#METABOLOMICS WORKBENCH Jinmei_20170812_050650 DATATRACK_ID:1217 STUDY_ID:ST000875 ANALYSIS_ID:AN001828 PROJECT_ID:PR000606 VERSION 1 CREATED_ON January 10, 2019, 1:15 pm #PROJECT PR:PROJECT_TITLE Metabolomic investigations on Nesterenkonia flava from different origins PR:PROJECT_TITLE revealed significant intraspecies differences between marine and terrestrial PR:PROJECT_TITLE actinomycetes PR:PROJECT_SUMMARY Marine is one of the most important resources of microorganisms, including PR:PROJECT_SUMMARY bacteria, actinomycetes, and fungi. As marine and terrestrial environments PR:PROJECT_SUMMARY differ a lot in many aspects it is not surprising that the species and PR:PROJECT_SUMMARY characteristics of microorganisms living there are very different. PR:PROJECT_SUMMARY Interestingly, many marine microorganisms can find their congeners of the same PR:PROJECT_SUMMARY species from terrestrial resources. The aim of this work is to evaluate the PR:PROJECT_SUMMARY intraspecies differences between marine and terrestrial actinomycetes on PR:PROJECT_SUMMARY metabolic level and to uncover the mechanism responsible for the differences. To PR:PROJECT_SUMMARY address this, we carried out comparative metabolomics study on Nesterenkonia PR:PROJECT_SUMMARY flava strains isolated from marine and terrestrial environments. The results PR:PROJECT_SUMMARY showed that marine strains were clearly distinguished from their terrestrial PR:PROJECT_SUMMARY congeners on the principal components analysis (PCA) scores plot of PR:PROJECT_SUMMARY intracellular metabolites. The markers responsible for the discrimination of PR:PROJECT_SUMMARY marine and terrestrial strains were figured out using loading plot from partial PR:PROJECT_SUMMARY least squares discrimination analysis (PLS-DA). Pathway analysis based on PR:PROJECT_SUMMARY PLS-DA, univariate analysis, and correlation analysis of metabolites showed that PR:PROJECT_SUMMARY the major differential metabolites between the terrestrial N. flava and the PR:PROJECT_SUMMARY marine ones were involved in osmotic regulation, redox balancing, and energy PR:PROJECT_SUMMARY metabolism. Together, these insights provide clues as to how the previous living PR:PROJECT_SUMMARY environment of microbes affect their current metabolic performances under PR:PROJECT_SUMMARY laboratory cultivation conditions. PR:INSTITUTE Third Institute of Oceanography, State Oceanic Administration PR:LAST_NAME Xia PR:FIRST_NAME Jinmei PR:ADDRESS 184 Daxue Road, Xiamen 361005, PR China PR:EMAIL xiajinmei@tio.org.cn PR:PHONE 86-13003995626 #STUDY ST:STUDY_TITLE Metabolomic investigations on Nesterenkonia flava from different origins ST:STUDY_TITLE revealed significant intraspecies differences between marine and terrestrial ST:STUDY_TITLE actinomycetes ST:STUDY_SUMMARY Marine is one of the most important resources of microorganisms, including ST:STUDY_SUMMARY bacteria, actinomycetes, and fungi. As marine and terrestrial environments ST:STUDY_SUMMARY differ a lot in many aspects it is not surprising that the species and ST:STUDY_SUMMARY characteristics of microorganisms living there are very different. ST:STUDY_SUMMARY Interestingly, many marine microorganisms can find their congeners of the same ST:STUDY_SUMMARY species from terrestrial resources. The aim of this work is to evaluate the ST:STUDY_SUMMARY intraspecies differences between marine and terrestrial actinomycetes on ST:STUDY_SUMMARY metabolic level and to uncover the mechanism responsible for the differences. To ST:STUDY_SUMMARY address this, we carried out comparative metabolomics study on Nesterenkonia ST:STUDY_SUMMARY flava strains isolated from marine and terrestrial environments. The results ST:STUDY_SUMMARY showed that marine strains were clearly distinguished from their terrestrial ST:STUDY_SUMMARY congeners on the principal components analysis (PCA) scores plot of ST:STUDY_SUMMARY intracellular metabolites. The markers responsible for the discrimination of ST:STUDY_SUMMARY marine and terrestrial strains were figured out using loading plot from partial ST:STUDY_SUMMARY least squares discrimination analysis (PLS-DA). Pathway analysis based on ST:STUDY_SUMMARY PLS-DA, univariate analysis, and correlation analysis of metabolites showed that ST:STUDY_SUMMARY the major differential metabolites between the terrestrial N. flava and the ST:STUDY_SUMMARY marine ones were involved in osmotic regulation, redox balancing, and energy ST:STUDY_SUMMARY metabolism. Together, these insights provide clues as to how the previous living ST:STUDY_SUMMARY environment of microbes affect their current metabolic performances under ST:STUDY_SUMMARY laboratory cultivation conditions. ST:INSTITUTE Third Institute of Oceanography, State Oceanic Administration ST:LAST_NAME Xia ST:FIRST_NAME Jinmei ST:ADDRESS 184 Daxue Road, Xiamen 361005, PR China ST:EMAIL xiajinmei@tio.org.cn ST:PHONE 86-13003995626 #SUBJECT SU:SUBJECT_TYPE NMR based metabolomics of microbes SU:SUBJECT_SPECIES Nesterenkonia flava SU:TAXONOMY_ID 469799 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Strain:1K00606 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 2 Strain:1K00606 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 3 Strain:1K00606 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 4 Strain:1K00606 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 5 Strain:1K00606 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 6 Strain:1K00606 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 7 Strain:1K00607 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 8 Strain:1K00607 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 9 Strain:1K00607 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 10 Strain:1K00607 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 11 Strain:1K00607 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 12 Strain:1K00607 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 13 Strain:1K00610 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 14 Strain:1K00610 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 15 Strain:1K00610 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 16 Strain:1K00610 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 17 Strain:1K00610 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 18 Strain:1K00610 | Strain source:Marine | Medium:A14 SUBJECT_SAMPLE_FACTORS - 19 Strain:1A10663 | Strain source:Terrestrial | Medium:A14 SUBJECT_SAMPLE_FACTORS - 20 Strain:1A10663 | Strain source:Terrestrial | Medium:A14 SUBJECT_SAMPLE_FACTORS - 21 Strain:1A10663 | Strain source:Terrestrial | Medium:A14 SUBJECT_SAMPLE_FACTORS - 22 Strain:1A10663 | Strain source:Terrestrial | Medium:A14 SUBJECT_SAMPLE_FACTORS - 23 Strain:1A10663 | Strain source:Terrestrial | Medium:A14 SUBJECT_SAMPLE_FACTORS - 24 Strain:1A10663 | Strain source:Terrestrial | Medium:A14 SUBJECT_SAMPLE_FACTORS - 25 Strain:1K00606 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 26 Strain:1K00606 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 27 Strain:1K00606 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 28 Strain:1K00606 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 29 Strain:1K00606 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 30 Strain:1K00606 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 31 Strain:1K00607 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 32 Strain:1K00607 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 33 Strain:1K00607 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 34 Strain:1K00607 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 35 Strain:1K00607 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 36 Strain:1K00607 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 37 Strain:1K00610 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 38 Strain:1K00610 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 39 Strain:1K00610 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 40 Strain:1K00610 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 41 Strain:1K00610 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 42 Strain:1K00610 | Strain source:Marine | Medium:A6 SUBJECT_SAMPLE_FACTORS - 43 Strain:1A10663 | Strain source:Terrestrial | Medium:A6 SUBJECT_SAMPLE_FACTORS - 44 Strain:1A10663 | Strain source:Terrestrial | Medium:A6 SUBJECT_SAMPLE_FACTORS - 45 Strain:1A10663 | Strain source:Terrestrial | Medium:A6 SUBJECT_SAMPLE_FACTORS - 46 Strain:1A10663 | Strain source:Terrestrial | Medium:A6 SUBJECT_SAMPLE_FACTORS - 47 Strain:1A10663 | Strain source:Terrestrial | Medium:A6 SUBJECT_SAMPLE_FACTORS - 48 Strain:1A10663 | Strain source:Terrestrial | Medium:A6 SUBJECT_SAMPLE_FACTORS - 49 Strain:1K00606 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 50 Strain:1K00606 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 51 Strain:1K00606 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 52 Strain:1K00606 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 53 Strain:1K00606 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 54 Strain:1K00606 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 55 Strain:1K00607 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 56 Strain:1K00607 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 57 Strain:1K00607 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 58 Strain:1K00607 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 59 Strain:1K00607 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 60 Strain:1K00607 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 61 Strain:1K00610 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 62 Strain:1K00610 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 63 Strain:1K00610 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 64 Strain:1K00610 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 65 Strain:1K00610 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 66 Strain:1K00610 | Strain source:Marine | Medium:A3 SUBJECT_SAMPLE_FACTORS - 67 Strain:1A10663 | Strain source:Terrestrial | Medium:A3 SUBJECT_SAMPLE_FACTORS - 68 Strain:1A10663 | Strain source:Terrestrial | Medium:A3 SUBJECT_SAMPLE_FACTORS - 69 Strain:1A10663 | Strain source:Terrestrial | Medium:A3 SUBJECT_SAMPLE_FACTORS - 70 Strain:1A10663 | Strain source:Terrestrial | Medium:A3 SUBJECT_SAMPLE_FACTORS - 71 Strain:1A10663 | Strain source:Terrestrial | Medium:A3 SUBJECT_SAMPLE_FACTORS - 72 Strain:1A10663 | Strain source:Terrestrial | Medium:A3 #COLLECTION CO:COLLECTION_SUMMARY The broth cultures (50 mL) were harvested via centrifugation at 7000 g for 10 CO:COLLECTION_SUMMARY min under 4 °C. The pellet was quenched using 10 mL of 60% cold methanol (−80 CO:COLLECTION_SUMMARY °C) containing 0.85% (wt./vol.) of NaCl for 30 min. The quenched cell pellets CO:COLLECTION_SUMMARY were re-suspended in 10 mL of cold PBS and were washed for 3 times. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY For each cell pellet sample, 5 mL of the mixture of methanol/water (1:0.9, v/v) TR:TREATMENT_SUMMARY was added and ultrasonicated for 25 min to break cells bathing in ice. The TR:TREATMENT_SUMMARY mixture was added with 5 mL of cold chloroform, vortexed and subjected to 10 min TR:TREATMENT_SUMMARY of ultrasonic extraction under the bath of ice. The mixture was then centrifuged TR:TREATMENT_SUMMARY at 9500 g for 8 min. The upper layer phase containing methanol and water was TR:TREATMENT_SUMMARY taken out and evaporated in the fume cupboard to remove methanol. The remaining TR:TREATMENT_SUMMARY water solution was freezed under −80 °C, and then freeze-dried to afford dry TR:TREATMENT_SUMMARY samples, which were stored under −80 °C before analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The intracellular extract was re-suspended in 550 µL of phosphate buffer SP:SAMPLEPREP_SUMMARY containing 1.5 M KH2PO4, 0.1% sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 SP:SAMPLEPREP_SUMMARY (TSP), and 10% D2O. Subsequently, all the samples were vortexed and centrifuged SP:SAMPLEPREP_SUMMARY at 12000 g for 15 min at 4°C to remove any insoluble components. The collected SP:SAMPLEPREP_SUMMARY supernatants (500 μL) were transferred to 5 mm NMR tubes for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance III 850 MHz spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 850 MHz NM:NMR_RESULTS_FILE ST000875_AN001828_Results.txt UNITS:ppm #END