#METABOLOMICS WORKBENCH Lu_Group_20210907_075703_mwtab.txt DATATRACK_ID:2828 STUDY_ID:ST001939 ANALYSIS_ID:AN003153 PROJECT_ID:000000 VERSION 1 CREATED_ON October 12, 2021, 6:32 pm #PROJECT PR:PROJECT_TITLE Metabolomics characterized concentration-dependent metabolic influence of PR:PROJECT_TITLE magnesium on biofilm formation in Escherichia coli PR:PROJECT_TYPE Targeted MS quantitative analysis PR:PROJECT_SUMMARY Metabolomics characterized concentration-dependent metabolic influence of PR:PROJECT_SUMMARY magnesium on biofilm formation in Escherichia coli PR:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University PR:DEPARTMENT Shanghai Center for Systems Biomedicine PR:LABORATORY Lu Group PR:LAST_NAME Lu PR:FIRST_NAME Haitao PR:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China PR:EMAIL haitao_lu@sjtu.edu.cn PR:PHONE 15221478139 #STUDY ST:STUDY_TITLE Metabolomics characterized concentration-dependent metabolic influence of ST:STUDY_TITLE magnesium on biofilm formation in Escherichia coli (Part 2) ST:STUDY_SUMMARY Biofilms are broadly formed by a diversity of microorganisms that enable them to ST:STUDY_SUMMARY adapt stressful environments. Biofilms often impose harmful influences in many ST:STUDY_SUMMARY niches, as they can cause food contamination, antibiotics resistance, and ST:STUDY_SUMMARY environmental issues. However, eradicating biofilms remains difficult since the ST:STUDY_SUMMARY formation mechanism of biofilms are still incompletely clarified. In this study, ST:STUDY_SUMMARY we aimed at exploring the regulatory role of magnesium (Mg2+) on biofilm ST:STUDY_SUMMARY formation in Escherichia coli (E. coli) using phenotype visualization combined ST:STUDY_SUMMARY with targeted metabolomics method. We found that Mg2+ could exert significant ST:STUDY_SUMMARY influence on biofilm formation in a concentration-dependent manner by regulating ST:STUDY_SUMMARY phenotypic morphology and triggering metabolic modifications of biofilm. ST:STUDY_SUMMARY Phenotypic imaging revealed that increasing concentration of Mg2+ gradually ST:STUDY_SUMMARY inhibited biofilm formation, Mg2+ was observed to restore the microstructure of ST:STUDY_SUMMARY E. coli strain in biofilms to that in the relevant planktonic cells. In ST:STUDY_SUMMARY addition, our metabolomics analysis characterized 20 differential metabolites ST:STUDY_SUMMARY and associated 2 metabolic pathways including nucleotide metabolism and amino ST:STUDY_SUMMARY acid metabolism that were notably modified during biofilm formation under the ST:STUDY_SUMMARY treatments of different concentrations of Mg2+. Altogether, our work provides a ST:STUDY_SUMMARY novel insight into the influence of Mg2+ on biofilm formation at a metabolic ST:STUDY_SUMMARY level, which are implicated in the novel solution to disturb biofilm formation ST:STUDY_SUMMARY through the regulation of Mg2+ and functional metabolite interaction, then ST:STUDY_SUMMARY biofilms associated harmful impacts in different niches could be well tangled ST:STUDY_SUMMARY accordingly. ST:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University ST:DEPARTMENT Shanghai Center for Systems Biomedicine ST:LABORATORY Lu Group ST:LAST_NAME Lu ST:FIRST_NAME Haitao ST:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China ST:EMAIL haitao_lu@sjtu.edu.cn ST:PHONE 15221478139 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Escherichia coli SU:TAXONOMY_ID 562 #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 - 20210227-3-C-1 Concentration of MgSO4(w/v):0.01% RAW_FILE_NAME=20210227-3-C-1 SUBJECT_SAMPLE_FACTORS - 20210227-3-C-2 Concentration of MgSO4(w/v):0.01% RAW_FILE_NAME=20210227-3-C-2 SUBJECT_SAMPLE_FACTORS - 20210227-3-C-3 Concentration of MgSO4(w/v):0.01% RAW_FILE_NAME=20210227-3-C-3 SUBJECT_SAMPLE_FACTORS - 20210227-3-C-4 Concentration of MgSO4(w/v):0.01% RAW_FILE_NAME=20210227-3-C-4 SUBJECT_SAMPLE_FACTORS - 20210227-3-C-5 Concentration of MgSO4(w/v):0.01% RAW_FILE_NAME=20210227-3-C-5 SUBJECT_SAMPLE_FACTORS - 20210227-3-C-6 Concentration of MgSO4(w/v):0.01% RAW_FILE_NAME=20210227-3-C-6 SUBJECT_SAMPLE_FACTORS - 20210227-3-30-1 Concentration of MgSO4(w/v):0.15% RAW_FILE_NAME=20210227-3-30-1 SUBJECT_SAMPLE_FACTORS - 20210227-3-30-2 Concentration of MgSO4(w/v):0.15% RAW_FILE_NAME=20210227-3-30-2 SUBJECT_SAMPLE_FACTORS - 20210227-3-30-3 Concentration of MgSO4(w/v):0.15% RAW_FILE_NAME=20210227-3-30-3 SUBJECT_SAMPLE_FACTORS - 20210227-3-30-4 Concentration of MgSO4(w/v):0.15% RAW_FILE_NAME=20210227-3-30-4 SUBJECT_SAMPLE_FACTORS - 20210227-3-30-5 Concentration of MgSO4(w/v):0.15% RAW_FILE_NAME=20210227-3-30-5 SUBJECT_SAMPLE_FACTORS - 20210227-3-30-6 Concentration of MgSO4(w/v):0.15% RAW_FILE_NAME=20210227-3-30-6 SUBJECT_SAMPLE_FACTORS - 20210227-3-50-1 Concentration of MgSO4(w/v):0.25% RAW_FILE_NAME=20210227-3-50-1 SUBJECT_SAMPLE_FACTORS - 20210227-3-50-2 Concentration of MgSO4(w/v):0.25% RAW_FILE_NAME=20210227-3-50-2 SUBJECT_SAMPLE_FACTORS - 20210227-3-50-3 Concentration of MgSO4(w/v):0.25% RAW_FILE_NAME=20210227-3-50-3 SUBJECT_SAMPLE_FACTORS - 20210227-3-50-4 Concentration of MgSO4(w/v):0.25% RAW_FILE_NAME=20210227-3-50-4 SUBJECT_SAMPLE_FACTORS - 20210227-3-50-5 Concentration of MgSO4(w/v):0.25% RAW_FILE_NAME=20210227-3-50-5 SUBJECT_SAMPLE_FACTORS - 20210227-3-50-6 Concentration of MgSO4(w/v):0.25% RAW_FILE_NAME=20210227-3-50-6 SUBJECT_SAMPLE_FACTORS - 20210227-3-80-1 Concentration of MgSO4(w/v):0.40% RAW_FILE_NAME=20210227-3-80-1 SUBJECT_SAMPLE_FACTORS - 20210227-3-80-2 Concentration of MgSO4(w/v):0.40% RAW_FILE_NAME=20210227-3-80-2 SUBJECT_SAMPLE_FACTORS - 20210227-3-80-3 Concentration of MgSO4(w/v):0.40% RAW_FILE_NAME=20210227-3-80-3 SUBJECT_SAMPLE_FACTORS - 20210227-3-80-4 Concentration of MgSO4(w/v):0.40% RAW_FILE_NAME=20210227-3-80-4 SUBJECT_SAMPLE_FACTORS - 20210227-3-80-5 Concentration of MgSO4(w/v):0.40% RAW_FILE_NAME=20210227-3-80-5 SUBJECT_SAMPLE_FACTORS - 20210227-3-80-6 Concentration of MgSO4(w/v):0.40% RAW_FILE_NAME=20210227-3-80-6 SUBJECT_SAMPLE_FACTORS - 20210227-3-100-1 Concentration of MgSO4(w/v):0.50% RAW_FILE_NAME=20210227-3-100-1 SUBJECT_SAMPLE_FACTORS - 20210227-3-100-2 Concentration of MgSO4(w/v):0.50% RAW_FILE_NAME=20210227-3-100-2 SUBJECT_SAMPLE_FACTORS - 20210227-3-100-3 Concentration of MgSO4(w/v):0.50% RAW_FILE_NAME=20210227-3-100-3 SUBJECT_SAMPLE_FACTORS - 20210227-3-100-4 Concentration of MgSO4(w/v):0.50% RAW_FILE_NAME=20210227-3-100-4 SUBJECT_SAMPLE_FACTORS - 20210227-3-100-5 Concentration of MgSO4(w/v):0.50% RAW_FILE_NAME=20210227-3-100-5 SUBJECT_SAMPLE_FACTORS - 20210227-3-100-6 Concentration of MgSO4(w/v):0.50% RAW_FILE_NAME=20210227-3-100-6 #COLLECTION CO:COLLECTION_SUMMARY After 72 hours of culture, the planktonic cells were isolated, and the biofilms CO:COLLECTION_SUMMARY were washed with PBS solvent for three times. Then, planktonic cells and biofilm CO:COLLECTION_SUMMARY solutions were centrifuged at 2000 rpm and 4°C for 15 minutes, to collect the CO:COLLECTION_SUMMARY strain pellets. CO:SAMPLE_TYPE Biofilm #TREATMENT TR:TREATMENT_SUMMARY Colony-forming antigen (CFA) medium (1% casamino acids, 0.15% yeast extract, TR:TREATMENT_SUMMARY 0.005% magnesium sulfate, and 0.0005% manganese chloride) was used to form TR:TREATMENT_SUMMARY biofilms. The E. coli strain was incubated in LB-agar plate for 12 hours, one TR:TREATMENT_SUMMARY colony was isolated to LB broth for further 4 hours incubation, then diluted the TR:TREATMENT_SUMMARY solution into CFA medium at a ratio of 1:1000 and the cultures were incubated TR:TREATMENT_SUMMARY for another 72 h at 30°C to form mature biofilms. To test the influence of TR:TREATMENT_SUMMARY MgSO4 on biofilm formation, the purified compound of MgSO4 was added to the CFA TR:TREATMENT_SUMMARY medium at different concentrations for the preparation of treated culture TR:TREATMENT_SUMMARY medium; The left cultural procedures were kept as the same as the protocol TR:TREATMENT_SUMMARY listed above. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each pellet sample was mixed with 2 mL of 80% ice-cold methanol, and the mixed SP:SAMPLEPREP_SUMMARY solution was homogenized on ice for 2 minutes and repeated the same procedure SP:SAMPLEPREP_SUMMARY for three times. Next, the collected samples were centrifuged at 12000 rpm and SP:SAMPLEPREP_SUMMARY 4°C for 15 minutes, to collect the supernatants. The supernatants were further SP:SAMPLEPREP_SUMMARY mixed with 800 μL of ice-cold acetonitrile for 20 minutes before further SP:SAMPLEPREP_SUMMARY centrifuge at 12000 rpm and 4°C for 15 minutes. At last, the isolated SP:SAMPLEPREP_SUMMARY supernatants were gone through a 0.22 μm filter membrane (nylon) before the SP:SAMPLEPREP_SUMMARY lyophilization. The lyophilized samples were stored at -80°C for metabolomics SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The chromatographic separation of biological samples was implemented on a Waters CH:CHROMATOGRAPHY_SUMMARY ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 μm) with an optimized CH:CHROMATOGRAPHY_SUMMARY gradient-elution program (mobile phase A and B were 0.1% formic acid in water CH:CHROMATOGRAPHY_SUMMARY and acetonitrile (v/v)): 0-2 minutes, 98% A; 2-10 minutes, 98%-65% A; 10-12 CH:CHROMATOGRAPHY_SUMMARY minutes, 65%-20% A; 12-14 minutes, 20%-2% A; 14-30 minutes, 2% A. The column CH:CHROMATOGRAPHY_SUMMARY temperature was at 40°C and the flow rate was set at 0.3 mL/ minutes. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter MS:MS_COMMENTS QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ) #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS - MS_METABOLITE_DATA_START Samples 20210227-3-C-1 20210227-3-C-2 20210227-3-C-3 20210227-3-C-4 20210227-3-C-5 20210227-3-C-6 20210227-3-30-1 20210227-3-30-2 20210227-3-30-3 20210227-3-30-4 20210227-3-30-5 20210227-3-30-6 20210227-3-50-1 20210227-3-50-2 20210227-3-50-3 20210227-3-50-4 20210227-3-50-5 20210227-3-50-6 20210227-3-80-1 20210227-3-80-2 20210227-3-80-3 20210227-3-80-4 20210227-3-80-5 20210227-3-80-6 20210227-3-100-1 20210227-3-100-2 20210227-3-100-3 20210227-3-100-4 20210227-3-100-5 20210227-3-100-6 Factors Concentration of MgSO4(w/v):0.01% Concentration of MgSO4(w/v):0.01% Concentration of MgSO4(w/v):0.01% Concentration of MgSO4(w/v):0.01% Concentration of MgSO4(w/v):0.01% Concentration of MgSO4(w/v):0.01% Concentration of MgSO4(w/v):0.15% Concentration of MgSO4(w/v):0.15% Concentration of MgSO4(w/v):0.15% Concentration of MgSO4(w/v):0.15% Concentration of MgSO4(w/v):0.15% Concentration of MgSO4(w/v):0.15% Concentration of MgSO4(w/v):0.25% Concentration of MgSO4(w/v):0.25% Concentration of MgSO4(w/v):0.25% Concentration of MgSO4(w/v):0.25% Concentration of MgSO4(w/v):0.25% Concentration of MgSO4(w/v):0.25% Concentration of MgSO4(w/v):0.40% Concentration of MgSO4(w/v):0.40% Concentration of MgSO4(w/v):0.40% Concentration of MgSO4(w/v):0.40% Concentration of MgSO4(w/v):0.40% Concentration of MgSO4(w/v):0.40% Concentration of MgSO4(w/v):0.50% Concentration of MgSO4(w/v):0.50% Concentration of MgSO4(w/v):0.50% Concentration of MgSO4(w/v):0.50% Concentration of MgSO4(w/v):0.50% Concentration of MgSO4(w/v):0.50% Citrate 265.624683 229.582648 101.6533597 202.2938781 78.21547351 166.1863813 116.3935181 114.7306693 87.60577131 141.6165639 161.9964823 95.43590407 118.7946599 87.36403033 41.04976171 102.6413214 115.2794131 71.26490955 24.12817163 48.67699242 79.83783761 61.82223922 148.8604223 27.10157944 101.4703045 36.99933658 105.4966118 74.43474508 80.07335833 62.58124827 Isocitric acid 273.2894311 185.334621 92.56521134 215.6236227 80.8531579 134.5427337 114.5221143 95.68503314 118.714412 134.3521161 164.8696992 92.49417252 109.8913976 89.63689412 41.53256461 80.07891671 98.82451032 58.11110161 31.77116835 44.95267889 68.86434712 75.45553159 102.968415 27.02255301 72.05102806 32.90157369 72.57385992 73.49203836 66.98590014 51.32835018 Methylmalonic acid 645.2408364 671.9398089 396.0831827 763.4151132 314.8070231 448.9304212 268.8262434 281.3520378 263.8100633 246.1142733 301.152173 434.0536531 164.6801904 252.3421861 122.8380661 228.1595418 208.8163602 181.6652954 51.65897361 170.6076465 216.5672238 316.0159023 371.9978358 255.2096014 157.9114133 139.0755396 273.2382258 270.4376582 395.6152924 280.6376582 3-Hydroxybenzoic acid 2.050217583 2.707146038 0.936584635 2.940541901 1.149922382 1.66367402 0.99235104 0.87149896 1.11352359 0.840508449 0.961541454 1.678010448 0.569078678 0.785394136 0.478478401 0.815054399 0.506824747 0.59738412 0.240014729 1.002712187 1.284286192 1.380328763 3.02836668 1.177275284 1.119118335 0.991513751 1.879346054 1.164526128 1.892517412 1.324998846 L-Lactate 1.165487497 1.067296454 0.488439063 2.349300438 0.974192226 1.668104563 0.39197932 0.700583921 0.733050998 0.494328456 0.544165978 1.245458087 0.337695969 0.49182746 0.204870392 0.512879242 0.481274041 0.577119993 0.356587644 1.405782971 2.947889564 2.009967827 4.934016772 1.581759911 0.662987863 0.339543965 1.053989404 2.401957305 2.579563368 2.925889059 4-Hydroxybenzoic acid 2.451854918 3.205460806 1.214484633 3.410496604 1.311195415 2.186007251 1.188758964 1.052273228 1.169972791 1.036456332 1.129741095 2.137656279 0.622888546 0.825785553 0.608555836 1.021629881 0.636141868 0.774593426 0.28695676 1.25045221 1.4941889 1.727932682 3.716894852 1.566019563 1.367417311 1.170323281 2.276882747 1.377811692 2.584939732 1.468292635 Azelaic acid 3.481191494 2.630131007 1.474156155 2.831528874 1.050852627 1.696553433 2.004102455 1.126636306 1.166322835 1.223599814 1.402394136 1.279713791 0.899791388 1.3328948 0.537828089 0.912186869 0.717747001 0.897004377 0.213640312 0.542717116 0.742751907 0.824641331 1.252232198 0.781356269 1.648847765 0.65415992 1.35541398 0.729131394 1.558344933 0.932171646 Sebacic acid 1.524846071 1.260470757 0.702676827 1.736153716 0.663629699 0.984550943 0.893518352 0.731648323 0.648982274 0.500780836 0.610601351 0.543257312 0.306453976 0.452004959 0.186125624 0.328731563 0.245941309 0.387661727 0.083773391 0.401464936 0.36625222 0.397617 0.565520078 0.332684382 0.401615495 0.265405411 0.515999026 0.462718656 0.862455477 0.365650284 N-Acetylglutamic acid 2132.000139 1959.225602 869.3346926 2465.370805 1104.495707 1558.090009 1014.901786 965.3267112 806.1389213 717.0080366 1050.758372 1064.122761 839.9665109 642.0451205 341.1699507 682.9184067 623.66011 713.7551539 176.9936361 425.4058936 611.7438714 779.0738351 885.7673749 408.8156238 576.6512307 360.2163174 748.2890344 642.5297336 986.6493931 632.5143382 Cis-Aconitic acid 3.770748326 3.878397655 1.780141426 4.405397734 1.941281653 2.273516872 1.131416602 1.13947344 1.108476857 1.125464797 1.198069874 0.710793161 1.271474245 0.863778631 0.365413452 0.938028978 0.897929587 0.79336169 0.259412984 0.382926432 0.863791625 0.638928551 0.895184164 0.36775229 0.818899985 0.307977106 0.703111949 0.675830718 1.066534364 0.62732054 Flavin adenine dinucleotide 44.69686265 43.22332208 18.04995573 43.55254822 16.56501895 27.21514844 31.04533891 25.656764 21.55078866 20.80378311 31.44418062 37.63047929 15.75704854 20.50275276 9.53004764 17.97088112 14.56317745 13.44452952 4.30292426 12.38016549 17.29005822 23.39702517 27.85651947 14.88850617 14.87310609 11.19162295 22.00719321 16.40557227 26.04200522 14.54995398 Fumarate 13.06415389 9.687641936 6.492306501 10.31413829 4.14429181 6.837628296 4.311879664 1.661890622 5.191220515 2.442149561 5.903763565 13.67979642 7.803828451 4.013882899 1.51306306 4.764638846 3.854180137 1.868513468 2.066579249 1.748065655 3.024941415 8.506860528 16.51208249 1.348742399 4.247555564 3.349980244 7.875460017 5.2406761 4.321302379 6.189757778 Malic acid 53.50016927 58.78505197 29.11744306 83.02273354 29.38000295 44.6787627 44.52527372 31.15278783 39.9723448 41.64340219 38.04715515 41.66844199 15.69559575 20.50726613 11.83179016 29.81509759 20.36597944 17.33590238 3.185441848 16.57850087 15.87567265 20.55489608 27.83321348 13.87492563 14.82082691 17.0892507 18.98118749 9.317527208 31.01978077 9.941222933 Succinic acid 638.3755233 660.4705851 403.3862549 742.4376414 310.2149463 412.7604474 275.0304945 271.1941966 267.4292253 232.458419 319.7624686 412.9087006 158.8840691 230.0703382 123.3607422 212.6960493 198.3210089 169.9888747 54.04907348 181.4821868 230.8644289 310.1056415 356.0858608 246.5879425 159.2309791 134.5281384 252.9701153 232.2537434 399.3276125 285.0983449 Phenyllactic acid 27.78708382 30.5006912 11.51866226 32.62891545 13.64199644 20.9209414 19.39325233 19.69308838 13.2785098 8.054105431 16.18490323 23.25313129 19.12682994 13.62674661 7.586936894 13.58582316 21.09540359 9.992675418 2.077202694 6.086413117 8.271234187 10.58884756 16.17061427 7.547686793 10.1789664 5.742648633 11.1447368 11.23898665 13.10733546 11.44492615 Citramalic acid 3.129708926 3.050388264 1.433791664 2.967060042 1.193391166 2.00777075 2.45333104 1.368994849 1.059375087 1.039844383 1.997330252 2.0213396 3.316076292 1.468659298 0.525262236 1.077014127 1.624410528 0.944568724 0.287755247 0.445627612 0.605663115 0.824563366 0.85644655 0.505577136 0.70028305 0.461779497 0.848730165 0.817137209 1.161011867 0.829778832 Glutaric acid 117.753355 126.719386 31.75172135 139.6706059 56.37972408 85.5140255 40.10995955 26.95023729 26.45766707 35.30531774 22.09283235 43.01207277 52.98039508 28.40836228 15.97103137 25.19897421 24.47701522 24.16719959 8.495573375 14.62399387 24.72060538 30.37909996 25.2179663 18.89045207 34.82879489 18.68279871 42.08024839 31.98175805 50.48198609 36.1646488 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z RT Citrate 191.11 1.48 Isocitric acid 191.02 1.47 Methylmalonic acid 117.02 1.8 3-Hydroxybenzoic acid 137.02 6.12 L-Lactate 89.02 1.35 4-Hydroxybenzoic acid 137.02 6.12 Azelaic acid 187.1 9.45 Sebacic acid 201.1 10.77 N-Acetylglutamic acid 188.05 1.64 Cis-Aconitic acid 173 1.54 Flavin adenine dinucleotide 784.15 6.03 Fumarate 115 1.72 Malic acid 133.01 1.11 Succinic acid 117.02 1.81 Phenyllactic acid 165.05 8.15 Citramalic acid 147.03 1.88 Glutaric acid 131.03 3.37 METABOLITES_END #END