#METABOLOMICS WORKBENCH amitch_20150910_9594951_mwtab.txt DATATRACK_ID:374 STUDY_ID:ST000247 ANALYSIS_ID:AN000389 PROJECT_ID:PR000199 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Utilization of a global and targeted metabolomics approach to probe the effects PR:PROJECT_TITLE nitric oxide on physiology of the pathogen Staphylococcus aureus PR:PROJECT_TYPE Targeted LCMS Metabolomics PR:PROJECT_SUMMARY Staphylococcus aureus is a significant cause of morbidity and mortality in both PR:PROJECT_SUMMARY settings and the community at-large, and methicillin-resistant S. aureus (MRSA) PR:PROJECT_SUMMARY been recently categorized by the CDC as a significant antibiotic-resistant PR:PROJECT_SUMMARY As such, there exists an on-going critical need to study S. aureus genes that PR:PROJECT_SUMMARY growth and resistance to host immune responses, so that their encoded products PR:PROJECT_SUMMARY eventually be evaluated for potential as novel antimicrobial targets. In this PR:PROJECT_SUMMARY S. aureus nitric-oxide (NO) synthase (saNOS) and NO-reductase (saNOR) have been PR:PROJECT_SUMMARY shown to dramatically affect the physiology of this pathogen. Specifically, nos PR:PROJECT_SUMMARY have displayed decreased virulence in both subcutaneous abscess and sepsis PR:PROJECT_SUMMARY of infection, and our own research has suggested that NO produced via saNOS has PR:PROJECT_SUMMARY negative effect on endogenous reactive oxygen species (ROS) accumulation under PR:PROJECT_SUMMARY conditions promoting aerobic respiration and may regulate metabolism in an PR:PROJECT_SUMMARY unknown fashion. Our research has also demonstrated a role for saNOR in PR:PROJECT_SUMMARY cellular NO levels when exposed to exogenous NO donor and may contribute to PR:PROJECT_SUMMARY metabolism under conditions of nitrosative stress. Therefore, overall PR:PROJECT_SUMMARY of this proposal is that modulation of exogenous and endogenous NO levels (by PR:PROJECT_SUMMARY and saNOS, respectively) represent novel metabolic adaptations that contribute PR:PROJECT_SUMMARY S. aureus survival during infection. To this end, a global untargeted PR:PROJECT_SUMMARY approach will be employed to complete two research aims: Aim 1: Compare the PR:PROJECT_SUMMARY of saNOR on S. aureus metabolism when grown in the presence and absence of PR:PROJECT_SUMMARY NO. Aim 2: Identify the effects of endogenous NO produced by saNOS on S. aureus PR:PROJECT_SUMMARY when grown under conditions promoting aerobic respiration. These proposed PR:PROJECT_SUMMARY will help catalogue the exact metabolic changes that occur as a function of PR:PROJECT_SUMMARY and saNOR, which will help unravel the upstream regulatory circuits and PR:PROJECT_SUMMARY cellular targets of both of these enzymes. PR:INSTITUTE University of Florida PR:DEPARTMENT Microbiology and Cell Science PR:LABORATORY Kelly Rice PR:LAST_NAME Rice PR:FIRST_NAME Kelly PR:ADDRESS Room 1130, 1355 Museum Dr, UF campus, 32611-0700 PR:EMAIL kcrice@ufl.edu PR:PHONE 352-392-1192 PR:FUNDING_SOURCE Southeastern Center for Integrated Metabolomics (SECIM) pilot and feasibility PR:FUNDING_SOURCE NIH U24 DK097209 #STUDY ST:STUDY_TITLE Determining the metabolic profile of wildtype and nos mutant Staphylococcus ST:STUDY_TITLE grown in media lacking glucose using targeted LC/MS ST:STUDY_TYPE Single time point ST:STUDY_SUMMARY Whole cells from wildtype, nos mutant, SrrAB mutant, SrrAB nos double mutant, ST:STUDY_SUMMARY complement strains will be isolated for targeted metabolite analysis. In ST:STUDY_SUMMARY supernatants (extracellular metabolites) will also be analyzed for their ST:STUDY_SUMMARY profile. ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LAST_NAME Rice ST:FIRST_NAME Kelly ST:ADDRESS - ST:EMAIL kcrice@ufl.edu ST:PHONE - ST:NUM_GROUPS 7 ST:TOTAL_SUBJECTS 21 #SUBJECT SU:SUBJECT_TYPE Bacterial cells SU:SUBJECT_SPECIES Staphylococcus aureus SU:TAXONOMY_ID 1280 SU:SPECIES_GROUP Microorganism #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 13A Strain:N/A | Sample type:Media Only Sample Type=Fresh TSB No Dextrose Media; Genus_Or_Species=N/A; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 13B Strain:N/A | Sample type:Media Only Sample Type=Fresh TSB No Dextrose Media; Genus_Or_Species=N/A; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 13C Strain:N/A | Sample type:Media Only Sample Type=Fresh TSB No Dextrose Media; Genus_Or_Species=N/A; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 9A Strain:nos complement | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos complement; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 9B Strain:nos complement | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos complement; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 9C Strain:nos complement | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos complement; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 3A Strain:nos complement | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos complement; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 3B Strain:nos complement | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos complement; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 3C Strain:nos complement | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos complement; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 8A Strain:nos mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 8B Strain:nos mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 8C Strain:nos mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 2A Strain:nos mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 2B Strain:nos mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 2C Strain:nos mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 12A Strain:nos SrrAB mutant nos complement | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant nos complement; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 12B Strain:nos SrrAB mutant nos complement | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant nos complement; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 12C Strain:nos SrrAB mutant nos complement | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant nos complement; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 6A Strain:nos SrrAB mutant nos complement | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant nos complement; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 6B Strain:nos SrrAB mutant nos complement | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant nos complement; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 6C Strain:nos SrrAB mutant nos complement | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant nos complement; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 11A Strain:nos SrrAB mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 11B Strain:nos SrrAB mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 11C Strain:nos SrrAB mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 5A Strain:nos SrrAB mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 5B Strain:nos SrrAB mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 5C Strain:nos SrrAB mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus nos SrrAB double mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 10A Strain:SrrAB mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus SrrAB mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 10B Strain:SrrAB mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus SrrAB mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 10C Strain:SrrAB mutant | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus SrrAB mutant; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 4A Strain:SrrAB mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus SrrAB mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 4B Strain:SrrAB mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus SrrAB mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 4C Strain:SrrAB mutant | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus SrrAB mutant; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 7A Strain:Wildtype | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus Wildtype; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 7B Strain:Wildtype | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus Wildtype; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 7C Strain:Wildtype | Sample type:Supernatant Sample Type=Supernatant metabolites in TSB No Dextrose; Genus_Or_Species=Staphylococcus aureus Wildtype; Volume=1; Units=ml SUBJECT_SAMPLE_FACTORS - 1A Strain:Wildtype | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus Wildtype; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 1B Strain:Wildtype | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus Wildtype; Volume=N/A; Units=Cell Pellet SUBJECT_SAMPLE_FACTORS - 1C Strain:Wildtype | Sample type:Whole Cells Sample Type=Dry Cell Pellet; Genus_Or_Species=Staphylococcus aureus Wildtype; Volume=N/A; Units=Cell Pellet #COLLECTION CO:COLLECTION_SUMMARY The wildtype S. aureus, nos mutant, SrrAB mutant, nos SrrAB double mutant, and CO:COLLECTION_SUMMARY strains will be grown in 40 ml trytic soy broth lacking dextrose for 4 hours. CO:COLLECTION_SUMMARY 4 hours growth, all 40 ml was isolated, centrifuged at 4500 rpms and 4C for 10 CO:COLLECTION_SUMMARY and then placed on ice. 1 ml of supernatant was removed in duplicate and saved CO:COLLECTION_SUMMARY supernatant. The rest of the supernatant was discarded and pellets were CO:COLLECTION_SUMMARY in 2 ml PBS, centrifuged at 4500 rpms for 3 minutes (4C). Supernatant was CO:COLLECTION_SUMMARY and samples were again resuspended in 2 ml PBS. These samples were split into 2 CO:COLLECTION_SUMMARY 1 ml aliquots (for metabolite and protein determination) and frozen with liquid CO:COLLECTION_SUMMARY Separate media only samples of Tryptic Soy Broth without dextrose were isolated CO:COLLECTION_SUMMARY each day (3 total) Samples were stored at -80C. A total of 39 samples (18 whole CO:COLLECTION_SUMMARY 18 supernatant, and 3 media) were isolated. CO:COLLECTION_PROTOCOL_FILENAME Rice_Collections_targeted_20150807.txt #TREATMENT TR:TREATMENT_SUMMARY nos mutation, SrrAB mutation, and nos SrrAB double mutant mutations were TR:TREATMENT_SUMMARY as well as complementation before growing each strain under the same TR:TREATMENT_SUMMARY All strains were grown in Tryptic soy broth without dextrose. No additional TR:TREATMENT_SUMMARY and/or variables are included in the experiment. TR:TREATMENT_PROTOCOL_FILENAME Rice_Treatments_targeted_20150807.txt #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Amino acid|Organic acid|NAD|NADH SP:SAMPLEPREP_PROTOCOL_FILENAME SB_AA_Assay.pdf;SB_OA_Assay.pdf;SB_NAD_Assay.pdf;SB_NADH_Assay.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Amino acid|Organic acid|NAD|NADH CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME Thermo Quantiva QQQ CH:COLUMN_NAME Waters Acquity HSS T3 (100 x 2.1mm, 1.8um) CH:METHODS_FILENAME SB_AA_Assay.pdf;SB_OA_Assay.pdf;SB_NAD_Assay.pdf;SB_NADH_Assay.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Sanford Burnham AN:DETECTOR_TYPE Triple Quadrupole #MS MS:INSTRUMENT_NAME Thermo Quantiva QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS NADH(Instrument:Thermo Scientific Quantiva) MS:MS_COMMENTS ATP #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg protein MS_METABOLITE_DATA_START Samples 13A 13B 13C 9A 9B 9C 3A 3B 3C 8A 8B 8C 2A 2B 2C 12A 12B 12C 6A 6B 6C 11A 11B 11C 5A 5B 5C 10A 10B 10C 4A 4B 4C 7A 7B 7C 1A 1B 1C Factors Strain:N/A | Sample type:Media Only Strain:N/A | Sample type:Media Only Strain:N/A | Sample type:Media Only Strain:nos complement | Sample type:Supernatant Strain:nos complement | Sample type:Supernatant Strain:nos complement | Sample type:Supernatant Strain:nos complement | Sample type:Whole Cells Strain:nos complement | Sample type:Whole Cells Strain:nos complement | Sample type:Whole Cells Strain:nos mutant | Sample type:Supernatant Strain:nos mutant | Sample type:Supernatant Strain:nos mutant | Sample type:Supernatant Strain:nos mutant | Sample type:Whole Cells Strain:nos mutant | Sample type:Whole Cells Strain:nos mutant | Sample type:Whole Cells Strain:nos SrrAB mutant nos complement | Sample type:Supernatant Strain:nos SrrAB mutant nos complement | Sample type:Supernatant Strain:nos SrrAB mutant nos complement | Sample type:Supernatant Strain:nos SrrAB mutant nos complement | Sample type:Whole Cells Strain:nos SrrAB mutant nos complement | Sample type:Whole Cells Strain:nos SrrAB mutant nos complement | Sample type:Whole Cells Strain:nos SrrAB mutant | Sample type:Supernatant Strain:nos SrrAB mutant | Sample type:Supernatant Strain:nos SrrAB mutant | Sample type:Supernatant Strain:nos SrrAB mutant | Sample type:Whole Cells Strain:nos SrrAB mutant | Sample type:Whole Cells Strain:nos SrrAB mutant | Sample type:Whole Cells Strain:SrrAB mutant | Sample type:Supernatant Strain:SrrAB mutant | Sample type:Supernatant Strain:SrrAB mutant | Sample type:Supernatant Strain:SrrAB mutant | Sample type:Whole Cells Strain:SrrAB mutant | Sample type:Whole Cells Strain:SrrAB mutant | Sample type:Whole Cells Strain:Wildtype | Sample type:Supernatant Strain:Wildtype | Sample type:Supernatant Strain:Wildtype | Sample type:Supernatant Strain:Wildtype | Sample type:Whole Cells Strain:Wildtype | Sample type:Whole Cells Strain:Wildtype | Sample type:Whole Cells ADP 33.0630 44.3227 48.4712 22.8207 15.5647 20.2849 33.3916 42.2665 27.9766 31.0975 28.2016 31.0458 30.2342 15.3497 28.7965 26.0310 19.1471 23.3225 AMP 7.8345 37.4850 18.8616 5.4021 12.5162 4.3169 7.9553 16.2918 10.9659 10.8553 11.7168 10.9759 5.8020 9.5182 8.3126 7.5527 35.9440 9.4644 ATP 59.0992 37.4789 66.2267 37.9747 11.3462 33.3250 69.4979 69.8353 46.0852 39.8965 31.0911 42.5207 61.5765 15.3722 46.6690 44.6990 8.5451 38.9800 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type ADP 428.0, 136.1 1.51 UFLORIDA_MS_RI 6022 C00008 UFLORIDA_FEATURE AMP 348.1, 136.1 1.51 UFLORIDA_MS_RI 6083 C00020 UFLORIDA_FEATURE ATP 508.1, 136.1 1.50 UFLORIDA_MS_RI 5957 C00002 UFLORIDA_FEATURE METABOLITES_END #END