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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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℃
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