Mgso4

C. crescentus strains were grown at 30°C in one of several types of media as indicated. The peptone-yeast extract (PYE) medium (0.2% [w/v] peptone (Fisher Bioreagents, Lot No. 225155), 0.1% [w/v] yeast extract (Fisher Bioreagents, Lot No. 220635), 1 mM MgSO₄ (Fisher Chemical, Lot No. 183674), 0.5 mM CaCl₂ (Fisher Chemical, Lot No. 117031)) is best described as a complex medium. In contrast M2X, is a defined medium based on M2 salt medium (6.1 mM Na₂HPO₄ (Aldrich Chemical Company, Inc., Lot No. 12030MN), 3.9 mM KH₂PO₄ (Fisher Bioreagents, Lot No. 141122), 9.3 mM NH₄Cl (Fisher Chemical, Lot No. 156427), 0.25 mM CaCl₂, 0.5 mM MgSO₄, 10 μM Fe•EDTA chelate (Sigma Life Science, Lot No. RNBD1641)) that has 0.15% (w/v) xylose (Acros Organics, Lot No. A0408426) added as a carbon source. The Fe•EDTA chelate in M2X, and occasionally used to supplement PYE, is a 1:1 molar mix of FeSO₄ and EDTA (ferrous sulfate chelate solution kept at 4°C, Sigma-Aldrich, F0518). However, the iron source in M2X was modified in some experiments, or omitted, as indicated. When ferric iron was used, a 100 mM FeCl₃ aqueous stock (pH ~3.0) kept at −20°C was thawed and used to make a working 10 mM stock that was added just before use to the final concentrations indicated. A 10 mM hemin stock solution was prepared in 1 M NaOH and diluted to the indicated final working concentrations. When Fe•EDTA was added to solid medium, it was added after autoclaving. When an iron source (Fe•EDTA, FeCl₃, hemin, desferrichrome (Ustilago sphaerogena; Sigma-Aldrich)) was added to liquid media, it was added to sterile media just before bacterial inoculation. The water used for the preparation of all media was ultra-purified using a Barnsted GenPure water purification system (ThermoFisher). E. coli strains were grown at 37°C in LB medium (1% [w/v] tryptone, 0.5% [w/v] yeast extract, 1% [w/v] NaCl). Growth media were solidified by the addition of 1.5% (w/v) agar when necessary. Antibiotics were used at the following concentrations in liquid and solid media as appropriate: C. crescentus, 5 μg/mL or 25 μg/mL kanamycin, 1 μg/mL or 2 μg/mL chloramphenicol, 1 μg/mL or 2 μg/ml tetracycline, 20 μg/mL nalidixic acid; E. coli, 50 μg/mL kanamycin, 20 μg/mL chloramphenicol, 10 μg/mL tetracycline.
Cells were also grown in environmental water samples from Lake Lansing and Northern Lake Huron (Hammond Bay). These samples were collected in high-density polyethylene analysis bottles provided by the Michigan Department of Environment, Great Lakes, and Energy (EGLE). Bottles were pre-rinsed with lake water from the sample site before sampling. Lake Huron waters were collected on November 10, 2023, in Presque Isle County, Michigan, USA, at 45°31’01.9”N 84°07’09.8”W. Lake Lansing waters were collected on February 14, 2024, from Ingham County, Michigan, USA, at 42°45’19.2”N 84°24’17.2”W. A quantitative elemental profile of these environmental samples was determined as described below.
Standard molecular biology techniques were used to construct all plasmids. Detailed information on strains, plasmids, and primers can be found in Table S6. To create plasmids for in-frame deletion allele replacements, the regions flanking the target gene were cloned into pNPTS138. For the generation of fluorescent transcriptional reporter plasmids, promoter regions (400–500 bp upstream of the open reading frame) were cloned into into the vector pPTM056 (81 (link)). For complementation, the entire coding sequences with their respective promoter regions were cloned, without generating a fluorescent fusion, into pMT603 (pXGFPC5) (21 (link)), which integrates at the xylX locus.
All plasmids were individually introduced into the C. crescentus CB15 strain either by electroporation or by tri-parental mating. For allele replacements, a double recombination strategy was used to select merodiploid strains harboring each relevant pNPTS-based plasmid with kanamycin resistance, followed by sacB counter selection on PYE plates containing 3% (w/v) sucrose. PCR was used to evaluate colonies that were sucrose-resistant and kanamycin-sensitive to identify those colonies harboring the null allele. For the construction of strains with multiple gene deletions for TonB-dependent transporter genes, counterselection was performed on PYE sucrose plates supplemented with 10 μM Fe•EDTA (Sigma-Aldrich, F0518).

All chemicals were purchased in the highest available purity from certified vendors: ¹³C-formate (> 99% ¹³C-enriched) was obtained as sodium formate from Sigma Aldrich, unlabelled sodium formate was obtained from Carl Roth chemicals; both were according to our knowledge not synthesised by electrochemical reduction. 2,5-Aminonitrophenol (2,5-ANP), 2,4-methoxy nitroaniline (2,4-MNA), 2,7-dihydroxynaphtalene (DHN), perchloric acid (70%), and formic acid (LC–MS grade) were purchased from Sigma Aldrich; NaCl, NH₄Cl, MgSO₄, KH₂PO₄, K₂HPO₄, CaCl₂, agarose, LB-agar, LB-medium, d-( +)-glucose, d-( +)-xylose, glycerol, iso-propyl-β-thiogalactopyranosid (IPTG), kanamycin, chloramphenicol, and glycine were purchased from Carl Roth; 2 × Phusion DNA polymerase master mix, acetonitrile (HPLC grade or LC–MS grade) were purchased from Thermo Fisher Scientific. Ethidium bromide was purchased from Merck KGaA, In-Fusion 5 × master mix was purchased from Takara Bio Inc.; high fidelity restriction enzymes, CutSmart buffer, T4-DNA Ligase, 10 × T4-DNA Ligase buffer, and 1 kb Plus DNA ladder were purchased from New England Biolabs; spectinomycin was purchased from VWR International.

Bacterial strains (Table S1, available in the online Supplementary Material) were grown in lysogeny broth (LB; Sigma-Aldrich, UK) or on solid l-agar [LB+1.5 % (w/v) agar (Sigma-Aldrich)] supplemented with relevant antibiotics unless otherwise stated. Bacteria were incubated at 37 °C (with aeration for liquid cultures) unless otherwise stated. Antibiotics (Sigma-Aldrich) were used at the following final concentrations for selection and plasmid maintenance: neomycin 50 µg ml⁻¹, chloramphenicol 30 µg ml⁻¹ and ampicillin 100 µg ml⁻¹. sucrose agar for quantification of pSTAB plasmid loss was made according to the following recipe: 10 g l⁻¹ tryptone (Sigma-Aldrich), 5 g l⁻¹ yeast extract (Fisher Scientific, UK), 10% (w/v) sucrose (Sigma-Aldrich) and 1.5% (w/v) agar. Where required for the growth of auxotrophs, diaminopimelic acid (DAP; Sigma-Aldrich) was used at 0.3 mM final concentration. PBS (Sigma-Aldrich) was used to wash and dilute bacterial cells. Minimal media were prepared from 5× M9 salts (Sigma-Aldrich), supplemented with 1 mM MgSO₄ (Sigma-Aldrich) and either 0.2% (v/v) glycerol (Sigma-Aldrich) or 0.2% (w/v) casamino acids (Fisher Scientific). Yeast extract casamino acids (YESCA) medium containing Congo red was prepared as follows: 10 g l⁻¹ casamino acids (Merck), 1 g l⁻¹ yeast extract and 2% (w/v) agar, supplemented with 0.005% (w/v) Congo red (Sigma) and 0.001% (w/v) Coomassie brilliant blue G (Sigma) after autoclaving.

DMEM/F12 (cat. No. G4610-500ML), DMEM (cat. No. G4511-500ML), L-glutamine (cat. No. G4211-100ML), HCl, Earle’s salts solution (cat. No. G4215-500ML), Hank’s salts solution (cat. No. G4204-500ML), Versene’s solution (cat. No. G4050-100ML), antibiotic/antimycotic mixture (penicillin, streptomycin, amphotericin B, cat. No. G4015-100ML), and trypsin (cat. No. G4012-100ML) were from Servicebio, Wuhan, China. Fetal bovine serum (cat. No. FBS-11A) was from Capricorn Scientific, Wuhan, China.
The cell lines MDA-MB-231 (HTB-26), MCF-10A (CRL-10317), MCF-7 (HTB-22), BT-474 (HTB-20), BT-20 (HTB-19), SK-BR-3 (HTB-30), and DU 145 (HTB-81) were purchased from ATCC, Manassas, VA, USA.
Antibodies anti-b-actin and anti-CB2 were from Abcam, Cambridge, UK. Anti-mouse IgG antibody was from Jackson ImmunoResearch, Cambridge, UK.
SR 144028, PSB CB5, ML-184, ML-193, capsazepine, and LPI was from Tocris Bioscience, Bristol, UK. DMSO, resazurin, D-glucose, glycylglycine, acetic acid, MgSO_4, EGTA, dithiothreitol, acrylamide, bis-acrylamide, Triton X-100, SDS, nitro blue tetrazolium, Tris, EDTA, agarose, bicinchoninic acid, bovine serum albumin, anti-rabbit IgG antibody, diclofenac, N-acetyl cysteine, and 5-Bromo-4-chloro-3-indolyl phosphate-toluoidine were from Sigma-Aldrich, St. Louis, MO, USA. The purity of all the used reagents was 95% or more.

Different commercial membranes were investigated: Fujifilm CEM
type-10 and type-12 (Fujifilm Manufacturing Europe BV, The Netherlands),
Selemion CMTE and CMVN (Asahi Glass Co., Japan), and Fumasep FKS-PET-130
and FKD-PK-75 (Fumatech BWT GmbH, Germany). The bare membrane properties,
e.g., membrane thickness, ionic charge density, and resistance, are
retrieved from our previous studies.^{24 (link),27 (link)} Solutions
were prepared using Milli-Q water (Millipore) and the salt(s) of interest
(reagent grade): Na₂SO₄, KCl, K₂SO₄, MgSO₄, MgCl₂, and CaCl₂ (Sigma-Aldrich). Sodium chloride was purchased from VWR Chemicals.
Poly(4-styrenesulfonic acid) solution (PSS, ∼ 75 kDa, 18 wt
% in H₂O, 1.11 g/mL at 25 °C) and poly(allylamine
hydrochloride) (PAH, ∼ 50 kDa) were purchased from Sigma-Aldrich.