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MB Sample ID: SA210089

Local Sample ID:codY ccpA 3h_1
Subject ID:SU002273
Subject Type:Bacteria
Subject Species:Staphylococcus aureus
Taxonomy ID:1280
Gender:Not applicable
Species Group:Bacteria

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Subject:

Subject ID:SU002273
Subject Type:Bacteria
Subject Species:Staphylococcus aureus
Taxonomy ID:1280
Gender:Not applicable
Species Group:Bacteria

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
codY ccpA 3h_1SA210089FL025637codY ccpAGenotype

Collection:

Collection ID:CO002266
Collection Summary:Strains were inoculated in 3 ml of TSB containing 14 mM glucose in glass tubes (Fisher Scientific, cat# 14-961-29) for 16 h and adjusted to an OD600 of 0.05 in 50 ml TSB containing 14 mM glucose in 500 ml flasks and grown at 250 rpm, 37oC for 3 h or 6 h. Culture volumes corresponding to OD600 of 10 optical units were harvested and rapidly filtered through a membrane (0.45 μm, Millipore Cat# MIHAWG100) using Microfil® Filtration Funnels (Millipore, Cat# HAWG047S6) and EZ-FITTM Manifold Base, 6 Place Stainless Steel (Millipore, Cat# EZFITBASE6). The cells on the membrane were washed twice with 5 ml of 0.6% saline and then immediately quenched in 5 ml of ice-cold 60% ethanol containing Br-ATP (final concentration 2 μM) as internal control. The cells were mechanically disrupted using 0.1 mm diameter Glass beads (biospec, Cat# 11079101) and bead homogenizer for 30 s at 6,800 rpm, 3 cycles, 10 sec pause, 4oC(Precellys Evolution, bertin technologies) and centrifuged at 13,000 rpm, 10 min at 4oC to remove cell debris. The supernatant containing intracellular metabolites were separated, and 3 ml was added to 17 ml DW, frozen, lyophilized using FreeZone 4.5 Liter (labconco, Catalog #: 720401050), , and stored in a ultra-low temperature freezer set at -80°C until further analysis.
Sample Type:Bacterial cells
Storage Conditions:-20℃

Treatment:

Treatment ID:TR002285
Treatment Summary:Staphylococcus aureus UAMS-1 wild-type strain (WT) Staphylococcus aureus UAMS-1 codY::ermC mutant (codY) Staphylococcus aureus UAMS-1 ccpA::tetM mutant (ccpA) Staphylococcus aureus UAMS-1 codY::ermC ccpA::tetM mutant (codY ccpA)

Sample Preparation:

Sampleprep ID:SP002279
Sampleprep Summary:Strains were inoculated in 3 ml of TSB containing 14 mM glucose in glass tubes (Fisher Scientific, cat# 14-961-29) for 16 h and adjusted to an OD600 of 0.05 in 50 ml TSB containing 14 mM glucose in 500 ml flasks and grown at 250 rpm, 37oC for 3 h or 6 h. Culture volumes corresponding to OD600 of 10 optical units were harvested and rapidly filtered through a membrane (0.45 μm, Millipore Cat# MIHAWG100) using Microfil® Filtration Funnels (Millipore, Cat# HAWG047S6) and EZ-FITTM Manifold Base, 6 Place Stainless Steel (Millipore, Cat# EZFITBASE6). The cells on the membrane were washed twice with 5 ml of 0.6% saline and then immediately quenched in 5 ml of ice-cold 60% ethanol containing Br-ATP (final concentration 2 μM) as internal control. The cells were mechanically disrupted using 0.1 mm diameter Glass beads (biospec, Cat# 11079101) and bead homogenizer for 30 s at 6,800 rpm, 3 cycles, 10 sec pause, 4oC(Precellys Evolution, bertin technologies) and centrifuged at 13,000 rpm, 10 min at 4oC to remove cell debris. The supernatant containing intracellular metabolites were separated, and 3 ml was added to 17 ml DW, frozen, lyophilized using FreeZone 4.5 Liter (labconco, Catalog #: 720401050), , and stored in a ultra-low temperature freezer set at -80°C until further analysis.
Processing Storage Conditions:On ice
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003581
Chromatography ID CH002648
MS ID MS003338
Analysis type MS
Chromatography type HILIC
Chromatography system Waters Acquity I-Class
Column Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name ABI Sciex 6500+ QTrap
Ion Mode UNSPECIFIED
Units CPS

Chromatography:

Chromatography ID:CH002648
Chromatography Summary:Triple-quadrupole-ion trap hybrid mass spectrometer viz., QTRAP6500+ (Sciex, USA) connected with ultra-performance liquid chromatography I-class (UPLC) system procured from Waters, USA was used for the metabolite analysis. The chromatographic separation was performed by liquid chromatography using XBridge Amide (150 × 2.1mm ID; 3.5µm particle size, Waters, USA) analytical column and a binary solvent system with a flow rate of 0.4 ml/min. A guard XBridge Amide column (20 × 2.1mm ID; 3.5µm particle size, Waters, USA) was connected in front of analytical column. Mobile phase A was composed of 10 mM ammonium acetate, 10mM ammonium hydroxide containing 5% acetonitrile in LC-MS grade water; mobile phase B was 100% LC-MS grade acetonitrile.
Instrument Name:Waters Acquity I-Class
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Flow Rate:0.4 ml/min
Solvent A:5% acetonitrile/95% water; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS003338
Analysis ID:AN003581
Instrument Name:ABI Sciex 6500+ QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:QTRAP6500+ operated in polarity switching mode was used for targeted quantitation of amino acids through Multiple Reaction Monitoring (MRM) process. Electrospray ionization (ESI) parameters were optimized are as follows: electrospray ion voltage of -4200V and 5500V in negative and positive mode respectively, source temperature of 400°C, curtain gas of 35, and gas 1 and 2 of 40 and 40 psi, respectively. Compound specific parameters were optimized for each compound using manual tuning. These parameters are declustering potential (DP) were 65V and -60V in positive and negative mode respectively, entrance potential (EP) was set at 10V and -10V in positive and negative mode respectively and collision cell exit potential (CXP) was maintained at 10V and -10V in positive and negative mode respectively. MRM data was analysed using Multiquant software.
Ion Mode:UNSPECIFIED
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