Sampling and analytical procedures:
V. harveyi cells were cultured at 30C and shaken at 200 RPM in 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 ).
For quantification of siderophores ~200 mL of V. harveyi overnight 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).
Amphi- enterobactins and breakdown products in the supernatant and pellets were determined by un-targeted HR- LC-MS/MS, using a C18 column (ACE 3 C18-AR, 1mm x 10cm, MAC MOD) coupled to an LTQ-Orbitrap XL mass spectrometer (ThermoFisher). Injected samples (20 μL) were separated (1 hr) under a gradient of solutions A and B (solution A: water + 0.1% FA + 0.1% acetic acid; solution B: acetonitrile + 0.1% FA + 0.1% acetic acid; gradient 0-100% B, flow rate 70 μL/min). Full-scan mass spectra were acquired in positive-ion mode (m/z = 160-1500) with an experimental resolving power of R=60000 (m/z=400). MS/MS spectra were simultaneously acquired using CID in the Orbitrap targeting the two most abundant species in the full-scan spectrum.
Location: Laboratory experiments conducted at Princeton University.
