Cytochrome c

The quantification of superoxide anion release was obtained following a standard protocol based on the reduction in cytochrome C [51 (link)], and the absorbance in culture supernatants was measured at 550 nm using the spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland). Specifically, 100 μL of cytochrome C (Merck Life Science, Rome, Italy) was added to all the wells, while 100 μL of superoxide dismutase (Merck Life Science, Rome, Italy) and 100 μL of cytochrome C were added to empty wells and the plate was then incubated for 30 min. After that, 100 μL was taken from each well and the absorbance was measured with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) at 550 nm. The O₂ rate was expressed as the mean ± SD (%) of nanomoles per reduced cytochrome C per microgram of protein compared to the control (0 line).

In order to characterize the surface charge of SA113 and its knock-outs, cytochrome c binding assays [66 (link)] were carried out. Bacterial cells were cultured to the exponential growth phase as outlined above. For the cytochrome c binding assays, bacterial cells from exponential growth phase cultures were collected by centrifugation, washed twice with morpholinepropanesulfonic acid (MOPS) buffer (20 mM, pH 7.0), and resuspended in the same buffer to an optical density at 600 nm (OD $${}_{600}$$ ) of 7. The resulting cell suspension were incubated for 10 min at RT with 0.25 mg/mL cytochrome c (Merck, Darmstadt, Germany), the bacterial cells were subsequently removed by centrifugation, and the amount of cytochrome c that remained in the supernatant was quantitated photometrically at 530 nm using a standard curve as the reference.

The determination of CPR (NADPH-cytochrome P450 reductase) activity was performed essentially as previously described [58 (link), 59 (link)]: The CPR activity was spectrophotometrically measured by the rate of reduction of cytochrome c in the presence of NADPH (Sigma Aldrich). 500 μg cytochrome c (Sigma Aldrich) in potassium phosphate buffer (50 mM, pH 7.5) were mixed with 100 μg microsomal protein and filled up with potassium phosphate buffer to 950 μL. The reaction was started by adding 50 μL of fresh aqueous NADPH solution (12 mM). The absorbance change was recorded at 550 nm for 20 seconds using SmartSpec Plus UV/Vis Spectrophotometer (Bio-Rad). The CPR activity was calculated using equation based on Beer Lambert law: ΔOD_550/min/ε, where OD₅₅₀ is the absorbance change measured at 550 nm, ε is extinction coefficient of 21 mM^-1 cm^-1. One enzyme unit is defined as μmol/min.

Cytochrome c reductase activity was determined for reaction mixtures containing Tris-HCl, pH 7.5 (50 mM), cytochrome c (50 μM, equine heart, Sigma) and redox protein (20 nM) as indicated. Reactions were started by the addition of NADPH to a final concentration of 100 μM. The final reaction volume was 1 ml. Cytochrome c reductase activity was determined by measuring the increase in absorbance at 550 nm52 (link). Reactions were carried out in triplicate and the data presented are the average of two independent experiments, with the activity of Fpr set to 100%. To calculate k_cat, a Δε₅₅₀ = 21,100 M⁻¹ cm⁻¹ was used for cytochrome c as provided by the manufacturer (Sigma53 (link)).

The determination of whole-cell surface charges was performed as described previously (Meehl et al., 2007 (link)). After culturing overnight, S. aureus BA01611 was diluted in the pre-warmed MH broth to ensure continued exponential growth during treatment. Cells were treated with and without colistin for 1, 2, and 4 h before harvesting at the mid-exponential phase. After washing twice by centrifugation in 20 mM MOPS buffer, pH 7, the cells were re-suspended in the neutral pH MOPS buffer to a final OD₆₀₀ of 0.6 before adding the positively charged cytochrome c (Sigma) at a concentration of 0.125 mg/mL. The mixture was incubated at room temperature for 10 min before centrifugation and the amount of unbound cationic cytochrome c in the supernatant was quantified photometrically at 530 nm. Each experiment was repeated three times.

DHODH activity and complex III activity were measured by spectrophotometry as previously described 59 (link). DHODH activity was determined spectrophotometrically at 37 °C by monitoring the decrease in absorbance at 600 nm of reduced 2,6-dichlorophenol-indophenol (DCPIP). Briefly, the reaction was initiated with 20 mM dihydroorotate (Sigma) in 1 ml of standard reaction buffer supplemented with 50 μM DCPIP (Sigma), 2 μg of rotenone (Sigma), 2 μg of antimycin A (Sigma), 5 mM NaN₃ (Sigma) and 0.1 mg of whole-cell lysate. The reaction was stopped by the addition of 2 μg leflunomide. Complex III activity was evaluated spectrophotometrically at 37 °C by monitoring the increase in absorbance at 550 nm of cytochrome c. Briefly, the reaction was initiated with 5 mM decylubiquinone (Sigma) to 1 mL of standard reaction buffer supplemented with 2 μg of rotenone (Sigma), 5 mM NaN₃, 60 μM cytochrome c (Sigma) and 0.1 mg of whole-cell lysate. The reaction was stopped by the addition of 2 μg antimycin A (Sigma). One unit was defined as nmol· min ^-1· μg ^-1 of protein. The fold changes were calculated using baseline values of control cells as a reference (set to 1).