Summary of Study ST000085

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 PR000075. The data can be accessed directly via it's Project DOI: 10.21228/M86K5H This work is supported by NIH grant, U2C- DK119886.

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Study IDST000085
Study TitleSalmonella Modulates Metabolism during Growth under Conditions that Induce Expression of Virulence Genes
Study Typegrowth conditions, timecourse
Study SummarySalmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative pathogen that uses complex mechanisms to invade and proliferate within mammalian host cells. To investigate possible contributions of metabolic processes to virulence in S. Typhimurium grown under conditions known to induce expression of virulence genes, we used a metabolomics-driven systems biology approach coupled with genome scale modeling. First, we identified distinct metabolite profiles associated with bacteria grown in either rich or virulence-inducing media and report the most comprehensive coverage of the S. Typhimurium metabolome to date. Second, we applied an omics-informed genome scale modeling analysis of the functional consequences of adaptive alterations in S. Typhimurium metabolism during growth under our conditions. Modeling efforts highlighted a decreased cellular capability to both produce and utilize intracellular amino acids during stationary phase culture in virulence conditions, despite significant abundance increases for these molecules as observed by our metabolomics measurements. Furthermore, analyses of omics data in the context of the metabolic model indicated rewiring of the metabolic network to support pathways associated with virulence. For example, cellular concentrations of polyamines were perturbed, as well as the predicted capacity for secretion and uptake.
Institute
Pacific Northwest National Laboratory
DepartmentBiological Separation and Mass Spectrometry
Last NameMetz
First NameThomas
Emailthomas.metz@pnnl.gov
Submit Date2014-06-25
Num Groups3
Total Subjects18
Raw Data AvailableYes
Raw Data File Type(s)cdf, d
Uploaded File Size245 M
Analysis Type DetailGC-MS
Release Date2014-08-08
Release Version1
Thomas Metz Thomas Metz
https://dx.doi.org/10.21228/M86K5H
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Treatment:

Treatment ID:TR000105
Treatment Summary:Luria-Bertani (LB) medium, hartvested after >2.5 hours | Low pH, low magnesium, low iron (LPM) medium, harvested after 20 h | Low pH, low magnesium, low iron (LPM) medium, harvested after 4 h | Low pH, low magnesium, low iron (LPM) medium, harvested after 20 h | Low pH, low magnesium, low iron (LPM) medium, harvested after 4 h
Treatment Protocol Comments:S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and were taken from a single colony on an agar plate, subsequently inoculated into 7 mL of Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7), and incubated overnight at 37°C. The overnight culture was centrifuged and the supernatant discarded. The cell pellets were suspended in LB media and centrifuged again, and the supernatant was discarded. For LB cultures, the cell pellets were subsequently suspended in 2 mL of LB media and used to inoculate 700 mL of LB in a 2.8 L flask. After 160 min of growth, cells were harvested via centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech) once cultures reached an OD600 of +1.0. / S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and were taken from a single colony on an agar plate, subsequently inoculated into 7 mL of Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7), and incubated overnight at 37°C. The overnight culture was centrifuged and the supernatant discarded. The cell pellets were suspended in LB media and centrifuged again, and the supernatant was discarded. To stimulate the Salmonella virulence program, cells were transferred to a low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8. Briefly, cell pellets from the 7 mL LB media were suspended in LPM media and centrifuged. The supernatant was discarded and cell pellets were suspended in 2 mL of LPM media and used to inoculate 700 mL of LPM in a 2.8 L flask. Cells were harvested after 20 h, then cells were harvested via centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech). Three biological replicates were performed for each of the conditions described above./ S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and were taken from a single colony on an agar plate, subsequently inoculated into 7 mL of Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7), and incubated overnight at 37°C. The overnight culture was centrifuged and the supernatant discarded. The cell pellets were suspended in LB media and centrifuged again, and the supernatant was discarded. To stimulate the Salmonella virulence program, cells were transferred to a low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8. Briefly, cell pellets from the 7 mL LB media were suspended in LPM media and centrifuged. The supernatant was discarded and cell pellets were suspended in 2 mL of LPM media and used to inoculate 700 mL of LPM in a 2.8 L flask. Cells were harvested after 4 h, then cells were harvested via centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech). Three biological replicates were performed for each of the conditions described above. || S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and were taken from a single colony on an agar plate, subsequently inoculated into 7 mL of Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7), and incubated overnight at 37°C. The overnight culture was centrifuged and the supernatant discarded. The cell pellets were suspended in LB media and centrifuged again, and the supernatant was discarded. To stimulate the Salmonella virulence program, cells were transferred to a low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8. Briefly, cell pellets from the 7 mL LB media were suspended in LPM media and centrifuged. The supernatant was discarded and cell pellets were suspended in 2 mL of LPM media and used to inoculate 700 mL of LPM in a 2.8 L flask. Cells were harvested after 20 h, then cells were harvested via centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech). Three biological replicates were performed for each of the conditions described above. || S. Typhimurium wild type cells (ATCC 14028) were used throughout this study and were taken from a single colony on an agar plate, subsequently inoculated into 7 mL of Luria-Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7), and incubated overnight at 37°C. The overnight culture was centrifuged and the supernatant discarded. The cell pellets were suspended in LB media and centrifuged again, and the supernatant was discarded. To stimulate the Salmonella virulence program, cells were transferred to a low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8. Briefly, cell pellets from the 7 mL LB media were suspended in LPM media and centrifuged. The supernatant was discarded and cell pellets were suspended in 2 mL of LPM media and used to inoculate 700 mL of LPM in a 2.8 L flask. Cells were harvested after 4 h, then cells were harvested via centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech). Three biological replicates were performed for each of the conditions described above.
Treatment:Abiotic
Treatment Route:Media
Cell Growth Container:700 mL of LB in a 2.8 L flask
Cell Media:Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% sodium chloride at pH 7) / low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8/ low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8 || low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8 || low pH, low magnesium, and low iron (LPM) medium comprised of 8 µM MgCL2, 0.5 µM ferric citrate, 5 mM KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v, 0.00001% thiamine, 337 µM H3PO4, and 80 mM 2-(N-morpholino)ethanesulfonic acid at pH 5.8
Cell Envir Cond:37° C
Cell Harvesting:centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech)
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