Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

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.

Peaks related to amphi-enterobactins were identified by a combination of their characteristic masses and their absorbance at 310 nm using MassHunter (Agilent).

BCO-DMO Data Manager Processing Notes:
* Imported data table from file "Vharveyi-SiderophoreConcentrations.csv" into the BCO-DMO data system.
* Renamed columns to meet BCO-DMO naming conventions: https://www.bco-dmo.org/page/bco-dmo-data-processing-conventions

NSF abstract:
Organic molecules that bind and transport iron are called siderophores. Because iron is an essential trace element for biological systems and exists at very, very low concentrations in the open ocean, siderophores perform a critical role in capturing iron for cellular function. It is known that marine bacteria can produce two different types of siderophores that either tightly bind iron or only weakly do so, with different ecological consequences. This researcher will leverage an exceptional career on metal-organism interactions to examine the unsolved question of exactly what environmental and biochemical conditions (for example the availability of iron) control bacterial production of various siderophores. Results will generate significant new understanding of a critical chemical oceanographic process, and cap this researcher's groundbreaking discoveries that have built to this project. Funding for this research will also support the advancement of women in science by both providing the highest quality training of a female scientist and providing the opportunity for her to host an oceanography booth at the Princeton Plasma Physics Lab's "Young Women in Science" conference.

This study will use Vibrio harveyi as a model organism to investigate a variety of questions surrounding the marine bacterial production of weak and strong siderophores. To start, the investigation will look into how siderophore production is controlled by varying iron availability and quorum sensing (i.e. a coordinated response correlated to population density and/or certain signaling molecules). This also includes in-depth investigation of the impact of life phase and biochemical changes with growth as they relate to coordinated use of weak and strong siderophores. Using established protocols for genetic manipulation of V. harveyi, the researcher plans to discover how varying combinations of weak and strong siderophores maximize the uptake of iron. The broader biogeochemical implications of this study to the field of chemical oceanography, with regard to the microbial use of, and cellular responses to, many essential micronutrients in the ocean would be to significantly influence understanding of elemental distributions beyond the specific study of iron and siderophore cycling in the ocean.