#METABOLOMICS WORKBENCH Jinmei_20171215_195714 DATATRACK_ID:1285 STUDY_ID:ST000914 ANALYSIS_ID:AN001484 PROJECT_ID:PR000632 VERSION 1 CREATED_ON January 9, 2018, 3:09 pm #PROJECT PR:PROJECT_TITLE Metabolomic adaptation of a deep sea Microbacterium sediminis to prolonged low PR:PROJECT_TITLE temperature under high pressure PR:PROJECT_SUMMARY Low temperature is the most wide-spread “hostile” environmental factor on PR:PROJECT_SUMMARY earth while at the same time the most common condition for marine organisms. PR:PROJECT_SUMMARY However, the unique adaptive mechanisms that enable the survival of marine PR:PROJECT_SUMMARY microorganisms under low temperature are unclear. Since low temperature is PR:PROJECT_SUMMARY always accompanied by high pressure and other adverse conditions in marine PR:PROJECT_SUMMARY environment, here we studied the metabolic adaptation of a marine strain to PR:PROJECT_SUMMARY prolonged low temperature under high pressure. The strain studied is a PR:PROJECT_SUMMARY psychrotolerant Microbacterium sediminis isolated from deep sea sediment. By PR:PROJECT_SUMMARY using 1H nuclear magnetic resonance (NMR)-based metabolomics approach, we PR:PROJECT_SUMMARY detected the spectral data of polar extracts of the strain M. sediminis, and PR:PROJECT_SUMMARY applied multivariate statistical analysis methods together with univariate PR:PROJECT_SUMMARY analysis to analyze metabolic profiles associated to different conditions. The PR:PROJECT_SUMMARY metabolic profiles of the M. sediminis strain cultivated under high pressure at PR:PROJECT_SUMMARY low temperature were distinctly different from those cultivated under high PR:PROJECT_SUMMARY pressure at normal temperature. We identified the differential metabolites which PR:PROJECT_SUMMARY were responsible for distinguishing the metabolic profiles and compared their PR:PROJECT_SUMMARY relative intensities between groups. We also compared the different adaptive PR:PROJECT_SUMMARY responses of the strain at different growth stages to the prolonged low PR:PROJECT_SUMMARY temperature under high pressure. We proposed that the low-temperature adapting PR:PROJECT_SUMMARY process of the M. sediminis strain involves, 1) the regulation of osmotic PR:PROJECT_SUMMARY pressure using amino acids as possible alternative osmolytes, and, 2) the PR:PROJECT_SUMMARY strengthen of glycolysis and the maintenance of TCA cycle via amino acids PR:PROJECT_SUMMARY anaplerotic reaction. We put forward that the main difference of adaptation to PR:PROJECT_SUMMARY low temperature for the strain at different growth stages was related to energy PR:PROJECT_SUMMARY metabolism. Our findings improved the understanding of the low-temperature PR:PROJECT_SUMMARY adaptive mechanisms for marine microorganisms under high pressure on the PR:PROJECT_SUMMARY metabolic level. 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 adaptation of a deep sea Microbacterium sediminis to prolonged low ST:STUDY_TITLE temperature under high pressure ST:STUDY_SUMMARY Low temperature is the most wide-spread “hostile” environmental factor on ST:STUDY_SUMMARY earth while at the same time the most common condition for marine organisms. ST:STUDY_SUMMARY However, the unique adaptive mechanisms that enable the survival of marine ST:STUDY_SUMMARY microorganisms under low temperature are unclear. Since low temperature is ST:STUDY_SUMMARY always accompanied by high pressure and other adverse conditions in marine ST:STUDY_SUMMARY environment, here we studied the metabolic adaptation of a marine strain to ST:STUDY_SUMMARY prolonged low temperature under high pressure. The strain studied is a ST:STUDY_SUMMARY psychrotolerant Microbacterium sediminis isolated from deep sea sediment. By ST:STUDY_SUMMARY using 1H nuclear magnetic resonance (NMR)-based metabolomics approach, we ST:STUDY_SUMMARY detected the spectral data of polar extracts of the strain M. sediminis, and ST:STUDY_SUMMARY applied multivariate statistical analysis methods together with univariate ST:STUDY_SUMMARY analysis to analyze metabolic profiles associated to different conditions. The ST:STUDY_SUMMARY metabolic profiles of the M. sediminis strain cultivated under high pressure at ST:STUDY_SUMMARY low temperature were distinctly different from those cultivated under high ST:STUDY_SUMMARY pressure at normal temperature. We identified the differential metabolites which ST:STUDY_SUMMARY were responsible for distinguishing the metabolic profiles and compared their ST:STUDY_SUMMARY relative intensities between groups. We also compared the different adaptive ST:STUDY_SUMMARY responses of the strain at different growth stages to the prolonged low ST:STUDY_SUMMARY temperature under high pressure. We proposed that the low-temperature adapting ST:STUDY_SUMMARY process of the M. sediminis strain involves, 1) the regulation of osmotic ST:STUDY_SUMMARY pressure using amino acids as possible alternative osmolytes, and, 2) the ST:STUDY_SUMMARY strengthen of glycolysis and the maintenance of TCA cycle via amino acids ST:STUDY_SUMMARY anaplerotic reaction. We put forward that the main difference of adaptation to ST:STUDY_SUMMARY low temperature for the strain at different growth stages was related to energy ST:STUDY_SUMMARY metabolism. Our findings improved the understanding of the low-temperature ST:STUDY_SUMMARY adaptive mechanisms for marine microorganisms under high pressure on the ST:STUDY_SUMMARY metabolic level. 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 Microbacterium sediminis SU:TAXONOMY_ID 904291 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 2A Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2B Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2C Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2D Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2E Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2F Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2G Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2H Treatment:NT-L SUBJECT_SAMPLE_FACTORS - 2I Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2J Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2K Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2L Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2M Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2N Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2O Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 2P Treatment:NT-S SUBJECT_SAMPLE_FACTORS - 4A Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4B Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4C Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4D Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4E Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4F Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4G Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4H Treatment:LT-L SUBJECT_SAMPLE_FACTORS - 4I Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4J Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4K Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4L Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4M Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4N Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4O Treatment:LT-S SUBJECT_SAMPLE_FACTORS - 4P Treatment:LT-S #COLLECTION CO:COLLECTION_SUMMARY A psychrotolerant strain of Microbacterium sediminis numbered as YLB-01 isolated CO:COLLECTION_SUMMARY from deep sea sediment of the Indian Ocean was used in this study. The strain CO:COLLECTION_SUMMARY can grow at temperatures ranging from 4 °C to 50 °C with an optimal growth CO:COLLECTION_SUMMARY temperature of 28 °C (Yu et al. 2013). Tryptone soy broth (TSB) medium, which CO:COLLECTION_SUMMARY contains 15 g/L tryptone, 5 g/L soy peptone, and 5 g/L NaCl, was used to CO:COLLECTION_SUMMARY cultivate the strain. The strain was maintained in glycerol tubes under -80 °C CO:COLLECTION_SUMMARY in a refrigerator and was activated using streaking inoculation on agar plates CO:COLLECTION_SUMMARY before use. For metabolomics research, a single colony was first inoculated from CO:COLLECTION_SUMMARY an agar plate to 32 test tubes containing 5 mL of TSB medium each and cultivated CO:COLLECTION_SUMMARY using a shaker under 28 °C for 12 hours. These seed cultures were then CO:COLLECTION_SUMMARY transferred to 150 mL Erlenmeyer flasks containing 100 mL of TSB medium each. In CO:COLLECTION_SUMMARY order to obtain a sufficient amount of cells, these cells were cultivated under CO:COLLECTION_SUMMARY normal temperature (28 °C) and atmospheric pressure (0.1 MPa) until logarithmic CO:COLLECTION_SUMMARY or stationary phase before transferred to lower temperature. Specifically, half CO:COLLECTION_SUMMARY of these samples were cultivated using a shaker under 28 °C for 18 hours to CO:COLLECTION_SUMMARY reach mid logarithmic phase (assigned as scenario L) and the other half were CO:COLLECTION_SUMMARY cultivated for 24 hours to reach stationary phase (assigned as scenario S) under CO:COLLECTION_SUMMARY the same condition. All samples were then transferred into 100 mL normal saline CO:COLLECTION_SUMMARY bags and put into a water-filled high-pressure chamber with the pressure set at CO:COLLECTION_SUMMARY 30 MPa. For half of the samples from a certain scenario (L or S) the temperature CO:COLLECTION_SUMMARY of the chamber was set at 28 °C. For the other half of the samples the CO:COLLECTION_SUMMARY temperature of the chamber was set at 4 °C. All samples were harvested after 7 CO:COLLECTION_SUMMARY days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their CO:COLLECTION_SUMMARY different cultivation conditions and sampling time CO:SAMPLE_TYPE cells #TREATMENT TR:TREATMENT_SUMMARY half of these samples were cultivated using a shaker under 28 °C for 18 hours TR:TREATMENT_SUMMARY to reach mid logarithmic phase (assigned as scenario L) and the other half were TR:TREATMENT_SUMMARY cultivated for 24 hours to reach stationary phase (assigned as scenario S) under TR:TREATMENT_SUMMARY the same condition. All samples were then transferred into 100 mL normal saline TR:TREATMENT_SUMMARY bags and put into a water-filled high-pressure chamber with the pressure set at TR:TREATMENT_SUMMARY 30 MPa. For half of the samples from a certain scenario (L or S) the temperature TR:TREATMENT_SUMMARY of the chamber was set at 28 °C. For the other half of the samples the TR:TREATMENT_SUMMARY temperature of the chamber was set at 4 °C. All samples were harvested after 7 TR:TREATMENT_SUMMARY days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their TR:TREATMENT_SUMMARY different cultivation conditions and sampling time #SAMPLEPREP SP:SAMPLEPREP_SUMMARY All 100 mL of the fermentation broth in a flask was harvested and poured into a SP:SAMPLEPREP_SUMMARY 250 mL centrifuge bottle and centrifuged at 6000g and 4 °C for 5 min. The SP:SAMPLEPREP_SUMMARY supernatant was discarded and the cell pellets were quenched using 100 mL of a SP:SAMPLEPREP_SUMMARY buffer composed of 3:2 methanol/water and 0.85% (wt./vol.) NaCl at -40 °C. The SP:SAMPLEPREP_SUMMARY resuspended mixture was again centrifuged at 6000g and 4 °C for 5 min. The cell SP:SAMPLEPREP_SUMMARY pellets were then washed using cold PBS for 3 times. The mixture was resuspended SP:SAMPLEPREP_SUMMARY and transferred into a 5 mL Eppendorf tube during the 3rd wash and then SP:SAMPLEPREP_SUMMARY centrifuged at 6000g and 4 °C for 5 min. The cell pellets were kept under -80 SP:SAMPLEPREP_SUMMARY °C until use. Intracellular metabolites were extracted using a procedure SP:SAMPLEPREP_SUMMARY adopted from Ye et al. (Ye et al. 2012). The frozen samples were homogenized in SP:SAMPLEPREP_SUMMARY 600 μL of cold 1:1 acetonitrile/water buffer. To destroy the bacterial cells, SP:SAMPLEPREP_SUMMARY the samples were further sonicated on wet ice for 180 cycles with each cycle SP:SAMPLEPREP_SUMMARY consisting of 2 s pulses and 3 s stops. The supernatant was collected by SP:SAMPLEPREP_SUMMARY centrifugation at 12000 g for 10 min at 4 °C. The remaining solid residues were SP:SAMPLEPREP_SUMMARY further extracted using the same extract solution and intensively homogenized SP:SAMPLEPREP_SUMMARY via vortexing. The second supernatant was collected after centrifugation and SP:SAMPLEPREP_SUMMARY pooled with the first one. The combined supernatants from the two extractions SP:SAMPLEPREP_SUMMARY were lyophilized, and stored at -80 °C. Immediately before 1H NMR measurements SP:SAMPLEPREP_SUMMARY were taken, the extract powder was redisclosed in 550 µL phosphate buffer (50 SP:SAMPLEPREP_SUMMARY mM K2HPO4/NaH2PO4, 10% D2O, 1mM 3-(Trimethylsilyl) propionate-2,2,3,3-d4 acid SP:SAMPLEPREP_SUMMARY sodium salt (TSP), pH7.4). Subsequently, all the samples were vortexed and SP:SAMPLEPREP_SUMMARY centrifuged at 12000 g for 15 min at 4 °C to remove any insoluble components. SP:SAMPLEPREP_SUMMARY Finally, aliquots of the supernatant were transferred into 5 mm NMR tubes SP:SAMPLEPREP_SUMMARY (Beckonert et al. 2007). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance III 600 MHz spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_RESULTS_FILE ST000914_AN001484_Results.txt UNITS:relative intensities #END