#METABOLOMICS WORKBENCH engelmab_20210414_233756 DATATRACK_ID:2576 STUDY_ID:ST001752 ANALYSIS_ID:AN002855 PROJECT_ID:PR001123 VERSION 1 CREATED_ON April 23, 2021, 5:56 pm #PROJECT PR:PROJECT_SUMMARY Bacterial small RNAs (sRNAs) are well-known to modulate gene expression by PR:PROJECT_SUMMARY base-pairing with trans-coded transcripts and are typically considered to be PR:PROJECT_SUMMARY non-coding. However, several sRNAs have been reported to also contain an open PR:PROJECT_SUMMARY reading frame and thus are considered dual-function regulators. We discovered a PR:PROJECT_SUMMARY dual-function regulator from Vibrio cholerae, called VcdRP, harboring a 29 amino PR:PROJECT_SUMMARY acid protein (VcdP), as well as a base-pairing sequence. In this study, we PR:PROJECT_SUMMARY measured the metabolite abundance of glycolytic and citric acid cycle PR:PROJECT_SUMMARY intermediates using LC-MS. PR:INSTITUTE Helmholtz Centre for Environmental Research PR:DEPARTMENT Molecular Systems Biology PR:LABORATORY Functional Metabolomics PR:FIRST_NAME Beatrice PR:ADDRESS Permoserstraße 15, Leipzipg, Saxony, 03418, Germany PR:EMAIL beatrice.engelmann@ufz.de PR:PHONE 00493412351099 PR:PROJECT_TITLE Dual RNA regulator VcdRP in V.cholera modulates central metabolism PR:LAST_NAME Engelmann #STUDY ST:STUDY_TITLE Dual RNA regulator VcdRP in V. cholerae modulates central metabolism ST:STUDY_SUMMARY Bacterial small RNAs (sRNAs) are well-known to modulate gene expression by ST:STUDY_SUMMARY base-pairing with trans-coded transcripts and are typically considered to be ST:STUDY_SUMMARY non-coding. However, several sRNAs have been reported to also contain an open ST:STUDY_SUMMARY reading frame and thus are considered dual-function regulators. We discovered a ST:STUDY_SUMMARY dual-function regulator from Vibrio cholerae, called VcdRP, harboring a 29 amino ST:STUDY_SUMMARY acid protein (VcdP), as well as a base-pairing sequence. In this study, we ST:STUDY_SUMMARY measured the metabolite abundance of glycolytic and citric acid cycle ST:STUDY_SUMMARY intermediates using LC-MS. ST:INSTITUTE Helmholtz Centre for Environmental Research - UFZ ST:DEPARTMENT Molecular Systems Biology ST:LABORATORY Functional Metabolomics ST:LAST_NAME Engelmann ST:FIRST_NAME Beatrice ST:ADDRESS Permoserstraße 15, Leipzig, Saxony, 03418, Germany ST:EMAIL beatrice.engelmann@ufz.de ST:PHONE 00493412351099 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Vibrio Cholerae SU:GENOTYPE_STRAIN V. cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961- SU:GENOTYPE_STRAIN access:2018.06.26) #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 1P Condition:Overnight | Strain:WT_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 2P Condition:Overnight | Strain:delta_VcdR_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 3P Condition:Overnight | Strain:delta_VcdR_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 4P Condition:Overnight | Strain:delta_VcdR_pVcdR RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 5P Condition:Overnight | Strain:delta_GltA_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 6P Condition:Overnight | Strain:delta_GltA_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 7P Condition:Overnight | Strain:WT_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 8P Condition:Overnight | Strain:delta_VcdR_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 9P Condition:Overnight | Strain:delta_VcdR_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 10P Condition:Overnight | Strain:delta_VcdR_pVcdR RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 11P Condition:Overnight | Strain:delta_GltA_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 12P Condition:Overnight | Strain:delta_GltA_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 13P Condition:Overnight | Strain:WT_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 14P Condition:Overnight | Strain:delta_VcdR_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 15P Condition:Overnight | Strain:delta_VcdR_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 16P Condition:Overnight | Strain:delta_VcdR_pVcdR RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 17P Condition:Overnight | Strain:delta_GltA_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 18P Condition:Overnight | Strain:delta_GltA_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 19P Condition:OD0.5 | Strain:WT_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 20P Condition:OD0.5 | Strain:delta_VcdR_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 21P Condition:OD0.5 | Strain:delta_VcdR_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 22P Condition:OD0.5 | Strain:delta_VcdR_pVcdR RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 23P Condition:OD0.5 | Strain:delta_GltA_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 24P Condition:OD0.5 | Strain:delta_GltA_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 25P Condition:OD0.5 | Strain:WT_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 26P Condition:OD0.5 | Strain:delta_VcdR_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 27P Condition:OD0.5 | Strain:delta_VcdR_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 28P Condition:OD0.5 | Strain:delta_VcdR_pVcdR RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 29P Condition:OD0.5 | Strain:delta_GltA_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 30P Condition:OD0.5 | Strain:delta_GltA_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 31P Condition:OD0.5 | Strain:WT_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 32P Condition:OD0.5 | Strain:delta_VcdR_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 33P Condition:OD0.5 | Strain:delta_VcdR_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 34P Condition:OD0.5 | Strain:delta_VcdR_pVcdR RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 35P Condition:OD0.5 | Strain:delta_GltA_pCtrl RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff SUBJECT_SAMPLE_FACTORS - 36P Condition:OD0.5 | Strain:delta_GltA_pVcdP RAW_FILE_NAME=VibrioCholerae_CentralCarbon.wiff #COLLECTION CO:COLLECTION_SUMMARY V. cholerae wild-type, ΔvcdRP and ΔgltA each harboring an empty control CO:COLLECTION_SUMMARY plasmid (pCtrl) and ΔvcdRP with pVcdRP expression plasmids (pVcdRP and pVcdP) CO:COLLECTION_SUMMARY as well as ΔgltA harboring pVcdP were grown in LB medium to exponential and CO:COLLECTION_SUMMARY stationary phase, and were subsequently quenched in ice-cold methanol. The CO:COLLECTION_SUMMARY quenched samples were centrifuged at 3000 x g for 10 min at -9°C. 1ml aliquot CO:COLLECTION_SUMMARY of supernatant was stored for leakage analysis, while the rest of the pellets CO:COLLECTION_SUMMARY were flash frozen in liquid nitrogen. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The frozen cell pellets were thawed on ice and resuspended in 500 µl methanol. SP:SAMPLEPREP_SUMMARY After three freeze (liquid nitrogen) and thaw cycles, the sample was centrifuged SP:SAMPLEPREP_SUMMARY (16000 x g, -4 °C and 5 min). The supernatant was transferred into a new tube SP:SAMPLEPREP_SUMMARY and stored on ice. The extraction step was repeated and the supernatants were SP:SAMPLEPREP_SUMMARY combined prior to evaporation. The samples were stored at -80°C until further SP:SAMPLEPREP_SUMMARY analysis. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Ion pair chromatography CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters XSelect HSS T3 XP (2.1 x 1500mm, 2.5 µm) CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 10 mM tributylamine, 10 mM acetic acid, 5% methanol and 2% 2-propanol (pH 7.1) CH:SOLVENT_A in water CH:SOLVENT_B 100% 2-propanol #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME AB Sciex 6500+ QTRAP MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS acquisition as well as data processing software was Analyst vs 1.7.0 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative abundance MS_METABOLITE_DATA_START Samples 1P 2P 3P 4P 5P 6P 7P 8P 9P 10P 11P 12P 13P 14P 15P 16P 17P 18P 19P 20P 21P 22P 23P 24P 25P 26P 27P 28P 29P 30P 31P 32P 33P 34P 35P 36P Factors Condition:Overnight | Strain:WT_pCtrl Condition:Overnight | Strain:delta_VcdR_pCtrl Condition:Overnight | Strain:delta_VcdR_pVcdP Condition:Overnight | Strain:delta_VcdR_pVcdR Condition:Overnight | Strain:delta_GltA_pCtrl Condition:Overnight | Strain:delta_GltA_pVcdP Condition:Overnight | Strain:WT_pCtrl Condition:Overnight | Strain:delta_VcdR_pCtrl Condition:Overnight | Strain:delta_VcdR_pVcdP Condition:Overnight | Strain:delta_VcdR_pVcdR Condition:Overnight | Strain:delta_GltA_pCtrl Condition:Overnight | Strain:delta_GltA_pVcdP Condition:Overnight | Strain:WT_pCtrl Condition:Overnight | Strain:delta_VcdR_pCtrl Condition:Overnight | Strain:delta_VcdR_pVcdP Condition:Overnight | Strain:delta_VcdR_pVcdR Condition:Overnight | Strain:delta_GltA_pCtrl Condition:Overnight | Strain:delta_GltA_pVcdP Condition:OD0.5 | Strain:WT_pCtrl Condition:OD0.5 | Strain:delta_VcdR_pCtrl Condition:OD0.5 | Strain:delta_VcdR_pVcdP Condition:OD0.5 | Strain:delta_VcdR_pVcdR Condition:OD0.5 | Strain:delta_GltA_pCtrl Condition:OD0.5 | Strain:delta_GltA_pVcdP Condition:OD0.5 | Strain:WT_pCtrl Condition:OD0.5 | Strain:delta_VcdR_pCtrl Condition:OD0.5 | Strain:delta_VcdR_pVcdP Condition:OD0.5 | Strain:delta_VcdR_pVcdR Condition:OD0.5 | Strain:delta_GltA_pCtrl Condition:OD0.5 | Strain:delta_GltA_pVcdP Condition:OD0.5 | Strain:WT_pCtrl Condition:OD0.5 | Strain:delta_VcdR_pCtrl Condition:OD0.5 | Strain:delta_VcdR_pVcdP Condition:OD0.5 | Strain:delta_VcdR_pVcdR Condition:OD0.5 | Strain:delta_GltA_pCtrl Condition:OD0.5 | Strain:delta_GltA_pVcdP acetyl coenzyme A 364000 721000 3640000 1900000 13700000 22300000 292000 1310000 5420000 1340000 19800000 21600000 326000 NA 4730000 2990000 14600000 25200000 894000 318000 12600000 1020000 4500000 3760000 66600 6310000 1380000 5510000 538000 11000000 546000 11900000 NA 3570000 2490000 425000 pyruvic acid 444000 713000 334000 344000 235000 195000 341000 516000 967000 490000 361000 329000 727000 667000 696000 624000 560000 634000 1660000 1240000 1050000 68700 1620000 434000 258000 2890000 622000 126000 1160000 932000 1030000 2860000 866000 142000 7582000 89800 citric acid 13600000 15500000 15600000 13200000 8690000 7530000 14700000 19500000 24500000 16700000 10700000 8070000 16300000 34000000 24200000 20900000 5600000 11400000 355000000 220000000 487000000 256000000 254000000 268000000 26800000 283000000 289000000 283000000 329000000 266000000 236000000 283000000 559000000 274000000 700000000 254000000 succinic acid NA 2620000 3350000 2530000 290000 242000 2070000 3180000 2160000 3280000 956000 916000 5620000 5260000 6480000 10200000 947000 1500000 13000000 7080000 14500000 9960000 8850000 9500000 881000 11000000 8750000 12100000 6910000 9680000 9650000 10400000 15400000 5750000 21900000 7860000 glutamic acid 168000000 75900000 236000000 56800000 38500000 32200000 44000000 45600000 298000000 66300000 34300000 28000000 78900000 57700000 256000000 61500000 43300000 34700000 292000000 202000000 318000000 246000000 222000000 250000000 43300000 244000000 227000000 274000000 283000000 235000000 211000000 237000000 305000000 219000000 431000000 222000000 cis-aconitic acid NA 27300000 7250000 19200000 6750000 5120000 24600000 20400000 15800000 26000000 3370000 1020000 29000000 23900000 4720000 16500000 4190000 2000000 14600000 8000000 19300000 12000000 12400000 11400000 1300000 17600000 11500000 16500000 16200000 13800000 9940000 14900000 26800000 16400000 26500000 9680000 glucose 6-phosphate 696000 1470000 1360000 1850000 688000 721000 669000 1910000 2500000 2390000 964000 1070000 1620000 1740000 1700000 1960000 1240000 1810000 613000 324000 568000 1190000 348000 292000 357000 329000 517000 2260000 543000 306000 300000 579000 525000 1490000 828000 282000 fructose 6-phosphate NA 4200000 3310000 6070000 2240000 2440000 3500000 4260000 6490000 6380000 2690000 2260000 4920000 4140000 4960000 7180000 2710000 4390000 6410000 2640000 5470000 3260000 3250000 3590000 608000 3550000 4270000 3460000 4790000 3040000 2930000 3160000 4070000 3230000 5930000 3380000 phosphoenolpyruvic acid NA 7860000 4750000 5650000 4530000 4870000 9480000 7420000 11900000 11800000 11200000 9430000 14400000 11900000 11200000 10900000 9380000 18300000 163000 93600 289000 312000 121000 165000 23600 435000 108000 432000 185000 369000 108000 456000 203000 750000 251000 318000 lactic acid 2890000 1350000 684000 800000 1170000 1430000 1290000 876000 1180000 1150000 885000 1690000 2800000 4120000 3550000 3740000 1340000 985000 48000000 37300000 50400000 38000000 39300000 41500000 8720000 40400000 39400000 39300000 44300000 41300000 37100000 40300000 47000000 40500000 62100000 36600000 aspartic acid 189000000 105000000 49200000 60800000 68300000 61900000 64300000 75100000 115000000 92500000 68200000 49800000 120000000 82700000 67900000 60100000 97600000 76400000 130000000 78700000 146000000 29500000 92100000 104000000 12400000 87600000 98800000 18700000 135000000 104000000 85500000 92000000 151000000 17000000 195000000 49600000 asparagine NA 1320000 972000 1180000 1300000 1090000 1220000 1020000 1410000 1240000 1410000 1070000 1550000 1280000 1080000 1320000 1420000 1390000 2550000 1880000 3860000 623000 2450000 3040000 413300 731000 3070000 731000 3700000 2470000 1820000 1520000 3710000 870000 3830000 880000 malic acid 2140000 5960000 5730000 4230000 1320000 2080000 1740000 6460000 5810000 3900000 5910000 5400000 3260000 4140000 9800000 6690000 6980000 10400000 109000000 65100000 112000000 54800000 73100000 85100000 10500000 77300000 62500000 55100000 88400000 72900000 70500000 74300000 97700000 2870000 149000000 128000000 glutamine NA 5700000 2640000 3700000 4660000 3770000 4830000 3620000 6010000 5130000 4340000 3510000 6540000 4310000 3210000 3040000 5320000 3950000 756000 550000 1330000 887000 747000 610000 988000 1610000 632000 1520000 666000 1160000 688000 1510000 1540000 2660000 1140000 1150000 alanine 397000 426000 303000 301000 262000 197000 126000 352000 406000 359000 280000 234000 190000 317000 364000 381000 282000 356000 295000 241000 333000 259000 258000 284000 81800 314000 284000 274000 313000 302000 265000 294000 312000 319000 425000 271000 fructose 1,6-bisphosphate NA 678000 873000 1230000 327000 326000 1410000 1000000 1260000 947000 633000 924000 849000 777000 1010000 1080000 703000 1560000 834000 473000 3550000 999000 1050000 808000 64000 473000 1560000 2080000 98600 1870000 465000 2930000 NA 1650000 1120000 460000 glucose NA 889000 746000 774000 1010000 927000 896000 810000 825000 900000 1220000 885000 956000 794000 714000 956000 1010000 1090000 627000 499000 634000 578000 464000 489000 32490 533000 542000 546000 646000 475000 485000 482000 586000 593000 716000 525000 alpha ketoglutaric acid 294000 253000 741000 160000 122000 256000 318000 282000 1110000 237000 163000 165000 506000 494000 1370000 639000 285000 307000 844000 260000 537000 369000 265000 60100 NA 438000 511000 248000 114000 335000 238000 152000 380000 210000 1980000 NA MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG acetyl coenzyme A C00024 pyruvic acid C00022 citric acid C00158 succinic acid C00042 glutamic acid C00025 cis-aconitic acid C00417 glucose 6-phosphate C00668 fructose 6-phosphate C05345 phosphoenolpyruvic acid C00074 lactic acid C00186 aspartic acid C00049 asparagine C00152 malic acid C00149 glutamine C00064 alanine C00041 fructose 1,6-bisphosphate C05378 glucose C00267 alpha ketoglutaric acid C00026 METABOLITES_END #END