File(s) | Type | Description | Action |
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vharveyi_conc.csv (1.92 KB) | Comma Separated Values (.csv) | Primary data file for dataset ID 861154 | Add to Cart Download |
Amphi-enterobactins and related siderophore concentrations found in Vibrio harveyi supernatants and pellets from laboratory experiments in 2017. These data were published in McRose et al. (2018, Fig. 4).
Sampling and analytical procedures:
V. harveyi cells were cultured at 30C with shaking at 200 RPM. Growth experiments were conducted using a fully chemically defined artificial seawater medium consisting of basic salts (3x10-1 M NaCl, 1.05x10-2 M CaCl2Ÿ2H2O, 5x10-2 M MgSO4Ÿ7H2O, 4.85x10-4 M H3BO3) as well as 1x10-4 M K2HPO4, 6.51x10-2 M glycerol, 2.65 x10-8 M riboflavin, 2.96 x 10-6 M thiamine and Aquil trace metals without added Fe. Aquil trace metals contain 100 M EDTA, background Fe concentrations were determined by inductively coupled plasma MS (ICP-MS) to be ~100 nM. Nitrogen was added as MEM essential and non-essential amino acids (Sigma M5550, 92 mL L-1 ; Sigma M7145, 46 mL L-1 ). All cells were pre-cultured for ~24 hours in low Fe medium before the start of experiments to exhaust background trace metal supplies.
For quantification of siderophores ~50 mL of V. harveyi culture was centrifuged at 16,000 xg for 6 minutes. Supernatant samples were decanted, filtered (0.2 m) and acidified with 0.1% formic acid. Samples were then extracted using Oasis HLB (Waters) columns with the following conditions: 20 mL methanol, 20 mL MilliQ H2O, 50 mL sample, 20 mL 0.03% trifluoroacetic acid, 10 mL 0.03% formic acid and final elution with 30 mL of 40% methanol. Cell pellets were extracted overnight (~18 hours) with 5 mL of 80% methanol with 0.1% formic acid. Four mL of the resulting supernatant was diluted to 20% methanol with acidic (0.1% formic acid) MilliQ and extracted using an HLB column: 20 mL methanol, 20 mL MilliQ, 16 mL sample, 20 mL MilliQ and elution with 30 mL of 100% methanol. Samples were dried under vacuum (SpeedVac, ThermoFisher) and resuspended in either 1 mL MilliQ (supernatants) or 1 mL of 80% methanol (pellets). Extracted samples were acidified (0.1% acetic acid and 0.1% formic acid) and analyzed using electrospray-ionization LC-MS (Agilent 6120, Agilent, Santa Clara, CA, USA), with a UV-vis diode array detector and a C18 column (Agilent 4 Eclipse Plus C18, 3.5 m, 4.6 mm x 100 mm). Injected samples (100 L) were separated using a gradient of solutions A and B (A: water, 1% formic acid, 1% acetic acid, 1% acetonitrile; solution B: acetonitrile, 1% formic acid, 1% acetic acid, 2% water; gradient 0-100% B) over 30 min, with a flow rate of 0.8 mL min-1 . Full-scan mass spectra were collected in both positive- and negative-ion (m/z=140-1400).
Location: Laboratory experiments conducted at Princeton University.
Morel, F. (2021) Amphi-enterobactins and related siderophore concentrations found in Vibrio harveyi supernatants and pellets from laboratory experiments in 2017. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2021-10-01 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.861154.1 [access date]
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