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.

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Study IDST001308
Study Title1H NMR metabolomics corroborates serine hydroxymethyltransferase as the primary target of 2-aminoacrylate in a ridA mutant of Salmonella enterica
Study TypeNMR metabolomics on Salmonella enterica
Study SummaryThe 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
DepartmentMicrobiology, Biochemistry, Complex Carbohydrate Research Center
LaboratoryEdison Lab and Downs lab
Last NameGouveia
First NameGoncalo
Address315 riverbend road, Complex Carbohydrate Research Centre, ATHENS, GA, 30605, USA
Emailgoncalog@uga.edu
Phone7063087500
Submit Date2020-01-22
Num Groups4
Total Subjects40
Raw Data AvailableYes
Raw Data File Type(s).par, pdata, info, .xml
Analysis Type DetailNMR
Release Date2020-03-03
Release Version1
Goncalo Gouveia Goncalo Gouveia
https://dx.doi.org/10.21228/M8S39G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
SA094489media_3480 +Gly 6mut_gly rid1-knockout glycine
SA094490media_3480 +Gly 10mut_gly rid1-knockout glycine
SA094491media_3480 +Gly 8mut_gly rid1-knockout glycine
SA0944923480 +Gly 1mut_gly rid1-knockout glycine
SA0944933480 +Gly 8mut_gly rid1-knockout glycine
SA094494media_3480 +Gly 3mut_gly rid1-knockout glycine
SA0944953480 +Gly 7mut_gly rid1-knockout glycine
SA0944963480 +Gly 9mut_gly rid1-knockout glycine
SA0944973480 +Gly 2mut_gly rid1-knockout glycine
SA0944983480 +Gly 5mut_gly rid1-knockout glycine
SA0944993480 +Gly 4mut_gly rid1-knockout glycine
SA094500media_3480 +Gly 5mut_gly rid1-knockout glycine
SA094501media_3480 +Gly 7mut_gly rid1-knockout glycine
SA094502media_3480 +Gly 2mut_gly rid1-knockout glycine
SA094503media_3480 +Gly 1mut_gly rid1-knockout glycine
SA0945043480 +Gly 3mut_gly rid1-knockout glycine
SA094505media_3480 +Gly 4mut_gly rid1-knockout glycine
SA094506media_3480 +Gly 9mut_gly rid1-knockout glycine
SA0945073480 +Gly 10mut_gly rid1-knockout glycine
SA0945083480 +Gly 6mut_gly rid1-knockout glycine
SA094509media_3480 +Ile 10mut_ile rid1-knockout isoleucine
SA094510media_3480 +Ile 6mut_ile rid1-knockout isoleucine
SA0945113480 +Ile 7mut_ile rid1-knockout isoleucine
SA094512media_3480 +Ile 5mut_ile rid1-knockout isoleucine
SA0945133480 +Ile 1mut_ile rid1-knockout isoleucine
SA0945143480 +Ile 4mut_ile rid1-knockout isoleucine
SA0945153480 +Ile 6mut_ile rid1-knockout isoleucine
SA0945163480 +Ile 5mut_ile rid1-knockout isoleucine
SA094517media_3480 +Ile 3mut_ile rid1-knockout isoleucine
SA094518media_3480 +Ile 9mut_ile rid1-knockout isoleucine
SA094519media_3480 +Ile 2mut_ile rid1-knockout isoleucine
SA094520media_3480 +Ile 1mut_ile rid1-knockout isoleucine
SA094521media_3480 +Ile 4mut_ile rid1-knockout isoleucine
SA0945223480 +Ile 8mut_ile rid1-knockout isoleucine
SA094523media_3480 +Ile 7mut_ile rid1-knockout isoleucine
SA094524media_3480 +Ile 8mut_ile rid1-knockout isoleucine
SA0945253480 +Ile 9mut_ile rid1-knockout isoleucine
SA0945263480 +Ile 2mut_ile rid1-knockout isoleucine
SA0945273480 +Ile 3mut_ile rid1-knockout isoleucine
SA0945283480 +Ile 10mut_ile rid1-knockout isoleucine
SA094529media_3480 Min 8mut_min rid1-knockout minimal
SA0945303480 Min 5mut_min rid1-knockout minimal
SA094531media_3480 Min 4mut_min rid1-knockout minimal
SA0945323480 Min 1mut_min rid1-knockout minimal
SA094533media_3480 Min 3mut_min rid1-knockout minimal
SA094534media_3480 Min 1mut_min rid1-knockout minimal
SA094535media_3480 Min 9mut_min rid1-knockout minimal
SA094536media_3480 Min 6mut_min rid1-knockout minimal
SA0945373480 Min 3mut_min rid1-knockout minimal
SA094538media_3480 Min 10mut_min rid1-knockout minimal
SA094539media_3480 Min 7mut_min rid1-knockout minimal
SA094540media_3480 Min 2mut_min rid1-knockout minimal
SA094541media_3480 Min 5mut_min rid1-knockout minimal
SA0945423480 Min 4mut_min rid1-knockout minimal
SA0945433480 Min 10mut_min rid1-knockout minimal
SA0945443480 Min 8mut_min rid1-knockout minimal
SA0945453480 Min 6mut_min rid1-knockout minimal
SA0945463480 Min 2mut_min rid1-knockout minimal
SA0945473480 Min 9mut_min rid1-knockout minimal
SA0945483480 Min 7mut_min rid1-knockout minimal
SA0945499404 +Gly 9wt_gly wild-type glycine
SA0945509404 +Gly 1wt_gly wild-type glycine
SA094551media_9404 +Gly 4wt_gly wild-type glycine
SA094552media_9404 +Gly 3wt_gly wild-type glycine
SA094553media_9404 +Gly 1wt_gly wild-type glycine
SA0945549404 +Gly 7wt_gly wild-type glycine
SA094555media_9404 +Gly 9wt_gly wild-type glycine
SA094556media_9404 +Gly 10wt_gly wild-type glycine
SA094557media_9404 +Gly 5wt_gly wild-type glycine
SA0945589404 +Gly 3wt_gly wild-type glycine
SA0945599404 +Gly 5wt_gly wild-type glycine
SA0945609404 +Gly 2wt_gly wild-type glycine
SA094561media_9404 +Gly 7wt_gly wild-type glycine
SA094562media_9404 +Gly 2wt_gly wild-type glycine
SA0945639404 +Gly 8wt_gly wild-type glycine
SA094564media_9404 +Gly 8wt_gly wild-type glycine
SA094565media_9404 +Gly 6wt_gly wild-type glycine
SA0945669404 +Gly 6wt_gly wild-type glycine
SA0945679404 +Gly 4wt_gly wild-type glycine
SA0945689404 +Gly 10wt_gly wild-type glycine
SA094569media_9404 +Ile 6wt_ile wild-type isoleucine
SA0945709404 +Ile 10wt_ile wild-type isoleucine
SA0945719404 +Ile 5wt_ile wild-type isoleucine
SA094572media_9404 +Ile 1wt_ile wild-type isoleucine
SA094573media_9404 +Ile 5wt_ile wild-type isoleucine
SA0945749404 +Ile 7wt_ile wild-type isoleucine
SA094575media_9404 +Ile 2wt_ile wild-type isoleucine
SA094576media_9404 +Ile 3wt_ile wild-type isoleucine
SA0945779404 +Ile 3wt_ile wild-type isoleucine
SA094578media_9404 +Ile 9wt_ile wild-type isoleucine
SA0945799404 +Ile 4wt_ile wild-type isoleucine
SA094580media_9404 +Ile 4wt_ile wild-type isoleucine
SA0945819404 +Ile 6wt_ile wild-type isoleucine
SA094582media_9404 +Ile 7wt_ile wild-type isoleucine
SA0945839404 +Ile 9wt_ile wild-type isoleucine
SA0945849404 +Ile 2wt_ile wild-type isoleucine
SA094585media_9404 +Ile 10wt_ile wild-type isoleucine
SA0945869404 +Ile 1wt_ile wild-type isoleucine
SA094587media_9404 +Ile 8wt_ile wild-type isoleucine
SA0945889404 +Ile 8wt_ile wild-type isoleucine
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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
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