In a microtiter plate the bacterial strains were inoculated in 150 µl of media at starting OD600 = 0.01. The media were LB supplemented with 5 µg/ml of streptonigrin (Sigma), and LB supplemented with 5 µg/ml of streptonigrin and 1 mM of FeCl3. The OD600 was recorded each 10 min.
Streptonigrin
Manufactured by Merck Group
Sourced in United States
Streptonigrin is a laboratory reagent used for various research and scientific applications. It functions as a potent antibiotic and antitumor agent. The core purpose of Streptonigrin is to serve as a tool for scientific investigation and experimentation.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Phospho p53, by Cell Signaling Technology (1 mentions)
Phospho histone h2a x, by Cell Signaling Technology (1 mentions)
α β tubulin, by Cell Signaling Technology (1 mentions)
Sumo2 3, by Thermo Fisher Scientific (1 mentions)
Sumo1, by Cell Signaling Technology (1 mentions)
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Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cl amidine, by Merck Group (1 mentions)
Ammonium iron 2 sulfate, by Merck Group (1 mentions)
Chelex 100, by Merck Group (1 mentions)
Paraquat methyl viologen, by Merck Group (1 mentions)
Fluostar optima plate reader, by BMG Labtech (1 mentions)
Flat bottom 96 well plate, by Greiner (1 mentions)
11 protocols using streptonigrin
1
Bacterial Growth in Streptonigrin Media
2
Intracellular Iron Quantification via Streptonigrin Assay
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For indirect quantification of the intracellular iron level, we performed a Streptonigrin sensitivity assay as described previously [34 (link)]. Briefly, R. anatipestifer CH-1pLMF03, R. anatipestifer CH-1ΔfurpLMF03 and R. anatipestifer CH-1ΔfurpLMF03::fur were grown to OD600 = 1.0 in GCB medium, GCB medium supplemented with 100 μM EDDHA, and GCB medium supplemented with 100 μM EDDHA and 200 μM Fe(NO3)3 at 37 °C in a shaking incubator. Cells were harvested by centrifugation at 6000 rpm for 10 min, and pellets were diluted with fresh PBS up to OD600 = 0.5 and aliquoted at 1 mL/tube. Streptonigrin (Sigma-Aldrich, St. Louis, USA) was diluted to 1 µg/mL with sterile PBS, 0 µL, 50 µL, and 80 µL was added to each tube of bacterial solution, the final concentration of Streptonigrin was 0 ng/mL, 50 ng/mL and 80 ng/mL, respectively. Then the samples were incubated in the static incubator at 37 °C for 30 min. After incubation, the bacterial solution was diluted and spread onto GCB plates for counting (T0, T50, and T80). After a 1-day incubation at 37 °C, the grown colonies were counted. The survival rate was calculated as (T50/T0) × 100% and (T80/T0) × 100%, and the experiments were performed in triplicate.
3
Trx2-Mediated Oxidative Stress Response
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Ginkgolic Acid [(GA,15:1) - CAS 22910-60-7, 345887] was purchased from Millipore. N-ethylmaleimide (NEM, SENPs inhibitor, E1271), streptonigrin (SN, SENPs inhibitor, S1014) were bought from Sigma. Protein A/G PLUS-Agarose (sc-2003) was obtained from Santa Cruz.
Antibodies for immunoblotting, immunoprecipitation, and immunostaining were: V5 (Cell Signaling Technology, 13202, Rabbit, WB 1:1000), Trx2 (Abcam, ab185544, Rabbit, WB, 1:10000), Trx2 (Santa Cruz, sc-133201, Mouse, IF, 1:50), Phospho-p53 (Cell Signaling Technology, 9284, Rabbit, WB 1:500), P21 (Cell Signaling Technology, 2947, Rabbit, WB 1:1000), Phospho-Histone H2A.X (Cell Signaling Technology, 9718, Rabbit, WB 1:200), β-Actin (Cell Signaling Technology, 4970, Rabbit, WB 1:1000), α/β-Tubulin (Cell Signaling Technology, 2148, Rabbit, WB 1:1000), TFAM (Cell Signaling Technology, 8076, Rabbit, IF 1:100), PMPCA (Santa Cruz, sc-390471, mouse, IP 1:50 WB 1:500), SUMO1 (Cell Signaling Technology, 4930, Rabbit, WB 1:200), SUMO2/3 (life technologies, 519100, Rabbit, 1:600), Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202, IF 1:200), Donkey anti-Rabbit IgG (H + L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, A-21207, IF 1:200), normal mouse IgG (Santa Cruz, sc-2025, IP 1:100).
Antibodies for immunoblotting, immunoprecipitation, and immunostaining were: V5 (Cell Signaling Technology, 13202, Rabbit, WB 1:1000), Trx2 (Abcam, ab185544, Rabbit, WB, 1:10000), Trx2 (Santa Cruz, sc-133201, Mouse, IF, 1:50), Phospho-p53 (Cell Signaling Technology, 9284, Rabbit, WB 1:500), P21 (Cell Signaling Technology, 2947, Rabbit, WB 1:1000), Phospho-Histone H2A.X (Cell Signaling Technology, 9718, Rabbit, WB 1:200), β-Actin (Cell Signaling Technology, 4970, Rabbit, WB 1:1000), α/β-Tubulin (Cell Signaling Technology, 2148, Rabbit, WB 1:1000), TFAM (Cell Signaling Technology, 8076, Rabbit, IF 1:100), PMPCA (Santa Cruz, sc-390471, mouse, IP 1:50 WB 1:500), SUMO1 (Cell Signaling Technology, 4930, Rabbit, WB 1:200), SUMO2/3 (life technologies, 519100, Rabbit, 1:600), Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202, IF 1:200), Donkey anti-Rabbit IgG (H + L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, A-21207, IF 1:200), normal mouse IgG (Santa Cruz, sc-2025, IP 1:100).
4
Cl-amidine and Streptonigrin Treatment
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Cl-amidine (Sigma-Aldrich, St. Louis, MO, USA) and streptonigrin (Sigma-Aldrich, St. Louis, MO, USA) were administered via intraperitoneal injection once daily on days 1 to 5 in the dose of 10 mg/kg and 0.4 mg/kg, respectively, in 100 μL of phosphate buffered saline (PBS).
5
In Vivo Mouse Eye Injury Model
6
Staphylococcus aureus Antibiotic Susceptibility
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Staphylococcus aureus strains were incubated overnight in tryptic soy broth (TSB) at 37 °C, shaking at 250 rpm. Overnight cultures were adjusted to an OD600 of 0.05 using TSB, and 100 µl of adjusted culture were added to 4 mL of 0.3% molten tryptic soy agar (TSA) maintained at 45 °C. The TSA‐culture was mixture and poured on top of a 1.5% TSA plate and allowed to solidify. The center of the soft agar plates was spotted with 2.5 µL of 1 mg mL‐1 streptonigrin (Sigma–Aldrich). The soft agar plates were incubated at 37 °C for 20 h. The diameter of the zone of inhibition diameter was determined. The assay was performed with five replicates.
7
Streptonigrin Resistance Assay in Riemerella anatipestifer
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The Streptonigrin resistance assay was performed as previously described (28 (link)). Briefly, the tested strains (R. anatipestifer ATCC 11845 pFY02, R. anatipestifer ATCC 11845 ΔfeoAB pFY02, and R. anatipestifer ATCC 11845 ΔfeoAB pFY02::feoAB) were cultured in TSB medium overnight, and then the bacteria were grown in fresh TSB at 37°C and 180 rpm until the exponential phase (OD600, approximately 1 to 1.5). Cells were harvested by centrifugation at 6,000 rpm for 10 min, and pellets were diluted with sterile PBS up to an OD600 of 0.5 in a volume of 1 mL. Streptonigrin (Sigma-Aldrich, St. Louis, USA) was diluted to 1 μg/mL with sterile PBS and added to each tube of bacterial solution at final concentrations of 0 ng/mL, 30 ng/mL, and 50 ng/mL. After incubation at 37°C for 30 min, the bacterial solution was diluted and plated on GCB plates for colony counting (designated T0, T30, and T50). After incubation at 37°C for 24 h, the grown colonies were counted. The survival rate was calculated as (T30/T0) × 100% and (T50/T0) × 100%. Experiments were performed in triplicate using three biological replicates.
8
Intracellular Iron Quantification by Streptonigrin Assay
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For indirect quantification of intracellular level of iron, we performed streptonigrin sensitivity assay in broth culture as described previously [62 (link)]. Briefly, different strains of Xcc were grown to OD600 1.0 in PS medium with respective antibiotics at 28°C and 200 rpm in shaking incubator. Cells were harvested by centrifugation at 4000 g for 6 min and pellets were diluted with fresh PS medium up to OD600 of 0.6. 100 μl of culture was then inoculated in 4 ml PS medium with streptonigrin (Sigma-Aldrich, St. Louis, USA) and sodium citrate and incubated in the static incubator at 28°C. OD600 was measured after 16 and 40 hrs of incubation.
9
Streptonigrin Survival Assays in Pneumococcus
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streptonigrin survival assays were essentially performed as described in ref. [25 (link)]. Iron-depleted medium was prepared by treating THY medium with 2% Chelex-100 (Sigma-Aldrich) overnight at 4°C followed by filter sterilization and addition of 2 mM MgSO4 and 100 μM CaCl2. Pneumococcal cells were grown in iron-depleted medium until the culture reached an OD600 of 0.2, whereupon cells where frozen in 1 ml aliquots at -80°C. Before challenge with streptonigrin, cells were quickly thawed, pelleted at 10,000 × g for 2 minutes and then resuspended in 1 ml of iron-depleted medium supplemented with 50 μM ammonium iron(II) sulfate (Sigma-Aldrich). The treated pneumococcal cells were then incubated at 37°C for 40 minutes prior to challenge with 4 μg/ml streptonigrin (Sigma-Aldirch). Samples were taken at various time-points and CFUs were determined by dilution and plating on TSA + 5% sheep's blood.
10
Streptonigrin sensitivity assay for Xoc
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For sensitivity assay on plate, cultures of Xoc were grown overnight and normalized to an OD600 of 0.6. Serial dilution of cultures were then spotted on PSA containing different concentration of streptonigrin (Sigma Aldrich, St. Louis, USA) in the range of 0.05 μg/ml to 0.15 μg/ml. Plates were then incubated at 28°C for 72 h to observe growth inhibition.
For streptonigrin sensitivity assay in broth culture was performed as described previously (Wilson et al., 1998) . Briefly, different strains of Xoc were grown to OD600 1.0 in PS medium with respective antibiotics. Cells were then pelleted down and resuspended in fresh PS medium at an OD600 of 0.6. 100μl of culture was then inoculated in 4 ml PS medium with or without streptonigrin. streptonigrin was added to a final concentration of 0.1 μg/ml. The dishes were incubated without shaking at 28°C. OD600 was measured after 16 and 42 h of incubation and growth was determined respective to the growth of the corresponding control cultures (PS grown culture without addition of streptonigrin).
For streptonigrin sensitivity assay in broth culture was performed as described previously (Wilson et al., 1998) . Briefly, different strains of Xoc were grown to OD600 1.0 in PS medium with respective antibiotics. Cells were then pelleted down and resuspended in fresh PS medium at an OD600 of 0.6. 100μl of culture was then inoculated in 4 ml PS medium with or without streptonigrin. streptonigrin was added to a final concentration of 0.1 μg/ml. The dishes were incubated without shaking at 28°C. OD600 was measured after 16 and 42 h of incubation and growth was determined respective to the growth of the corresponding control cultures (PS grown culture without addition of streptonigrin).
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