics corroborates serine hydroxymethyltransferase as the primary target of 2-aminoacrylate in a ridA mutant of Salmonella enterica |
| Study Type | NMR metabolomics on Salmonella enterica |
| Study Summary | The reactive intermediate deaminase RidA (EC: 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When S. enterica ridA mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5’-phosphate (PLP)- dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted 1H NMR metabolomics to determine whether the metabolic state of a S. enterica ridA mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a ridA mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the shifts in the metabolome of a ridA mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled. |
| Institute | University of Georgia |
| Department | Microbiology, Biochemistry, Complex Carbohydrate Research Center |
| Laboratory | Edison Lab and Downs lab |
| Last Name | Gouveia |
| First Name | Goncalo |
| Address | 315 riverbend road, Complex Carbohydrate Research Centre, ATHENS, GA, 30605, USA |
| goncalog@uga.edu | |
| Phone | 7063087500 |
| Submit Date | 2020-01-22 |
| Num Groups | 4 |
| Total Subjects | 40 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | ft |
| Analysis Type Detail | NMR |
| Release Date | 2020-03-03 |
| Release Version | 1 |
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Project:
| Project ID: | PR000889 |
| Project DOI: | doi: 10.21228/M8S39G |
| Project Title: | 1H NMR metabolomics confirms serine hydroxymethyltransferase is the primary target of 2-aminoacrylate in a ridA mutant of Salmonella enterica |
| Project Type: | NMR metabolomics Salmonella enterica |
| Project Summary: | The reactive intermediate deaminase RidA (EC: 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When S. enterica ridA mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5’-phosphate (PLP)- dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted 1H NMR metabolomics to determine whether the metabolic state of a S. enterica ridA mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a ridA mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the shifts in the metabolome of a ridA mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled. |
| Institute: | University of Georgia |
| Department: | Microbiology, Biochemistry, Complex Carbohydrate Research Center |
| Laboratory: | Edison Lab and Downs lab |
| Last Name: | Edison |
| First Name: | Arthur |
| Address: | 315 riverbend road, Complex Carbohydrate Research Centre, ATHENS, GA, 30605, USA |
| Email: | aedison@uga.edu |
| Phone: | na |
| Contributors: | Andrew J. Borchert, Goncalo J. Gouveia, Arthur S. Edison, and Diana M. Downs |
Subject:
| Subject ID: | SU001382 |
| Subject Type: | Bacteria |
| Subject Species: | Salmonella enterica serovar Typhimurium |
| Taxonomy ID: | 90371 |
| Genotype Strain: | LT2 |
| Gender: | Not applicable |
| Subject Comments: | The wild type as describe, also used a ridA null mutant (DM3480, ridA3::MudJ1734), DM9404 |
Factors:
Subject type: Bacteria; Subject species: Salmonella enterica serovar Typhimurium (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample_group | Genotype | Suplement |
|---|---|---|---|---|
| SA094489 | media_3480 +Gly 6 | mut_gly | rid1-knockout | glycine |
| SA094490 | media_3480 +Gly 10 | mut_gly | rid1-knockout | glycine |
| SA094491 | media_3480 +Gly 8 | mut_gly | rid1-knockout | glycine |
| SA094492 | 3480 +Gly 1 | mut_gly | rid1-knockout | glycine |
| SA094493 | 3480 +Gly 8 | mut_gly | rid1-knockout | glycine |
| SA094494 | media_3480 +Gly 3 | mut_gly | rid1-knockout | glycine |
| SA094495 | 3480 +Gly 7 | mut_gly | rid1-knockout | glycine |
| SA094496 | 3480 +Gly 9 | mut_gly | rid1-knockout | glycine |
| SA094497 | 3480 +Gly 2 | mut_gly | rid1-knockout | glycine |
| SA094498 | 3480 +Gly 5 | mut_gly | rid1-knockout | glycine |
| SA094499 | 3480 +Gly 4 | mut_gly | rid1-knockout | glycine |
| SA094500 | media_3480 +Gly 5 | mut_gly | rid1-knockout | glycine |
| SA094501 | media_3480 +Gly 7 | mut_gly | rid1-knockout | glycine |
| SA094502 | media_3480 +Gly 2 | mut_gly | rid1-knockout | glycine |
| SA094503 | media_3480 +Gly 1 | mut_gly | rid1-knockout | glycine |
| SA094504 | 3480 +Gly 3 | mut_gly | rid1-knockout | glycine |
| SA094505 | media_3480 +Gly 4 | mut_gly | rid1-knockout | glycine |
| SA094506 | media_3480 +Gly 9 | mut_gly | rid1-knockout | glycine |
| SA094507 | 3480 +Gly 10 | mut_gly | rid1-knockout | glycine |
| SA094508 | 3480 +Gly 6 | mut_gly | rid1-knockout | glycine |
| SA094509 | media_3480 +Ile 10 | mut_ile | rid1-knockout | isoleucine |
| SA094510 | media_3480 +Ile 6 | mut_ile | rid1-knockout | isoleucine |
| SA094511 | 3480 +Ile 7 | mut_ile | rid1-knockout | isoleucine |
| SA094512 | media_3480 +Ile 5 | mut_ile | rid1-knockout | isoleucine |
| SA094513 | 3480 +Ile 1 | mut_ile | rid1-knockout | isoleucine |
| SA094514 | 3480 +Ile 4 | mut_ile | rid1-knockout | isoleucine |
| SA094515 | 3480 +Ile 6 | mut_ile | rid1-knockout | isoleucine |
| SA094516 | 3480 +Ile 5 | mut_ile | rid1-knockout | isoleucine |
| SA094517 | media_3480 +Ile 3 | mut_ile | rid1-knockout | isoleucine |
| SA094518 | media_3480 +Ile 9 | mut_ile | rid1-knockout | isoleucine |
| SA094519 | media_3480 +Ile 2 | mut_ile | rid1-knockout | isoleucine |
| SA094520 | media_3480 +Ile 1 | mut_ile | rid1-knockout | isoleucine |
| SA094521 | media_3480 +Ile 4 | mut_ile | rid1-knockout | isoleucine |
| SA094522 | 3480 +Ile 8 | mut_ile | rid1-knockout | isoleucine |
| SA094523 | media_3480 +Ile 7 | mut_ile | rid1-knockout | isoleucine |
| SA094524 | media_3480 +Ile 8 | mut_ile | rid1-knockout | isoleucine |
| SA094525 | 3480 +Ile 9 | mut_ile | rid1-knockout | isoleucine |
| SA094526 | 3480 +Ile 2 | mut_ile | rid1-knockout | isoleucine |
| SA094527 | 3480 +Ile 3 | mut_ile | rid1-knockout | isoleucine |
| SA094528 | 3480 +Ile 10 | mut_ile | rid1-knockout | isoleucine |
| SA094529 | media_3480 Min 8 | mut_min | rid1-knockout | minimal |
| SA094530 | 3480 Min 5 | mut_min | rid1-knockout | minimal |
| SA094531 | media_3480 Min 4 | mut_min | rid1-knockout | minimal |
| SA094532 | 3480 Min 1 | mut_min | rid1-knockout | minimal |
| SA094533 | media_3480 Min 3 | mut_min | rid1-knockout | minimal |
| SA094534 | media_3480 Min 1 | mut_min | rid1-knockout | minimal |
| SA094535 | media_3480 Min 9 | mut_min | rid1-knockout | minimal |
| SA094536 | media_3480 Min 6 | mut_min | rid1-knockout | minimal |
| SA094537 | 3480 Min 3 | mut_min | rid1-knockout | minimal |
| SA094538 | media_3480 Min 10 | mut_min | rid1-knockout | minimal |
| SA094539 | media_3480 Min 7 | mut_min | rid1-knockout | minimal |
| SA094540 | media_3480 Min 2 | mut_min | rid1-knockout | minimal |
| SA094541 | media_3480 Min 5 | mut_min | rid1-knockout | minimal |
| SA094542 | 3480 Min 4 | mut_min | rid1-knockout | minimal |
| SA094543 | 3480 Min 10 | mut_min | rid1-knockout | minimal |
| SA094544 | 3480 Min 8 | mut_min | rid1-knockout | minimal |
| SA094545 | 3480 Min 6 | mut_min | rid1-knockout | minimal |
| SA094546 | 3480 Min 2 | mut_min | rid1-knockout | minimal |
| SA094547 | 3480 Min 9 | mut_min | rid1-knockout | minimal |
| SA094548 | 3480 Min 7 | mut_min | rid1-knockout | minimal |
| SA094549 | 9404 +Gly 9 | wt_gly | wild-type | glycine |
| SA094550 | 9404 +Gly 1 | wt_gly | wild-type | glycine |
| SA094551 | media_9404 +Gly 4 | wt_gly | wild-type | glycine |
| SA094552 | media_9404 +Gly 3 | wt_gly | wild-type | glycine |
| SA094553 | media_9404 +Gly 1 | wt_gly | wild-type | glycine |
| SA094554 | 9404 +Gly 7 | wt_gly | wild-type | glycine |
| SA094555 | media_9404 +Gly 9 | wt_gly | wild-type | glycine |
| SA094556 | media_9404 +Gly 10 | wt_gly | wild-type | glycine |
| SA094557 | media_9404 +Gly 5 | wt_gly | wild-type | glycine |
| SA094558 | 9404 +Gly 3 | wt_gly | wild-type | glycine |
| SA094559 | 9404 +Gly 5 | wt_gly | wild-type | glycine |
| SA094560 | 9404 +Gly 2 | wt_gly | wild-type | glycine |
| SA094561 | media_9404 +Gly 7 | wt_gly | wild-type | glycine |
| SA094562 | media_9404 +Gly 2 | wt_gly | wild-type | glycine |
| SA094563 | 9404 +Gly 8 | wt_gly | wild-type | glycine |
| SA094564 | media_9404 +Gly 8 | wt_gly | wild-type | glycine |
| SA094565 | media_9404 +Gly 6 | wt_gly | wild-type | glycine |
| SA094566 | 9404 +Gly 6 | wt_gly | wild-type | glycine |
| SA094567 | 9404 +Gly 4 | wt_gly | wild-type | glycine |
| SA094568 | 9404 +Gly 10 | wt_gly | wild-type | glycine |
| SA094569 | media_9404 +Ile 6 | wt_ile | wild-type | isoleucine |
| SA094570 | 9404 +Ile 10 | wt_ile | wild-type | isoleucine |
| SA094571 | 9404 +Ile 5 | wt_ile | wild-type | isoleucine |
| SA094572 | media_9404 +Ile 1 | wt_ile | wild-type | isoleucine |
| SA094573 | media_9404 +Ile 5 | wt_ile | wild-type | isoleucine |
| SA094574 | 9404 +Ile 7 | wt_ile | wild-type | isoleucine |
| SA094575 | media_9404 +Ile 2 | wt_ile | wild-type | isoleucine |
| SA094576 | media_9404 +Ile 3 | wt_ile | wild-type | isoleucine |
| SA094577 | 9404 +Ile 3 | wt_ile | wild-type | isoleucine |
| SA094578 | media_9404 +Ile 9 | wt_ile | wild-type | isoleucine |
| SA094579 | 9404 +Ile 4 | wt_ile | wild-type | isoleucine |
| SA094580 | media_9404 +Ile 4 | wt_ile | wild-type | isoleucine |
| SA094581 | 9404 +Ile 6 | wt_ile | wild-type | isoleucine |
| SA094582 | media_9404 +Ile 7 | wt_ile | wild-type | isoleucine |
| SA094583 | 9404 +Ile 9 | wt_ile | wild-type | isoleucine |
| SA094584 | 9404 +Ile 2 | wt_ile | wild-type | isoleucine |
| SA094585 | media_9404 +Ile 10 | wt_ile | wild-type | isoleucine |
| SA094586 | 9404 +Ile 1 | wt_ile | wild-type | isoleucine |
| SA094587 | media_9404 +Ile 8 | wt_ile | wild-type | isoleucine |
| SA094588 | 9404 +Ile 8 | wt_ile | wild-type | isoleucine |
Collection:
| Collection ID: | CO001377 |
| Collection Summary: | Bacterial cells were grown in media. Each sample was centrifuged and the spent media was collected as well as the pelleted bacterial cells, both flash frozen in liquid nitrogen. |
| Collection Protocol Filename: | Cells_NMR_AJB_12_16_2018 |
| Sample Type: | Bacterial cells |
| Collection Method: | Centrifuge and freeze |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001397 |
| Treatment Summary: | Ten biologically independent wild-type and ridA mutant cultures were grown overnight in NB medium shaking at 37 °C and used to inoculate (1% inoculum) 250 mL non-baffled flasks holding 125 mL of medium. Each culture inoculated one each of the three media types (minimal medium, minimal medium with 1 mM L-isoleucine, and minimal medium with 1 mM glycine), for a total of 60 flasks. Flasks were randomly arranged in an Innova®44 incubator and cultures allowed to grow 16 h at 37 °C, shaking at 180 RPM. Cultures were chilled 5 min on ice and then harvested by centrifugation at 7,000 x G for 10 min at 4 °C. The supernatant was decanted, with 10 mL transferred to sterile 15 mL conical tubes and flash-frozen using liquid nitrogen for downstream analysis of external metabolites. Cell pellets were transferred to sterile 15 mL conical tubes after resuspension in 10 mL ddH2O, prior to a second pelleting at 7,000 x G for 10 min at 4 °C. The supernatant was decanted and pellets were flash-frozen using liquid nitrogen and stored at -80 °C. |
Sample Preparation:
| Sampleprep ID: | SP001390 |
| Sampleprep Summary: | Preparation of growth medium samples. Spent media from each bacterial culture was lyophilized (VirTis Benchtop K) for 48 h. Once dry, each lyophilized sample was reconstituted in 150 L of 100 mM sodium phosphate buffer (Cambridge Isotope Laboratories), pH 7.0, containing 1/3 mM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid, Cambridge Isotope Laboratories) as an internal standard. Each sample was centrifuged at 20,000 X G for 30 min and 50 L of supernatant was transferred by a Bruker SamplePro liquid handler into 1.7 mm SampleJet NMR tubes (Bruker Biospin). Metabolite extraction from bacterial pellets. Each frozen bacterial pellet was thawed on ice and 1 mL of ice cold 80/20 methanol/water together with approximately 200 mL of 0.7mm silica beads (BioSpec products). Homogenization was carried out using a FastPrep 96 (MPBIO). The samples and extraction blanks went through three cycles of homogenization at 1800 rpm for 300 s each. At the end of each cycle samples and controls were centrifuged at 20000 x G for 30 min. Each supernatant was transferred to a new tube and 1 mL of ice-cold methanol/water added to the original tubes before each new cycle. The combined supernatants from each cycle were pooled and concentrated overnight using a CentriVap Benchtop Vacuum Concentrator (Labconco) down to 0.1 mL. The samples were then diluted with 0.5 mL of methanol/water and transferred into 0.6 mL centrifuge tube and concentrated to dryness. The extracts were reconstituted in 150 uL of deuterated 100 mM sodium phosphate buffer containing 1/3 mM of the internal standard DSS (d6 4,4-dimethyl-4-silapentane-1-sulfonic acid) at pH 7.0 and vortex mixed for 5 min. Each sample was centrifuged at 20000 x G for 30 min and transferred by a Bruker SamplePro liquid handler into 1.7 mm SampleJet NMR tubes. Extraction blanks were prepared following the same procedure except the biological material was replaced with an equal volume of water. Solvent blanks consisted of the reconstituting NMR buffer (deuterated sodium phosphate buffer with DSS). |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Analysis:
| Analysis ID: | AN002177 |
| Laboratory Name: | Edison Lab - Complex Carbohydrate Research Center |
| Analysis Type: | NMR |
| Acquisition Date: | jan-4-2019 |
| Operator Name: | Goncalo Gouveia |
| Detector Type: | FT-NMR |
| Num Factors: | 6 |
| Num Metabolites: | 21 |
| Units: | Area under the curve |
NMR:
| NMR ID: | NM000160 |
| Analysis ID: | AN002177 |
| Instrument Name: | 600Mhz Avance III HD |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Spectrometer Frequency: | 600 |
| NMR Probe: | TCI - cryo-probe |
| NMR Solvent: | D2O |
| NMR Tube Size: | 1.7 mm |
| Shimming Method: | TopShim |
| Pulse Sequence: | PROJECT (periodic refocusing of J evolution by coherence transfer) |
| Water Suppression: | presat |
| Receiver Gain: | 203 |
| Number Of Scans: | 64 |
| Dummy Scans: | 16 |
| Acquisition Time: | 1.3107200 |
| Spectral Width: | 20.8 ppm |
| Num Data Points Acquired: | 32768 |
| Line Broadening: | 2 |
| Apodization: | Exponential |
| Baseline Correction Method: | Polynomial order 3 |
| Chemical Shift Ref Std: | 0.00 ppm |
