#METABOLOMICS WORKBENCH petern_20160203_9687291_mwtab.txt DATATRACK_ID:499 amitch_20160212_075938 STUDY_ID:ST000351 ANALYSIS_ID:AN000567 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 Amino Acid method CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters AccQ Tag (100 x 2mm, 1.7um) #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 7 17 27 9 19 29 8 18 28 10 20 30 C1-1 C1-2 C1-3 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 Strain:UA159 wild type | Sample type:Supernatant | Growth condition:anaerobically Strain:UA159 wild type | Sample type:Supernatant | Growth condition:anaerobically Strain:UA159 wild type | Sample type:Supernatant | Growth condition:anaerobically Strain:UA159 wild type | Sample type:Supernatant | Growth condition:aerobically Strain:UA159 wild type | Sample type:Supernatant | Growth condition:aerobically Strain:UA159 wild type | Sample type:Supernatant | Growth condition:aerobically Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:anaerobically Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:anaerobically Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:anaerobically Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:aerobically Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:aerobically Strain:lrgAB mutant | Sample type:Supernatant | Growth condition:aerobically Strain:N/A | Sample type:Media only | Growth condition:N/A Strain:N/A | Sample type:Media only | Growth condition:N/A Strain:N/A | Sample type:Media only | Growth condition:N/A Alanine 63.59 65.52 93.61 67 55.42 28.73 77.92 63.05 57.37 71.62 53.2 66.56 2764.22 6189.19 6759.07 4967.78 3754.9 8305.34 4556.43 6223.76 6181.98 3618.12 5644.7 4033.19 7671.28 8075.1 6542.88 Arginine 26.36 26.46 27.29 20.14 18.66 9.5 21.87 26.99 21.35 15.2 9.17 19.41 1523.96 3004.7 3391.08 2216.57 1845.71 3166.86 2518.71 2958.1 2865.49 1871.51 2368.81 1729.62 3710.99 2906.95 2759.67 Asparagine 15.4 13.93 20.05 17.36 10.38 5.12 15.78 10.69 13.1 14.34 7.36 14.1 726 1777.44 2240.23 1281.14 1121.23 1839.29 1062.78 1973.34 1582.44 913.43 1442.58 944.73 1947.95 1834.33 1808.1 Aspartic Acid 34.22 35.39 44.66 21.38 13.14 5.79 25.85 24.29 36.05 37.39 29.36 20.96 1258.76 2450.64 2686.85 2179.03 1485.94 2851.08 1628.99 2666.43 2484.89 1384.81 2226.04 1459.77 3209.61 3288.16 2425.34 Citrulline BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ 51.71 101.81 139.77 71.73 67.34 104.26 71.09 92.57 97.72 57.11 84.98 58.87 133.35 108.45 100.35 Glutamic Acid 180.72 157.71 231.23 233.66 153.61 93.85 204.77 142.14 184.56 210.48 162.52 261.42 2861.44 5764.86 7120.55 4635.08 3153.79 6431.74 3642.75 6679.37 5712.8 3324.45 4687.93 2809.9 7160.8 6638.77 5465.76 Glutamine 40.1 42.95 47.97 49.95 44.63 22.99 40.06 35.84 30.17 75.24 44.69 38.46 217.06 1466.22 750.73 291.7 126.27 628.76 525.95 382.82 747.98 157.39 269.99 392.02 386.5 363.23 373.03 Glycine 12.66 11.63 15.02 13.9 10.36 4.01 11.34 11.12 13.76 20.06 10.73 16.78 1297.86 2418.86 2935.12 1906.37 1560.66 2638.31 1697.67 2307.89 2149.71 1315.08 1766.34 1253.34 2676.35 2565.68 2257.13 Histidine 5.7 4.39 5.25 4.09 3.64 2.11 5.33 4.11 4.03 5.42 2.6 4.73 338.47 677.18 762.62 501.45 468.96 781.05 489.65 820.38 656.9 386.83 621.33 432.91 813.9 844.46 655.36 Isoleucine 5.7 6.93 8.8 6.96 5.83 3.16 7.29 6.45 6.84 7.19 6.72 6.13 1649.92 3277.65 4071.57 2654.8 1915.18 3838.92 2394.5 3729.55 3158.03 1782.01 3134.96 1981.96 3926.18 3497.43 3395.17 Leucine 17.5 18.34 27.8 14.83 18.22 9.11 20.41 17.6 17.81 16.68 13.67 14.03 4356.19 10327.36 11004.05 7788.71 5151.96 9453.97 5596.2 10952.9 8930.55 4229.27 7599.4 5570.03 12301.86 11867.64 7939.2 Lysine 62.74 72.16 67.62 60.85 45.54 20.66 59.5 50.1 68.07 84.16 35.36 60 3704.87 7680.9 9779.02 5660.48 4100.94 8417.01 4749.27 8536.56 7465.01 3700.51 6192.09 3824.93 8487.13 8504.99 6594.41 Methionine 6.86 7.39 9.32 6.54 5.68 3.15 7.3 5.78 6.32 6.22 5.02 6.67 739.29 1342.75 1649.82 1175.52 876.78 1769.53 1035.38 1616.35 1323.12 844.27 1166.23 943.43 1570.22 1601.39 1467.07 Ornithine 3.63 3.29 3.48 2.93 2.47 2.34 3.7 3.06 2.93 2.27 1.75 2.41 205.4 547.61 597.52 356.7 282.87 560.72 311.13 478.41 478.81 259.59 388.45 250.75 493.91 466.96 431.37 Phenylalanine 8.77 9.86 10.9 7.92 9.03 4.72 10.77 8.87 10.63 7.76 7.38 8.49 1745.85 3810.04 4308 3452.09 2570.33 4837.92 2477.64 3853.38 3746.45 2479.44 3413.31 2531.55 4994.28 3984.23 3655.32 Proline 4.96 4.52 6.32 5.08 4.1 1.85 4.86 3.87 4.2 8.82 4.01 5.94 551.43 1196.26 1514.45 905.57 730.56 1305.38 949.38 1399.84 1223.53 680.18 1059.11 787.23 1579.17 1330.42 1238.24 Serine 13.01 13.43 17.67 20.6 11.26 5.1 12.74 11.12 12.09 20.07 11.78 14.97 1756.6 3601.82 3708.88 3329.27 1781.81 4203.32 2057.89 3239.32 3211.2 2053.08 2917.44 1834.56 3690.11 3756.73 3282.58 Threonine 26.58 24.48 32.53 21.21 17.39 9.31 28.34 21.26 20.54 22.44 16.67 22.74 1281.24 2640.8 3264.53 2173.82 1292.23 3335.46 2144.62 3196.7 2896.58 1649.73 2984.75 1565.96 3607.06 3421.15 3006.43 Tryptophan 3.76 3.34 4.28 3.25 3.43 2.09 3.95 3.79 3.47 3.91 2.65 3.74 388.38 734.16 939.59 640.16 499.24 884.18 521.14 929.64 845.2 469.27 638.16 554.19 1007.64 933.78 872.19 Tyrosine 2.91 2.55 3.78 3.22 3.1 1.43 3.1 2.87 2.84 4.13 2.36 3.35 454.84 912.05 961.42 744.47 508.63 1068.96 714.91 1035.46 857.09 577.04 924.57 539.37 1247.57 1251.13 885.5 Valine 12.41 12.35 14.89 9.73 7.91 3.44 12.4 10.57 11.03 11.07 6.6 9.9 2362.54 4182.18 5767.51 3623.66 2684.55 5239.14 2937.41 4948.94 3999.9 2292 4091.7 2965.81 5861.79 5207.83 4480.71 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Index Moverz Quant PubChem CID Alanine 4.16 260.17, 171 5950 Arginine 2.68 345.21, 170.9 6322 Asparagine 2.25 303.13, 170.9 6267 Aspartic Acid 2.99 304.11, 171 5960 Citrulline 3.33 346.21, 171 9750 Glutamic Acid 3.35 318.11, 171.1 33032 Glutamine 2.65 317.21, 171 5961 Glycine 2.81 246.08, 170.9 750 Histidine 2.07 326.21, 110 6274 Isoleucine 8.15 302.21, 170.9 6306 Leucine 8.26 302.21, 170.9 6106 Lysine 6.66 487.21, 170.9 5962 Methionine 6.87 320.21, 170.9 6137 Ornithine 5.93 473.22, 170.9 6262 Phenylalanine 8.4 336.21, 170.9 6140 Proline 4.84 286.16, 171 145742 Serine 2.6 276.11, 170.9 5951 Threonine 3.74 290.11, 170.9 6288 Tryptophan 8.54 375.21, 171 6305 Tyrosine 6.66 352.21, 170.9 6057 Valine 7.09 288.21, 171 6287 METABOLITES_END #END