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MB Sample ID: SA004292
| Local Sample ID: | SBEP_STM_LPM4_3 |
| 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 |
Select appropriate tab below to view additional metadata details:
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:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| SBEP_STM_LPM4_3 | SA004292 | FL001226 | Low pH, low magnesium, low iron (LPM) medium | Growth Conditions |
| SBEP_STM_LPM4_3 | SA004292 | FL001226 | 4hour | Time Harvested |
Collection:
| Collection ID: | CO000087 |
| Collection Summary: | Cells were harvested via centrifugation (4,000×g) and washed twice with Dulbeccos 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 Dulbeccos 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 Dulbeccos 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 Dulbeccos 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 Dulbeccos 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 Dulbeccos 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 Dulbeccos 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 |
