The S(IV) concentrations were determined using the method described by Humphrey et al. using DTNB (5,5 0 -dithiobis-(2-nitro-benzoic acid)) as the colorimetric agent. 13, 19 Fe(II) and total iron concentrations were determined with 1,10-phenanthroline as described in detail previously. 16, 20 ATR-FTIR spectroscopy ATR-FTIR spectroscopy measurements of S(IV) oxidation were taken using a Thermo-Nicolet spectrometer equipped with an MCT/A detector. An evenly coated sample thin lm (FA, g-Fe 2 O 3 , ATD) was deposited onto a Ge crystal element in a horizontal ATR cell (Pike Technologies, Inc.). One mL of Optima water (Fisher) was pipetted onto the thin lm and a background spectrum was collected. An aqueous solution of S(IV) was prepared by dissolving Na 2 SO 3 (Fisher) in Optima water (Fisher) and the pH was adjusted to 5 using dilute HCl. Aer pipetting off the water, 1 mL of the aqueous S(IV) solution (50 mM for FA and g-Fe 2 O 3 , 100 mM for ATD) was added to the surface. A glass slide was placed on the horizontal cell to prevent evaporation. A total of 200 scans were acquired for each spectrum and spectra were collected every 5 minutes for 3 hours.
Optima water
Manufactured by Thermo Fisher Scientific
The Optima water system is a laboratory water purification device that produces high-purity water to support a variety of applications in scientific research and analysis. The core function of the Optima water system is to remove impurities and contaminants from the input water, resulting in the production of purified water that meets the specified quality standards.
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Lab products found in correlation
Diethylene glycol, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
L rhamnose, by Merck Group (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
D mannose, by Merck Group (1 mentions)
D ribose, by Merck Group (1 mentions)
L phenylalanine, by Merck Group (1 mentions)
D galactose, by Merck Group (1 mentions)
Oasis mcx cartridge, by Waters Corporation (1 mentions)
2 deoxy d ribose, by Merck Group (1 mentions)
L asparagine, by Merck Group (1 mentions)
L methionine, by Merck Group (1 mentions)
L valine, by Merck Group (1 mentions)
L tyrosine, by Merck Group (1 mentions)
L proline, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
L ascorbic acid, by Thermo Fisher Scientific (1 mentions)
8 protocols using optima water
1
Spectroscopic Analysis of Sulfite Oxidation
2
Carbohydrate and Amino Acid Analysis
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Tetramethoxysilane (TMOS),
vinyltrimethoxysilane (VTMS), urea, polyethylene glycol (PEG, Mn = 10,000), 4-vinylphenylboronic acid (VPBA),
4-vinylphenylsulfonic acid sodium salt (VPSA), α,α′-azoisobutyronitrile
(AIBN), 1-phenyl-3-methyl-5-pyrazolone (PMP), formic acid, ammonium
acetate, 1-butanol (containing 3 M hydrogen chloride), standards of
monosaccharides [d -ribose (Rib), 2-deoxy-d -ribose
(Deo),l -rhamnose (Rha), d -mannose (Man), d -glucose (Glu), d -galactose (Gal), and d -fructose
(Fru)] and amino acids [l -asparagine (Asp), l -proline
(Pro),l -valine (Val), l -glutamine (Gln), l -tyrosine (Tyr), l -methionine (Met), and l -phenylalanine
(Phe)], and a deuterated standard (d -glu-13C6) were purchased from Sigma-Aldrich (St. Louis,
MO). The other three deuterated standards (l -val-d1, l -val-d8, and d -glu-d2) were purchased
from C/D/N isotopes (Pointe-Claire, Quebec, Canada). Optima water,
methanol, acetic acid, chloroform, diethylene glycol, and acetonitrile
were purchased from Fisher Scientific (Fair Lawn, NJ). Oasis MCX cartridges
(6 cc, 150 mg) were purchased from Waters (Milford, MA). Empty syringe
cartridges (60 mL) were purchased from Agilent Technologies (Santa
Clara, CA). The melamine–formaldehyde sponges (RioRand) were
purchased from Amazon.
vinyltrimethoxysilane (VTMS), urea, polyethylene glycol (PEG, Mn = 10,000), 4-vinylphenylboronic acid (VPBA),
4-vinylphenylsulfonic acid sodium salt (VPSA), α,α′-azoisobutyronitrile
(AIBN), 1-phenyl-3-methyl-5-pyrazolone (PMP), formic acid, ammonium
acetate, 1-butanol (containing 3 M hydrogen chloride), standards of
monosaccharides [
(Deo),
(Fru)] and amino acids [
(Pro),
(Phe)], and a deuterated standard (
MO). The other three deuterated standards (
from C/D/N isotopes (Pointe-Claire, Quebec, Canada). Optima water,
methanol, acetic acid, chloroform, diethylene glycol, and acetonitrile
were purchased from Fisher Scientific (Fair Lawn, NJ). Oasis MCX cartridges
(6 cc, 150 mg) were purchased from Waters (Milford, MA). Empty syringe
cartridges (60 mL) were purchased from Agilent Technologies (Santa
Clara, CA). The melamine–formaldehyde sponges (RioRand) were
purchased from Amazon.
3
Radiolabeling and Formulation of 212Pb-DOTAMTATE
4
Intracellular Iron and Magnesium in M. avium
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M. avium subsp. paratuberculosis strains were grown in triplicate, subjected to different (namely, 0%, 0.1%, and 1.0% [wt/vol]) ferric ammonium citrate (FAC) supplementations, and harvested 48 h after inoculation. Total iron (Fe) and magnesium (Mg) levels were determined by inductively coupled plasma mass spectrometry (ICP-MS; Concordia University, Montreal, Quebec, Canada) as described elsewhere (26 (link)). Briefly, bacterial pellets were washed twice in Optima water (Fisher Scientific), transferred to acid-washed Falcon tubes (Corning), digested with 65% nitric acid at 80°C for 1 h, and diluted with trace-analysis-grade water to a final concentration of 2% nitric acid. All plastic consumables that came in contact with nitric acid were acid washed prior to experiments (27 (link)). The abundance of each metal was quantified using an external standard, and the intracellular Fe:Mg ratio of each sample was calculated. Raw data are provided in Table S2 in the supplemental material.
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5
Proteomic Analysis of Pseudomonas aeruginosa
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The SDS-insoluble pellet was washed three times with Optima water (Fisher Scientific), solubilized in 100 µL of 98% formic acid at room temperature for 1 h, spin-vacuum dried for 1.5 h, washed with 50% methanol and water, and then dissolved in 2% RapiGest (Waters Corp.) with 6 mM DTT. Samples were then sonicated, boiled, and cooled. Cysteines were reduced and alkylated before proteins were digested with trypsin47 (link). Liquid Chromatography mass/spectrometry (120 min runs) was performed with the Synapt G2 (Waters Corp.) in positive resolution/ion mobility mode with proteins identified with ProteinLynx Global Server (PLGS)48 (link). Proteins were also identified and semi-quantitatively measured with a Q Exactive HF (Orbitrap, Thermo Scientific) with Mascot V. 2.549 (link). Identification of the most abundant proteins in these preparations by the Q Exactive HF were confirmed by the qualitative orthogonal method (Synapt G2 ion mobility/PLGS analysis). The reference proteome UP000000653 for P. aeruginosa strain PA14 from UniProt (Release 2015_10, 11/10/2015) was used for all database searches. All raw MS data files are publicly available at the MassIVE data repository (https://massive.ucsd.edu ).
6
Peptide Desalting and Tryptic Digestion
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Peptides were desalted with Sep-Pak C18 1 cc Vac Cartridges (Waters) over a vacuum manifold. Tryptic digests were first acidified by addition of 16.6 μL trifluoroacetic acid (TFA, Acros) to a final concentration of 1% (vol/vol). Cartridges were first conditioned (1 mL 80% ACN, 0.5% TFA) and equilibrated (4 × 1 mL 0.5% TFA) before loading the sample slowly under a diminished vacuum (ca. 1 mL/min). The columns were then washed (4 × 1 mL 0.5% TFA), and peptides were eluted by addition of 1 mL elution buffer (80% ACN, 0.5% TFA). During elution, vacuum cartridges were suspended above 15 mL conical tubes, placed in a swing-bucket rotor (Eppendorf 5910 R), and spun for 3 min at 350 × g. Eluted peptides were transferred from Falcon tubes back into microfuge tubes and dried using a vacuum centrifuge (Eppendorf Vacufuge). Dried peptides were stored at −80 °C until analysis. For analysis, samples were vigorously resuspended in 0.1% FA in Optima water (ThermoFisher) to a final concentration of 0.5 mg mL−1.
7
Amino Acid Sourcing and Preparation
8
Crustacean Physiological Saline and SIFamide
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C. borealis physiological saline was composed of 440 mM NaCl, 26 mM MgCl2, 13 mM CaCl2, 11 mM KCl, 10 mM Trizma base, 5 mM maleic acid (pH 7.4-7.6). Squid internal electrode solution contained 10 mM MgCl2, 400 mM potassium D-gluconic acid, 10 mM HEPES, 15 mM Na2SO4, 20 mM NaCl, pH 7.45 (Hooper et al., 2015) . Gly 1 -SIFamide (GYRKPPFNG-SIFamide, custom peptide synthesis: Genscript) (Blitz et al., 2019; Dickinson et al., 2008; Huybrechts et al., 2003; Yasuda et al., 2004) was prepared by dissolving it in optima water (Thermo Fisher Scientific) at 10 mM, and aliquoting and storing it at -20°C. Aliquots were diluted in physiological saline to a final concentration of 5 µM before use in experiments.
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