#METABOLOMICS WORKBENCH petern_20160203_9687291_mwtab.txt DATATRACK_ID:499 amitch_20160212_075938 STUDY_ID:ST000351 ANALYSIS_ID:AN000571 PROJECT_ID:PR000280 VERSION 1 CREATED_ON February 18, 2016, 3:29 pm #PROJECT PR:PROJECT_TITLE Application of a targeted metabolomic profiling for the delineation of PR:PROJECT_TITLE mechanisms of autolytic systems in Streptococcus mutans cells under oxidative PR:PROJECT_TITLE stresses PR:PROJECT_SUMMARY Streptococcus mutans is capable of withstanding a variety of stressors PR:PROJECT_SUMMARY encountered in the oral cavity. The ability to efficiently and rapidly adjust to PR:PROJECT_SUMMARY the changing environment is essential for its pathogenic lifestyle. Oxidative PR:PROJECT_SUMMARY stress is one of the most important environmental variables affecting the PR:PROJECT_SUMMARY pathogenic potential of S. mutans, as high oxygen concentrations disfavor growth PR:PROJECT_SUMMARY of S. mutans and other oral bacteria. In order to fulfill its role as a major PR:PROJECT_SUMMARY constituent of cariogenic oral biofilms and sustain virulence, S. mutans must PR:PROJECT_SUMMARY overcome oxidative stress. Oxidation sensing and response is a complex yet PR:PROJECT_SUMMARY highly-regulated process, particularly because it is cross-regulated with other PR:PROJECT_SUMMARY metabolic pathways, stress responses and virulence physiology. In addition, we PR:PROJECT_SUMMARY previously showed that exposure of S. mutans to oxygen strongly inhibits biofilm PR:PROJECT_SUMMARY formation and alters cell surface biogenesis, possibly through AtlA, a major PR:PROJECT_SUMMARY autolysin of the organism. More recently, we have also shown that S. mutans PR:PROJECT_SUMMARY Cid/Lrg system, potentially mediating cell lysis/death in a programmed fashion, PR:PROJECT_SUMMARY uniquely leads a global readjustment in central metabolism and virulence PR:PROJECT_SUMMARY processes, particularly in coping with oxidative stress. To this end, a targeted PR:PROJECT_SUMMARY metabolomics approach will be employed to understand how these autolytic systems PR:PROJECT_SUMMARY influence the metabolism of S. mutans, ultimately leading to a more complete PR:PROJECT_SUMMARY appreciation of how S. mutans survives and persists in hostile host PR:PROJECT_SUMMARY microenvironment. PR:INSTITUTE University of Florida PR:DEPARTMENT College of Dentistry PR:LAST_NAME Ahn PR:FIRST_NAME Sang-Joon PR:ADDRESS 1395 Center Drive, Room D5-29 Gainesville FL 32610 PR:EMAIL sahn@dental.ufl.edu PR:PHONE 352-273-8834 PR:FUNDING_SOURCE NIH R03 #STUDY ST:STUDY_TITLE Determining the metabolic profile of wildtype, lrgAB, and atlA mutant ST:STUDY_TITLE Steptococcus mutans grown aerobically and anaerobically ST:STUDY_TYPE Single time point, aerobic vs. anaerobic cultures ST:STUDY_SUMMARY Whole cells from both aerobic and anaerobic cultures of wild-type,lrgAB and atlA ST:STUDY_SUMMARY strain will be isolated for analysis of the whole cytosolic metabolome. ST:STUDY_SUMMARY Supernatants will be also analyzed for their metabolite profile in wild type and ST:STUDY_SUMMARY lrgAB cultures. ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LAST_NAME Ahn ST:FIRST_NAME Sang-Joon ST:ADDRESS 1395 Center Drive, Room D5-29 Gainesville FL 32610 ST:EMAIL sahn@dental.ufl.edu ST:PHONE 352-273-8834 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 36 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Streptococcus mutans #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Strain:UA159 wild type | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 2 Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 3 Strain:atlA mutant | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 4 Strain:UA159 wild type | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 5 Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 6 Strain:atlA mutant | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 7 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 8 Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 9 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 10 Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 11 Strain:UA159 wild type | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 12 Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 13 Strain:atlA mutant | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 14 Strain:UA159 wild type | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 15 Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 16 Strain:atlA mutant | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 17 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 18 Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 19 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 20 Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 21 Strain:UA159 wild type | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 22 Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 23 Strain:atlA mutant | Sample type:Whole cells | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 24 Strain:UA159 wild type | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 25 Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 26 Strain:atlA mutant | Sample type:Whole cells | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 27 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 28 Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:anaerobically SUBJECT_SAMPLE_FACTORS - 29 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - 30 Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:aerobically SUBJECT_SAMPLE_FACTORS - C1-1 Strain:N/A | Sample type:Media only | Growth condition:N/A SUBJECT_SAMPLE_FACTORS - C1-2 Strain:N/A | Sample type:Media only | Growth condition:N/A SUBJECT_SAMPLE_FACTORS - C1-3 Strain:N/A | Sample type:Media only | Growth condition:N/A #COLLECTION CO:COLLECTION_SUMMARY Bacterial cells were harvested at mid-exponential phase of growth by CO:COLLECTION_SUMMARY centrifugation. The cell pellets were washed with PBS buffer and then flash CO:COLLECTION_SUMMARY frozen in liquid nitrogen. The culture supernatants were filtered and then flash CO:COLLECTION_SUMMARY frozen in liquid nitrogen CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Bacterial cells were grown under aerobic and anaerobic conditions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY see attached protocols #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY CoA method CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2mm, 1.7um) CH:COLUMN_NAME 186002350 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg MS_METABOLITE_DATA_START Samples 1 11 21 4 14 24 2 12 22 5 15 25 Factors Strain:UA159 wild type | Sample type:Whole cells | Growth condition:anaerobically Strain:UA159 wild type | Sample type:Whole cells | Growth condition:anaerobically Strain:UA159 wild type | Sample type:Whole cells | Growth condition:anaerobically Strain:UA159 wild type | Sample type:Whole cells | Growth condition:aerobically Strain:UA159 wild type | Sample type:Whole cells | Growth condition:aerobically Strain:UA159 wild type | Sample type:Whole cells | Growth condition:aerobically Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:anaerobically Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:anaerobically Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:anaerobically Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:aerobically Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:aerobically Strain:lrgAB mutant | Sample type:Whole cells | Growth condition:aerobically Acetyl-CoA 537.44 757.39 929.62 654.6 1232.8 1087.18 569.64 791.56 474.72 797.34 895.11 665.55 Malonyl-CoA BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 6.64 6.07 BLOQ MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Moverz Quant PubChem CID Acetyl-CoA 1.37 810.1, 303.2 444493 Malonyl-CoA 1.16 854.1, 347.2 644066 METABOLITES_END #END