Summary of Study ST000247

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 PR000199. The data can be accessed directly via it's Project DOI: 10.21228/M8P304 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000247
Study TitleDetermining the metabolic profile of wildtype and nos mutant Staphylococcus aureus grown in media lacking glucose using targeted LC/MS
Study TypeSingle time point
Study SummaryWhole cells from wildtype, nos mutant, SrrAB mutant, SrrAB nos double mutant, and complement strains will be isolated for targeted metabolite analysis. In parallel, supernatants (extracellular metabolites) will also be analyzed for their metabolic profile.
Institute
University of Florida
DepartmentSECIM
Last NameRice
First NameKelly
Emailkcrice@ufl.edu
Submit Date2015-02-17
Num Groups7
Total Subjects21
Raw Data File Type(s)raw(Thermo), d
Analysis Type DetailLC-MS
Release Date2019-01-22
Release Version1
Kelly Rice Kelly Rice
https://dx.doi.org/10.21228/M8P304
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000199
Project DOI:doi: 10.21228/M8P304
Project Title:Utilization of a global and targeted metabolomics approach to probe the effects of nitric oxide on physiology of the pathogen Staphylococcus aureus
Project Type:Targeted LCMS Metabolomics
Project Summary:Staphylococcus aureus is a significant cause of morbidity and mortality in both hospital settings and the community at-large, and methicillin-resistant S. aureus (MRSA) has been recently categorized by the CDC as a significant antibiotic-resistant threat. As such, there exists an on-going critical need to study S. aureus genes that promote growth and resistance to host immune responses, so that their encoded products can eventually be evaluated for potential as novel antimicrobial targets. In this respect, S. aureus nitric-oxide (NO) synthase (saNOS) and NO-reductase (saNOR) have been recently shown to dramatically affect the physiology of this pathogen. Specifically, nos mutants have displayed decreased virulence in both subcutaneous abscess and sepsis models of infection, and our own research has suggested that NO produced via saNOS has a negative effect on endogenous reactive oxygen species (ROS) accumulation under growth conditions promoting aerobic respiration and may regulate metabolism in an as-yet unknown fashion. Our research has also demonstrated a role for saNOR in modulating cellular NO levels when exposed to exogenous NO donor and may contribute to cellular metabolism under conditions of nitrosative stress. Therefore, overall hypothesis of this proposal is that modulation of exogenous and endogenous NO levels (by saNOR and saNOS, respectively) represent novel metabolic adaptations that contribute to S. aureus survival during infection. To this end, a global untargeted metabolomics approach will be employed to complete two research aims: Aim 1: Compare the effect of saNOR on S. aureus metabolism when grown in the presence and absence of exogenous NO. Aim 2: Identify the effects of endogenous NO produced by saNOS on S. aureus metabolism when grown under conditions promoting aerobic respiration. These proposed studies will help catalogue the exact metabolic changes that occur as a function of saNOS and saNOR, which will help unravel the upstream regulatory circuits and downstream cellular targets of both of these enzymes.
Institute:University of Florida
Department:Microbiology and Cell Science
Laboratory:Kelly Rice
Last Name:Rice
First Name:Kelly
Address:Room 1130, 1355 Museum Dr, UF campus, 32611-0700
Email:kcrice@ufl.edu
Phone:352-392-1192
Funding Source:Southeastern Center for Integrated Metabolomics (SECIM) pilot and feasibility funding, NIH U24 DK097209

Subject:

Subject ID:SU000267
Subject Type:Bacterial cells
Subject Species:Staphylococcus aureus
Taxonomy ID:1280
Species Group:Microorganism

Factors:

Subject type: Bacterial cells; Subject species: Staphylococcus aureus (Factor headings shown in green)

mb_sample_id local_sample_id Strain Sample type
SA01133913BN/A Media Only
SA01134013AN/A Media Only
SA01134113CN/A Media Only
SA0113669Anos complement Supernatant
SA0113679Cnos complement Supernatant
SA0113689Bnos complement Supernatant
SA0113693Anos complement Whole Cells
SA0113703Cnos complement Whole Cells
SA0113713Bnos complement Whole Cells
SA0113728Cnos mutant Supernatant
SA0113738Bnos mutant Supernatant
SA0113748Anos mutant Supernatant
SA0113752Anos mutant Whole Cells
SA0113762Cnos mutant Whole Cells
SA0113772Bnos mutant Whole Cells
SA01135412Anos SrrAB mutant nos complement Supernatant
SA01135512Cnos SrrAB mutant nos complement Supernatant
SA01135612Bnos SrrAB mutant nos complement Supernatant
SA0113576Cnos SrrAB mutant nos complement Whole Cells
SA0113586Anos SrrAB mutant nos complement Whole Cells
SA0113596Bnos SrrAB mutant nos complement Whole Cells
SA01136011Bnos SrrAB mutant Supernatant
SA01136111Cnos SrrAB mutant Supernatant
SA01136211Anos SrrAB mutant Supernatant
SA0113635Bnos SrrAB mutant Whole Cells
SA0113645Cnos SrrAB mutant Whole Cells
SA0113655Anos SrrAB mutant Whole Cells
SA01134210CSrrAB mutant Supernatant
SA01134310ASrrAB mutant Supernatant
SA01134410BSrrAB mutant Supernatant
SA0113454BSrrAB mutant Whole Cells
SA0113464CSrrAB mutant Whole Cells
SA0113474ASrrAB mutant Whole Cells
SA0113487CWildtype Supernatant
SA0113497BWildtype Supernatant
SA0113507AWildtype Supernatant
SA0113511CWildtype Whole Cells
SA0113521BWildtype Whole Cells
SA0113531AWildtype Whole Cells
Showing results 1 to 39 of 39

Collection:

Collection ID:CO000258
Collection Summary:The wildtype S. aureus, nos mutant, SrrAB mutant, nos SrrAB double mutant, and complement strains will be grown in 40 ml trytic soy broth lacking dextrose for 4 hours. At 4 hours growth, all 40 ml was isolated, centrifuged at 4500 rpms and 4C for 10 minutes and then placed on ice. 1 ml of supernatant was removed in duplicate and saved as supernatant. The rest of the supernatant was discarded and pellets were resuspended in 2 ml PBS, centrifuged at 4500 rpms for 3 minutes (4C). Supernatant was discarded and samples were again resuspended in 2 ml PBS. These samples were split into 2 separate 1 ml aliquots (for metabolite and protein determination) and frozen with liquid nitrogen. Separate media only samples of Tryptic Soy Broth without dextrose were isolated for each day (3 total) Samples were stored at -80C. A total of 39 samples (18 whole cell, 18 supernatant, and 3 media) were isolated.
Collection Protocol Filename:Rice_Collections_targeted_20150807.txt
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR000278
Treatment Summary:nos mutation, SrrAB mutation, and nos SrrAB double mutant mutations were completed; as well as complementation before growing each strain under the same conditions. All strains were grown in Tryptic soy broth without dextrose. No additional treatment and/or variables are included in the experiment.
Treatment Protocol Filename:Rice_Treatments_targeted_20150807.txt

Sample Preparation:

Sampleprep ID:SP000272
Sampleprep Summary:Amino acid|Organic acid|NAD|NADH
Sampleprep Protocol Filename:SB_AA_Assay.pdf
SB_OA_Assay.pdf
SB_NAD_Assay.pdf
SB_NADH_Assay.pdf

Combined analysis:

Analysis ID AN000387 AN000388 AN000389 AN000390
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000 Agilent 1290 Infinity
Column Waters Acquity BEH C18 (100 x 2mm,1.7um) Thermo Hypercarb (50 x 2.1mm,3um) Waters Acquity HSS T3 (100 x 2.1mm,1.8um) Waters AccQ Tag (100 x 2mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name Thermo Quantiva QQQ Thermo Quantiva QQQ Thermo Quantiva QQQ Agilent 6490 QQQ
Ion Mode POSITIVE POSITIVE POSITIVE POSITIVE
Units nmol/mg protein nmol/mg protein nmol/mg protein nmol/mg protein

Chromatography:

Chromatography ID:CH001331
Methods Filename:SB_OA_Assay.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:45
Flow Rate:0.3 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH001332
Methods Filename:SB_NAD_Assay.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Thermo Hypercarb (50 x 2.1mm,3um)
Column Temperature:30
Flow Rate:0.65 mL/min
Solvent A:100% water; 10 mM ammonium acetate, pH 9.5
Solvent B:100% acetonitrile
Chromatography Type:Reversed phase
  
Chromatography ID:CH001333
Methods Filename:SB_NADH_Assay.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters Acquity HSS T3 (100 x 2.1mm,1.8um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001334
Methods Filename:SB_AA_Assay.pdf
Instrument Name:Agilent 1290 Infinity
Column Name:Waters AccQ Tag (100 x 2mm,1.7um)
Column Temperature:55
Flow Rate:0.7 mL/min
Solvent A:AccQ·Tag Eluent A
Solvent B:accQ·Tag Eluent B
Chromatography Type:Reversed phase

MS:

MS ID:MS000331
Analysis ID:AN000387
Instrument Name:Thermo Quantiva QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Organic Acids (Instrument:Thermo Scientific Quantiva)
Ion Mode:POSITIVE
  
MS ID:MS000332
Analysis ID:AN000388
Instrument Name:Thermo Quantiva QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:NAD(Instrument:Thermo Scientific Quantiva)
Ion Mode:POSITIVE
  
MS ID:MS000333
Analysis ID:AN000389
Instrument Name:Thermo Quantiva QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:NADH(Instrument:Thermo Scientific Quantiva)
Ion Mode:POSITIVE
  
MS ID:MS000334
Analysis ID:AN000390
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Amino Acids(Instrument:Agilent 6490)
Ion Mode:POSITIVE
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