2 2 dipyridyl

The wild-type and gene silenced P. plecoglossicida were grown at 28°C in LB overnight and then the OD_600nm of the cultures were adjusted to 0.2. The wild-type and gene silenced P. plecoglossicida were then incubated at 18°C in the presence or absence of 100 μM 2,2′-dipyridyl (Sigma) (n = 3). The values of OD_600nm were measured at 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28 h after the addition of 2,2′-dipyridyl. From the OD_600nm data, growth curves were plotted to compare the wild-type and silenced strains in the presence and absence of 2,2′-dipyridyl.

H. pylori N6 wild type and tlpD mutant were tested for growth defects and behaviour in agar diffusion assays and steady-state tracking assays under iron-limited conditions by adding the iron-chelator 2,2-dipyridyl (Sigma-Aldrich, St. Louis, USA). For agar diffusion assays, bacteria grown on blood agar plates for about 20 h were resuspended in BHI medium to an OD₆₀₀ of 0.07. 100 μl of the bacterial suspension were plated on Blood Agar Base No. 2 plates (Oxoid) supplemented with 5% horse serum. 10 μl of 10 mM, 20 mM and 40 mM 2,2-dipyridyl (dissolved in ethanol) as iron chelator or 10 μl of appropriate dilutions of ethanol in PBS (negative control) were applied to the plates on paper discs (diameter 5 mm). The diameter of the growth inhibition zone was measured upon incubation of bacteria at standard conditions for 48 to 72 h. Tracking assays under iron-limited conditions were performed as described above. After equilibrating the bacteria in liquid medium (see above), 0.5 mM or 5 mM 2,2-dipyridyl was added to the medium or the bacteria were left untreated. Movies were recorded 15 min and 30 min after the addition of 2,2-dipyridyl and steady-state behaviour was evaluated as described above. Steady-state assays with paraquat (10 μM, 100 μM) were carried out according to the same procedure.

Potato tuber extracts were prepared as previously described, with slight modifications (Mattinen et al., 2007 (link)). In summary, five grams of surface sterilized potato tubers (flesh and peel) were ground in 100 ml of distilled water using a blender. Plant tissue was removed by repeated centrifugation at 5000 rpm for 30 min at 4°C. The clarified plant extract was filter sterilized twice using a 0.2 μm filters, and aliquots stored at −16°C. Iron was selectively removed from potato tuber extracts by adding 200 μM of 2,2′-dipyridyl (Sigma Aldrich) dissolved in chloroform in a 1:1 ratio. The resulting mixture was vortexed for 2 min and iron free supernatant collected by centrifugation at 5000 rpm for 10 min at 4°C. The M9 agar plates were then supplemented with a 1:10 dilution of either plant extracts or plant extracts pre-treated with 2,2′-dipyridyl.

E. coli K88 and E. coli K12 were grown overnight in iron-limiting conditions (LB supplemented with 0.2 mM 2,2-Dipyridyl, Sigma-Aldrich) at 37°C with aeration. Approximately 5,000 or 8,000 CFU were inoculated into the supernatant collected from the apical or basolateral side of the Transwell plate 24 h after stimulation with IL-17 and IL-22 or mock control. CFU were enumerated by plating serial dilution 5 h after inoculation. Recombinant human Lcn2 was obtained from R&D Systems and utilized at the final concentration of 0.1, 0.5 and 1.0 µg/mL, respectively.

ABTS (2,2′-azinobis-[3-ethylbenzthiazoline-6-sulfonic acid] diammonium salt), Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), catechin, Folin and Ciocalteu’s phenol reagent, sodium carbonate (Na₂CO₃), gallic acid, vanillin, catechin, sodium nitrite (NaNO₂), aluminum chloride hexahydrate (AlCl₃ × 6 H₂O), DTT (dithiothreitol), NEM (N-ethylmaleimide), 2,2′-dipyridyl, iron chloride hexahydrate (FeCl₃ x 6 H₂O), ascorbic acid, as well as all HPLC-grade solvents were purchased from Sigma–Aldrich (Milan, Italy). High purity standards for the qualitative-quantitative determination of fatty acids (palmitic, stearic, oleic, linoleic, punicic), organic acid (citric, malic, succinic), sugars (glucose and fructose) were purchased from Sigma–Aldrich (Milan, Italy). Anthocyanin (cyaniding 3-glucoside, cyaniding 3,5-diglucoside, delphinidin 3-glucoside, delphinidin 3,5-diglucoside) standards were purchased from Extrasynthase (GenayCedex, France).

G. anatis 12656-12 hemolytic, virulent and F149^T non-hemolytic and non-virulent varieties were grown in brain heart infusion (BHI) medium for 24 h at 37°C under agitation. E. coli DH5α was used to made constructs. E. coli and Pseudomonas aeruginosa PAO1 were cultured in LB-broth to test siderophores production. G. anatis F114 and ESV34 strains hemolytic varieties (Montes-García et al., 2016 (link)) were also included in this study. Several fur-mutant strains were obtained in this work, but the mutant assayed in vivo here was Ω fur 126.13.
To eliminate the iron from the glassware, the material was soaked overnight in 0.5% ethylenediaminetetraacetic acid (EDTA) and then washed with deionized water, as previously described (Abascal et al., 2009 (link)); deionized water was used for the preparation of all solutions and culture media.
To evaluate the effect of iron on protein expression, an overnight culture of G. anatis was diluted 1:100 in flasks containing fresh BHI and incubated at 37°C under shaking until the cultures reached 0.1 optical density units at 600 nm. Next, the specific iron-chelating agent, 2,2′-dipyridyl (Sigma Aldrich, St Louis, MO, United States), was added at 0.25 mM (final concentration), keeping one flask as a control without addition. All assays were done in triplicate at least 3 times.

Arabidopsis Col-0 seeds were surface-sterilized and sown on solid standard medium (1/2 MS, 0.25% sucrose, 1% agar). After vernalization (4 °C, 3d, dark), plates were placed into an incubator (22 °C, 16/8h day/night regime). Twelve-day-old Arabidopsis seedlings were transferred to 12-well-plates containing 1 mL liquid standard medium (1 mL, 5 seedlings per well) and adapted overnight. Stresses were applied by adding 1 volume of 2 µM flg22 or 500 mM NaCl. For wounding, seedlings were punched with serrated forceps. To study prolyl hydroxylation, seedlings were incubated (4, 8, 24 h) in standard medium (1/2 MS) with or without 100 µM 2,2-dipyridyl (Sigma, St. Louis, MO, USA). Plants were shock-frozen and processed for protein extraction and immunoblot analyses.
Transient Agrobacterium-mediated transformation of N. benthamiana and T. majus was performed as previously described [29 (link),30 (link)]. For long-distance movement studies of AZI1, independent, similar-sized N. benthamiana plants (nine-leaf-stage) were used for each AZI1 derivative. Material from the infiltrated leaf (“local”), the adjacent area as well as from the immediately following upper leaf (“distal”) was collected for immunoblot analysis.