M8631

The melanin pigments of M. sextelata were analyzed using the HPLC method and compared with standard eumelanin (M8631, Sigma-Aldrich, St. Louis, MO, United States) (Ma et al., 2023 (link)). The pigments and the standard eumelanin were dissolved in a 0.5 M NaOH solution at a final concentration of 50 mg/L. The chromatographic analysis was conducted using an Agilent 1,290 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, United States) with a Waters C18 column (300 mm × 7.8 mm, 5 μm, Milford, MA, United States). The mobile phases consisted of 1% acetic acid (pH 2.8) and methanol in a volume ratio of 97:3, with a flow rate of 0.2 mL/min and an injection volume of 20 μL. Detection was performed at a wavelength of 210 nm, while the column temperature was maintained at 25°C.

For Western blot analysis of secreted ASIP, detached cells and debris were removed from culture medium by centrifugation and 50 μL of the cleared supernatant was electrophoresed on a 9% SDS-polyacrylamide gel under reducing conditions and then analyzed by immunoblot probed with anti-mouse ASIP antiserum (generous gift from Dr. V. J. Hearing, NIH) using ECL chemiluminescence (Pierce). Cell lysates were prepared by washing confluent monolayers with PBS to remove excess serum albumin and lysing in MNT buffer [40 mM 2-(N-morpholino)ethanesulfonic acid (MES), 60 mM Tris, 200 mM NaCl, 2.5 mM EDTA, adjusted to pH 7.4] containing 1% Triton X-100. Proteins from approximately 7×10⁴ cell-equivalents were separated by SDS-PAGE and subjected to immunoblot analysis as described above.
The relative melanin content in cell culture supernatants was measured as described by Watts and colleagues [53 (link)] except that absorbance at 355 nm was used because spectra measured on serial dilutions of purified melanin pigment (Sigma-Aldrich, catalog #M8631) prepared in growth medium showed an absorbance peak at 355 nm which was directly proportional to the melanin concentration.

The melanin-producing ability of strain GR-TSA-9^T was confirmed by growth on TSA medium supplemented with 0 and 10mg/ml of l-tyrosine followed by incubation at 25°C. Production was performed in TSB consisting of 0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/ml l-tyrosine, and after inoculation with 1ml of the cell suspension (OD₆₀₀=1.0) in 100ml TSB, shaking at 25°C and 150rpm for 1–7days, and melanin production in the broth was measured at OD₄₀₀ using the standard synthetic melanin (Sigma-Aldrich, M8631) calibration curve method (Singh et al., 2018 (link)). Melanin was purified as previously described (El-Naggar and El-Ewasy, 2017 (link)). Briefly, the supernatant was centrifuged at 8,000rpm to remove the cells, followed by adjustment of the pH of the supernatant to 2.0 using 6M HCl; the samples were then allowed to stand for 4h and centrifuged at 8,000rpm to collect the precipitate. The melanin pellets were washed with distilled water three times and then centrifuged at 8,000rpm for 10min to obtain melanin. The purified melanin was freeze-dried for further use. In vitro-synthesized pyomelanin was produced by auto-oxidation of 10mM HGA (Sigma-Aldrich, H0751) solution at pH 10 with constant stirring for 3days as described by Schmaler-Ripcke et al. (2009) (link).