{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000914","ANALYSIS_ID":"AN001484","VERSION":"1","CREATED_ON":"January 9, 2018, 3:09 pm"},

"PROJECT":{"PROJECT_TITLE":"Metabolomic adaptation of a deep sea Microbacterium sediminis to prolonged low temperature under high pressure","PROJECT_SUMMARY":"Low temperature is the most wide-spread “hostile” environmental factor on earth while at the same time the most common condition for marine organisms. However, the unique adaptive mechanisms that enable the survival of marine microorganisms under low temperature are unclear. Since low temperature is always accompanied by high pressure and other adverse conditions in marine environment, here we studied the metabolic adaptation of a marine strain to prolonged low temperature under high pressure. The strain studied is a psychrotolerant Microbacterium sediminis isolated from deep sea sediment. By using 1H nuclear magnetic resonance (NMR)-based metabolomics approach, we detected the spectral data of polar extracts of the strain M. sediminis, and applied multivariate statistical analysis methods together with univariate analysis to analyze metabolic profiles associated to different conditions. The metabolic profiles of the M. sediminis strain cultivated under high pressure at low temperature were distinctly different from those cultivated under high pressure at normal temperature. We identified the differential metabolites which were responsible for distinguishing the metabolic profiles and compared their relative intensities between groups. We also compared the different adaptive responses of the strain at different growth stages to the prolonged low temperature under high pressure. We proposed that the low-temperature adapting process of the M. sediminis strain involves, 1) the regulation of osmotic pressure using amino acids as possible alternative osmolytes, and, 2) the strengthen of glycolysis and the maintenance of TCA cycle via amino acids anaplerotic reaction. We put forward that the main difference of adaptation to low temperature for the strain at different growth stages was related to energy metabolism. Our findings improved the understanding of the low-temperature adaptive mechanisms for marine microorganisms under high pressure on the metabolic level.","INSTITUTE":"Third Institute of Oceanography, State Oceanic Administration","LAST_NAME":"Xia","FIRST_NAME":"Jinmei","ADDRESS":"184 Daxue Road, Xiamen 361005, PR China","EMAIL":"xiajinmei@tio.org.cn","PHONE":"86-13003995626"},

"STUDY":{"STUDY_TITLE":"Metabolomic adaptation of a deep sea Microbacterium sediminis to prolonged low temperature under high pressure","STUDY_SUMMARY":"Low temperature is the most wide-spread “hostile” environmental factor on earth while at the same time the most common condition for marine organisms. However, the unique adaptive mechanisms that enable the survival of marine microorganisms under low temperature are unclear. Since low temperature is always accompanied by high pressure and other adverse conditions in marine environment, here we studied the metabolic adaptation of a marine strain to prolonged low temperature under high pressure. The strain studied is a psychrotolerant Microbacterium sediminis isolated from deep sea sediment. By using 1H nuclear magnetic resonance (NMR)-based metabolomics approach, we detected the spectral data of polar extracts of the strain M. sediminis, and applied multivariate statistical analysis methods together with univariate analysis to analyze metabolic profiles associated to different conditions. The metabolic profiles of the M. sediminis strain cultivated under high pressure at low temperature were distinctly different from those cultivated under high pressure at normal temperature. We identified the differential metabolites which were responsible for distinguishing the metabolic profiles and compared their relative intensities between groups. We also compared the different adaptive responses of the strain at different growth stages to the prolonged low temperature under high pressure. We proposed that the low-temperature adapting process of the M. sediminis strain involves, 1) the regulation of osmotic pressure using amino acids as possible alternative osmolytes, and, 2) the strengthen of glycolysis and the maintenance of TCA cycle via amino acids anaplerotic reaction. We put forward that the main difference of adaptation to low temperature for the strain at different growth stages was related to energy metabolism. Our findings improved the understanding of the low-temperature adaptive mechanisms for marine microorganisms under high pressure on the metabolic level.","INSTITUTE":"Third Institute of Oceanography, State Oceanic Administration","LAST_NAME":"Xia","FIRST_NAME":"Jinmei","ADDRESS":"184 Daxue Road, Xiamen 361005, PR China","EMAIL":"xiajinmei@tio.org.cn","PHONE":"86-13003995626"},

"SUBJECT":{"SUBJECT_TYPE":"NMR based metabolomics of microbes","SUBJECT_SPECIES":"Microbacterium sediminis","TAXONOMY_ID":"904291"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"2A",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2B",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2C",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2D",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2E",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2F",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2G",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2H",
"Factors":{"Treatment":"NT-L"}
},
{
"Subject ID":"-",
"Sample ID":"2I",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2J",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2K",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2L",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2M",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2N",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2O",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"2P",
"Factors":{"Treatment":"NT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4A",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4B",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4C",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4D",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4E",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4F",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4G",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4H",
"Factors":{"Treatment":"LT-L"}
},
{
"Subject ID":"-",
"Sample ID":"4I",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4J",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4K",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4L",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4M",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4N",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4O",
"Factors":{"Treatment":"LT-S"}
},
{
"Subject ID":"-",
"Sample ID":"4P",
"Factors":{"Treatment":"LT-S"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"A psychrotolerant strain of Microbacterium sediminis numbered as YLB-01 isolated from deep sea sediment of the Indian Ocean was used in this study. The strain can grow at temperatures ranging from 4 °C to 50 °C with an optimal growth temperature of 28 °C (Yu et al. 2013). Tryptone soy broth (TSB) medium, which contains 15 g/L tryptone, 5 g/L soy peptone, and 5 g/L NaCl, was used to cultivate the strain. The strain was maintained in glycerol tubes under -80 °C in a refrigerator and was activated using streaking inoculation on agar plates before use. For metabolomics research, a single colony was first inoculated from an agar plate to 32 test tubes containing 5 mL of TSB medium each and cultivated using a shaker under 28 °C for 12 hours. These seed cultures were then transferred to 150 mL Erlenmeyer flasks containing 100 mL of TSB medium each. In order to obtain a sufficient amount of cells, these cells were cultivated under normal temperature (28 °C) and atmospheric pressure (0.1 MPa) until logarithmic or stationary phase before transferred to lower temperature. Specifically, half of these samples were cultivated using a shaker under 28 °C for 18 hours to reach mid logarithmic phase (assigned as scenario L) and the other half were cultivated for 24 hours to reach stationary phase (assigned as scenario S) under the same condition. All samples were then transferred into 100 mL normal saline bags and put into a water-filled high-pressure chamber with the pressure set at 30 MPa. For half of the samples from a certain scenario (L or S) the temperature of the chamber was set at 28 °C. For the other half of the samples the temperature of the chamber was set at 4 °C. All samples were harvested after 7 days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their different cultivation conditions and sampling time","SAMPLE_TYPE":"cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"half of these samples were cultivated using a shaker under 28 °C for 18 hours to reach mid logarithmic phase (assigned as scenario L) and the other half were cultivated for 24 hours to reach stationary phase (assigned as scenario S) under the same condition. All samples were then transferred into 100 mL normal saline bags and put into a water-filled high-pressure chamber with the pressure set at 30 MPa. For half of the samples from a certain scenario (L or S) the temperature of the chamber was set at 28 °C. For the other half of the samples the temperature of the chamber was set at 4 °C. All samples were harvested after 7 days. The samples were grouped as NT-L, LT-L, NT-S, and LT-S based on their different cultivation conditions and sampling time"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"All 100 mL of the fermentation broth in a flask was harvested and poured into a 250 mL centrifuge bottle and centrifuged at 6000g and 4 °C for 5 min. The supernatant was discarded and the cell pellets were quenched using 100 mL of a buffer composed of 3:2 methanol/water and 0.85% (wt./vol.) NaCl at -40 °C. The resuspended mixture was again centrifuged at 6000g and 4 °C for 5 min. The cell pellets were then washed using cold PBS for 3 times. The mixture was resuspended and transferred into a 5 mL Eppendorf tube during the 3rd wash and then centrifuged at 6000g and 4 °C for 5 min. The cell pellets were kept under -80 °C until use. Intracellular metabolites were extracted using a procedure adopted from Ye et al. (Ye et al. 2012). The frozen samples were homogenized in 600 μL of cold 1:1 acetonitrile/water buffer. To destroy the bacterial cells, the samples were further sonicated on wet ice for 180 cycles with each cycle consisting of 2 s pulses and 3 s stops. The supernatant was collected by centrifugation at 12000 g for 10 min at 4 °C. The remaining solid residues were further extracted using the same extract solution and intensively homogenized via vortexing. The second supernatant was collected after centrifugation and pooled with the first one. The combined supernatants from the two extractions were lyophilized, and stored at -80 °C. Immediately before 1H NMR measurements were taken, the extract powder was redisclosed in 550 µL phosphate buffer (50 mM K2HPO4/NaH2PO4, 10% D2O, 1mM 3-(Trimethylsilyl) propionate-2,2,3,3-d4 acid sodium salt (TSP), pH7.4). Subsequently, all the samples were vortexed and centrifuged at 12000 g for 15 min at 4 °C to remove any insoluble components. Finally, aliquots of the supernatant were transferred into 5 mm NMR tubes (Beckonert et al. 2007)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"-","INSTRUMENT_NAME":"-","COLUMN_NAME":"-"},

"ANALYSIS":{"ANALYSIS_TYPE":"NMR"},

"NM":{"INSTRUMENT_NAME":"Bruker Avance III 600 MHz spectrometer","INSTRUMENT_TYPE":"FT-NMR","NMR_EXPERIMENT_TYPE":"1D-1H","SPECTROMETER_FREQUENCY":"600 MHz","NMR_RESULTS_FILE":"ST000914_AN001484_Results.txt UNITS:relative intensities"}

}