#METABOLOMICS WORKBENCH Jinmei_20230629_031017 DATATRACK_ID:4124 STUDY_ID:ST002770 ANALYSIS_ID:AN004507 PROJECT_ID:PR001727 VERSION 1 CREATED_ON July 5, 2023, 10:17 pm #PROJECT PR:PROJECT_TITLE Bacillus-Derived Compounds Against a Vibrio PR:PROJECT_SUMMARY Metabolomics was employed in conjunction with bioactivity screening to identify PR:PROJECT_SUMMARY the antibacterial compounds from Bacillus strains against the highly lethal PR:PROJECT_SUMMARY Vibrio strain vp-HL. PR:INSTITUTE Third Institute of Oceanography, Ministry of Natural Resources PR:LAST_NAME Xia PR:FIRST_NAME Jinmei PR:ADDRESS Daxue Rd, Xiamen, China. PR:EMAIL xiajinmei@tio.org.cn PR:PHONE 13003995626 #STUDY ST:STUDY_TITLE Metabolomics and Molecular Networking-Guided Screening of Bacillus-Derived ST:STUDY_TITLE Bioactive Compounds Against a Highly Lethal Vibrio Species ST:STUDY_SUMMARY During our search for active substances capable of inhibiting a newly discovered ST:STUDY_SUMMARY highly lethal Vibrio strain vp-HL, we found that the fermentation broth of ST:STUDY_SUMMARY multiple Bacillus strains exhibited antibacterial activity. However, the ST:STUDY_SUMMARY substances responsible for the activity remained unclear. Metabolomics was ST:STUDY_SUMMARY employed in conjunction with bioactivity screening to identify the antibacterial ST:STUDY_SUMMARY compounds from Bacillus strains. The Ethyl Acetate extracts of the broth of 20 ST:STUDY_SUMMARY Bacillus strains were analyzed using UPLC-MS/MS. Principal Component Analysis ST:STUDY_SUMMARY (PCA) and Orthogonal Partial Least Square - Discriminant Analysis (OPLS-DA) was ST:STUDY_SUMMARY used for pattern recognition. The strains fall into two groups in the scores ST:STUDY_SUMMARY plot from PCA. The S-plot from OPLS-DA offered information on biomarkers. ST:INSTITUTE Third Institute of Oceanography, Ministry of Natural Resources ST:LAST_NAME Xia ST:FIRST_NAME Jinmei ST:ADDRESS Daxue Rd, Xiamen, China. ST:EMAIL xiajinmei@tio.org.cn ST:PHONE 13003995626 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Bacillus pumilus #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 - 2 Group:B1 RAW_FILE_NAME=2.mzml SUBJECT_SAMPLE_FACTORS - 4 Group:B1 RAW_FILE_NAME=4.mzml SUBJECT_SAMPLE_FACTORS - 7 Group:B1 RAW_FILE_NAME=7.mzml SUBJECT_SAMPLE_FACTORS - 14 Group:B1 RAW_FILE_NAME=14.mzml SUBJECT_SAMPLE_FACTORS - 15 Group:B1 RAW_FILE_NAME=15.mzml SUBJECT_SAMPLE_FACTORS - 20 Group:B1 RAW_FILE_NAME=20.mzml SUBJECT_SAMPLE_FACTORS - 22 Group:B1 RAW_FILE_NAME=22.mzml SUBJECT_SAMPLE_FACTORS - 23 Group:B1 RAW_FILE_NAME=23.mzml SUBJECT_SAMPLE_FACTORS - 24 Group:B1 RAW_FILE_NAME=24.mzml SUBJECT_SAMPLE_FACTORS - 27 Group:B1 RAW_FILE_NAME=27.mzml SUBJECT_SAMPLE_FACTORS - 34 Group:B2 RAW_FILE_NAME=34.mzml SUBJECT_SAMPLE_FACTORS - 35 Group:B2 RAW_FILE_NAME=35.mzml SUBJECT_SAMPLE_FACTORS - 38 Group:B2 RAW_FILE_NAME=38.mzml SUBJECT_SAMPLE_FACTORS - 39 Group:B2 RAW_FILE_NAME=39.mzml SUBJECT_SAMPLE_FACTORS - 43 Group:B2 RAW_FILE_NAME=43.mzml SUBJECT_SAMPLE_FACTORS - 44 Group:B2 RAW_FILE_NAME=44.mzml SUBJECT_SAMPLE_FACTORS - 46 Group:B2 RAW_FILE_NAME=46.mzml SUBJECT_SAMPLE_FACTORS - 47 Group:B2 RAW_FILE_NAME=47.mzml SUBJECT_SAMPLE_FACTORS - 50 Group:B2 RAW_FILE_NAME=50.mzml SUBJECT_SAMPLE_FACTORS - 56 Group:B2 RAW_FILE_NAME=56.mzml #COLLECTION CO:COLLECTION_SUMMARY The broth cultures were centrifuged at 7000 rpm for 10 min under 4 °C and the CO:COLLECTION_SUMMARY supernatants were collected. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY The full-length 16S rRNA gene sequence was obtained for the studied Bacillus TR:TREATMENT_SUMMARY strains. The phylogenetic tree of the 16S rRNA gene showed that the strains TR:TREATMENT_SUMMARY formed two groups, which were defined as Group B1 and B2. The antagonistic TR:TREATMENT_SUMMARY activities of the Bacillus broth against the vp-HL strain were evaluated by the TR:TREATMENT_SUMMARY agar well diffusion method. The DI (diameters of the inhibition zones) values of TR:TREATMENT_SUMMARY the 20 Bacillus strains against the vp-HL strain were measured. All strains TR:TREATMENT_SUMMARY belonging to Group B2 showed DI values greater than those of strains from Group TR:TREATMENT_SUMMARY B1. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The broth cultures were centrifuged at 7000 rpm for 10 min under 4 °C and the SP:SAMPLEPREP_SUMMARY supernatants were collected. Ethyl acetate (EtOAc) with 2 × volume was added SP:SAMPLEPREP_SUMMARY and sonicated for 10 min. The samples were then centrifuged at 7000 rpm at 4 °C SP:SAMPLEPREP_SUMMARY for 10 min and the organic phase was collected and dried using a rotary SP:SAMPLEPREP_SUMMARY evaporator. The extracts were resuspended in 1:1 acetonitrile: H2O with 1% SP:SAMPLEPREP_SUMMARY formic acid. These samples were diluted 20 times using methanol. All samples SP:SAMPLEPREP_SUMMARY were filtrated through 0.22 µm syringe filters. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Low pH polar CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um) CH:SOLVENT_A Water with 0.1% (vol/vol) formic acid CH:SOLVENT_B Acetonitrile with 0.1% (vol/vol) formic acid CH:FLOW_GRADIENT 5%B was kept for 0.5 min, 0.5-2.5 min, 5% to 30% B; 2.5-3.5 min, 30% to 40% B; CH:FLOW_GRADIENT 3.5-9 min, 40% to 99% B; 9-10 min, 99% to 5% B; and 5%B was kept for 3 min. CH:FLOW_RATE 0.35 mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Vion IMS QTof MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS UNIFI software (v. 1.8, Waters) MS:MS_RESULTS_FILE ST002770_AN004507_Results.txt UNITS:intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END