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).ini,.INI, txt
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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Project:

Project ID:PR000075
Project DOI:doi: 10.21228/M86K5H
Project Title:Systems Biology for EnteroPathogens
Project Type:MS analysis
Project Summary:sysbep.org
Institute:Pacific Northwest National Laboratory
Department:Biological Separation and Mass Spectrometry
Last Name:Joshua
First Name:Adkins
Email:Joshua.Adkins@pnnl.gov

Subject:

Subject ID:SU000104
Subject Type:Bacterial cells
Subject Species:Salmonella typhimurium
Taxonomy ID:90371
Genotype Strain:CDC 6516-60
Cell Biosource Or Supplier:ATCC 14028
Species Group:Microorganism

Factors:

Subject type: Bacterial cells; Subject species: Salmonella typhimurium (Factor headings shown in green)

mb_sample_id local_sample_id Growth Conditions Time Harvested
SA004289SBEP_STM_LPM20_1Low pH, low magnesium, low iron (LPM) medium 20hour
SA004290SBEP_STM_LPM20_2Low pH, low magnesium, low iron (LPM) medium 20hour
SA004291SBEP_STM_LPM20_3Low pH, low magnesium, low iron (LPM) medium 20hour
SA004292SBEP_STM_LPM4_3Low pH, low magnesium, low iron (LPM) medium 4hour
SA004293SBEP_STM_LPM4_2Low pH, low magnesium, low iron (LPM) medium 4hour
SA004294SBEP_STM_LPM4_1Low pH, low magnesium, low iron (LPM) medium 4hour
SA004295SBEP_STM_LB_2Luria-Bertani (LB) medium >2.5hour
SA004296SBEP_STM_LB_3Luria-Bertani (LB) medium >2.5hour
SA004297SBEP_STM_LB_1Luria-Bertani (LB) medium >2.5hour
Showing results 1 to 9 of 9

Collection:

Collection ID:CO000087
Collection Summary:Cells were harvested via centrifugation (4,000×g) and washed twice with Dulbecco’s PBS (Mediatech)
Sample Type:Cell
Collection Method:Centrifugation (4,000 x g)
Collection Frequency:Once
Collection Time:variable, see Treatments
Volumeoramount Collected:All
Storage Conditions:-80° C

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)

Sample Preparation:

Sampleprep ID:SP000100
Sampleprep Summary:Cell pellets resuspended in ammonium bicarbonate, lysed via bead beating, extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v)
Sampleprep Protocol Comments:Cell pellets were stored at -80°C prior to thawing and were subsequently resuspended in an appropriate volume of 100 mM ammonium bicarbonate according to their wet weight to compensate for any differences in cell numbers. The cells were lysed by bead-beating, and the lysates were transferred into new tubes. Subsequently, 100 µL aliquots of cell lysates were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the aqueous phases after centrifugation were transferred to glass vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA). All samples were kept at -80°C prior to chemical derivatization for GC-MS analysis. Dried metabolite extracts were briefly dried again to remove any residual water prior to chemical derivatization. To protect carbonyl groups and reduce the number of tautomeric isomers, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by incubation at 37°C with generous shaking for 90 min. To derivatize hydroxyl and amine groups to trimethylsilyated (TMS) forms, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37°C with shaking for 30 min. The samples were allowed to cool to room temperature and were then analyzed by GC-MS in random order. For technical replicates, each of the derivatized samples was split into two different vials and analyzed separately.
Processing Method:Lysed via bead-beating
Extraction Method:four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the aqueous phases after centrifugation were transferred to glass vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA)
Extract Concentration Dilution:chloroform/methanol (2:1, v/v)
Extract Enrichment:dried in vacuo
Extract Storage:dried in vacuo, stored at -80° C
Sample Resuspension:20 µL of methoxyamine in pyridine (30 mg/mL)
Sample Derivatization:20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS)

Combined analysis:

Analysis ID AN000137
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890A
Column Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5975C
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH000097
Chromatography Summary:Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation
Chromatography Comments:Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min.
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Flow Rate:0.450.5 mL/min
Injection Temperature:250 C
Sample Injection:1 L, splitless
Analytical Time:37.5 min
Oven Temperature:60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C
Sample Syringe Size:10 L
Chromatography Type:GC

MS:

MS ID:MS000113
Analysis ID:AN000137
Instrument Name:Agilent 5975C
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis.
Ion Mode:POSITIVE
Scan Range Moverz:50-550 m/z
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