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2 2 dipyridyl dip

Manufactured by Merck Group
Sourced in United States, Germany

2,2′-dipyridyl (DIP) is a chemical compound used in various laboratory applications. It is a heterocyclic organic molecule consisting of two pyridine rings. DIP serves as a chelating agent, capable of forming stable complexes with metal ions.

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11 protocols using 2 2 dipyridyl dip

1

Cultivation and Manipulation of E. coli and R. anatipestifer

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Hemin, δ-aminolevulinic acid (δ-ala) and 2,2’-dipyridyl (Dip) were obtained from Sigma Chemical (Sigma, China). Hemin was dissolved immediately before use in 0.1 N NaOH, Dip was dissolved in ethanol, and δ-ala was dissolved in distilled water. Hemin and δ-ala were filter-sterilized with 0.22-μm pore-size Millipore filters. E. coli strains were grown on LB medium (Sigma-Aldrich, Product Number: L3522) aerobically at 37°C. When required, δ-ala was used at a concentration of 50 μg/ml. Solid media contained 1.5% Difco agar. Iron-depleted medium for E. coli was obtained by the addition of Dip at a final concentration of 150 μM. Antibiotics for E. coli were added to the following final concentrations (μg/ml): Ampicillin (Amp), 100; Kanamycin (Km), 50; Spectinomycin (Spc), 50; Gentamicin (Gen), 20. Arabinose was added at 0.02% for induction of the pBAD promoter. IPTG was added at 0.5 mM for induction of pAM238 expression. R. anatipestifer was grown on LB plates supplemented with 5% defibrinated sheep blood [4 (link)] or TSA plates (Tryptone soy broth, TSB, containing 1.5% agar) [9 (link)] at 37°C under 5% CO2 atmosphere.
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2

Growth Assay of E. coli Strains

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All strains used in this study are derivative of E. coli MG1655 and are listed in Table S1 in the supplemental material. Strains were grown in LB broth (Sigma), containing various concentrations of 2,2′-dipyridyl (DIP) (Sigma) when stated. Marked mutations were moved between strains using classical P1 phage transduction as previously described (38 ). The plasmid library and the plac and pRyhB plasmids used in this study are described in reference 25 (link). Transformations were carried out as described previously (39 (link)). PCR amplifications were carried out using the GoTaq DNA polymerase from Promega.
For the growth assay presented in Fig. 7, overnight cultures of the different strains were diluted (1/1,000) in individual wells of a 96-well microtiter plate in 100 µl LB containing increasing concentrations of DIP. Cells were then grown at 37°C, with agitation, in a Tecan Infinite 200 microtiter plate reader. OD600 was measured every 15 min without removing the plate from the machine, and growth was followed for 14 h.
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3

Evaluating PPAP 23 Efficacy with Iron

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2× MIC PPAP 23 (4.6 μM) was added to a 48-well microplate containing varied concentrations of FeCl3 at 32, 128, and 512 μM. Early exponential S. aureus HG001 was then added to the microplate wells and incubated at 37°C with continuous agitation in BioTekTM microplate spectrophotometer for 24 h. A common iron chelator 2,2′-dipyridyl (DIP; Sigma-Aldrich) was added to the medium at varied final concentrations of 20, 80, and 500 μM to induce iron restriction growth condition. 0.5× MIC (1.15 μM) of PPAP 23 was added simultaneously to early exponential S. aureus in 48-well microplate. The plate was incubated at 37°C with shaking for 24 h in BioTekTM microplate spectrophotometer.
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4

P. gingivalis Growth Conditions

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P. gingivalis wild-type (A7436, ATCC 33277), pgfur mutant (TO6, TO16), complemented mutant (TO6 + pgfur, TO16 + pgfur), and control (TO6 + pTIO-tetQ, TO16 + pTIO-tetQ) strains were grown anaerobically at 37 °C for 3–10 days on blood agar plates (ABA) (Biocorp) as described previously [18 (link), 31 (link)]. These cultures were used as the inoculum for growth in liquid basal medium (BM) comprising 3% trypticase soy broth (Biocorp), containing 0.5% yeast extract (Biocorp), 0.05% cysteine (Sigma), 0.5 mg/l menadione (Fluka), and supplemented with 7.7 μM hemin (Hm) or 0.1–1.0 mM ZnCl2 (Zn). To mimic low-iron/heme conditions, bacteria were grown in BM in the absence of heme and in the presence of an iron chelator, 160 μM 2,2-dipyridyl (DIP) (Sigma) or 0.5 mM EDTA (EDTA) (Sigma). To examine the influence of oxidative stress, bacteria were grown in BM lacking cysteine and supplemented with 0.05 or 0.25 mM H2O2. TO6 and TO16 strains were maintained in the presence of 1 μg/ml erythromycin, whereas complemented mutant (TO6 + pgfur, TO16 + pgfur) and control (TO6 + pTIO-tetQ, TO16 + pTIO-tetQ) strains in the presence of 1 μg/ml erythromycin and 1 μg/ml tetracycline. Bacterial growth in liquid culture media was determined by measuring optical density at 600 nm (OD600) and on ABA plates by visual inspection.
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5

Isolation and Characterization of Stenotrophomonas maltophilia Mutants

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The reference strain Stenotrophomonas maltophilia K279a, which genome is fully sequenced (GenBank: AM743169.1) was used in this study (Crossman et al., 2008 (link)). The spontaneous fur mutant, F60, derivative from the wild-type (wt) strain S. maltophilia K279a, was isolated in this study. Xanthomonas campestris pv. campestris (Xcc) 8004 and Xcc 8523 (rpfF mutant) were used in the DSF bioassay described by Barber et al. (1997 (link)). Strains were kept frozen at −20°C in 15% glycerol. Before use, bacteria were cultured on tryptone soya agar (TSA; Oxoid Ltd, Basingstoke, Hampshire, UK) for 24 h at 35°C. Unless otherwise stated, all cultures were grown in tryptone soya broth (TSB, Oxoid Ltd) in the presence or absence of 200 μM 2,2′-dipyridyl (Dip; Sigma-Aldrich), and incubated for 48 h at 35°C. When required, the cultures were vigorously aerated on a gyratory water bath shaker (Model G75, New Brunswick Scientific Co. Edison NJ, USA) at 200 r.p.m.
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6

Effect of Iron Chelator on E. coli Growth

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The donor E. coli EC302/04, DH5α (pEC302/04) transconjugant and recipient DH5α were grown in LB broth overnight (16–18 h) at 37°C. Cultures were diluted to OD600nm of 0.05–0.1 into LB broth and LB broth supplemented with iron chelator 2'2-dipyridyl (DIP) (Sigma-Aldrich, Steinheim, Germany) (at concentrations of 100, 200, and 300 μM). The growth assay was performed in 96 well plates at 37°C with readings at 600 nm taken every 15 min for 24 h using a Spectramax spectrophotometer (Molecular Devices, USA). Growth kinetics for all 3 strains in each type of the media was determined using the average values of three independent biological and three technical replicates. The growth rates of each replicate for all three strains in different media were determined using GrowthRates (Hall et al., 2014 (link)).
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7

Bacterial Culture Maintenance Protocol

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Bacterial strains utilized in this study are listed in S4 Table. Bacterial cultures were routinely maintained in Luria-Bertani (LB; Lennox) broth (BD Difco) or in N-minimal medium containing 80 mM MES (for pH 5.8) or 100 mM Tris-HCl (for pH 7.0), 5 mM KCl, 7.5 mM (NH4)SO4, 0.5 mM K2SO4, 337 μM K2HPO4/KH2PO4, 20 mM MgCl2, 38 mM glycerol, and 0.1% Casamino acids as indicated and were plated onto LB or xylose lysine deoxycholate (XLD; BD Difco) agar plates. To chelate Fe2+, 2,2'-dipyridyl (Dip; Sigma-Aldrich) was added to LB at final concentration of 0.2 mM. Aerobic cultures were inoculated in 2 ml medium and grown in 15 ml glass tubes with vigorous shaking (250 RPM). Microaerobic cultures were grown by diluting 1:100 over-day aerobic culture into 10 ml medium transferred into 15 ml tubes that were incubated for 16 h without shacking, with the lid loosely screwed in. When appropriate, antibiotics were added to the medium as follows: tetracycline (20 μg/ml), kanamycin (50 μg/ml), ampicillin (100 μg/ml) and chloramphenicol (25 μg /ml).
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8

Iron Depletion Impacts APEC Growth

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Growth of all strains in iron-depleted or supplemented medium was examined as previously described (20 (link)). Avian pathogenic E. coli E516 and its isogenic mutants were cultured overnight in LB broth. Cultures were washed twice and diluted in 10 ml LB, iron-depleted medium [M9 minimum salts, 0.05 mg/ml thiamine, 20% glucose, 0.02 mg/ml L-Tryptopham, 5 mg/ml Casamino acids, 0.1 mM CaCl2, 2 mM MgSO4 and 200 μM 2,2-dipyridyl (DIP) (Sigma, St. Louis, MO, USA)], iron-depleted medium supplemented with 100 μM FeCl3, and the cell density was estimated by spectrophotometry to achieve an approximate starting concentration (OD600 = 0.05). Bacterial growth was measured at 1-h intervals over 12 h by spectrophotometry (OD600). The experiment was performed in triplicate.
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9

Bacterial Growth Under Iron Conditions

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A few single colonies were picked from the agar plates and inoculated into brain–heart infusion (BHI) broth. Followed by overnight incubation, cultures were centrifuged for 5 min at 10,000 rpm. The cell pellets were resuspended in phosphate buffered saline (PBS), centrifuged and washed twice. The cell suspensions in PBS were adjusted to 0.2 with an Eppendorf BioPhotometer Plus (Hamburg, Germany) at an optical density (OD) of 600 nm. One milliliter of a standardized suspension was used to inoculate the media prepared under two conditions. An iron-depleted condition was achieved by adding an iron chelator, 100 µM 2,2′-dipyridyl (DIP) (Sigma Aldrich, Darmstadt, Germany) to BHI broth (BHI-DIP), while the iron-repleted condition was further defined by the addition of 100 µM ferric chloride (Sigma Aldrich) to the BHI-DIP. The tubes were incubated aerobically (37 °C for clinical and 30 °C for environmental isolates) for 48 h on an incubator shaker (Model IKA® KS 4000 i control, IKA® Works (Asia) Sdn Bhd, Selangor, Malaysia) at 200 rpm to ensure stationary phase bacterial growth [21 (link)]. All glassware were treated with 3M HCl followed by extensive washing with deionized water to remove any iron from the labware before proceeding with the experiments [59 (link)].
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10

Staphylococcus lugdunensis Growth Assay

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The sequenced S. lugdunensis N920143 strain was used in this study (GenBank FR870271) [4 ]. Overnight cultures of S. lugdunensis were diluted 2:100 in Brain Heart Infusion (BHI) without or with 350 μM 2,2′-dipyridyl (DIP) (Sigma-Aldrich, Saint Louis, Mo, USA) as previously described [2 (link), 27 (link)]. For complementation experiments, 2 mM FeSO4 were added. To test the oxidative stress response, 0.4 mM H2O2 was added. These bacterial suspensions were used to fill the wells of a 96-wells flat-bottom sterile polystyrene microplates. Growth measurements (OD at 600 nm, every 10 min) were performed using the microtiter plate reader Tecan infinite 200 pro (Tecan, Männedorf, Switzerland) and p-values were determined using a variance Student t-test.
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