Fig 5.

Association of iron with bacterial HGA-melanin. Wild-type strain 130b (black bars) and lly mutant strain NU408 (bars with horizontal lines) were grown in CDMP supplemented with FeCl₃ for 3 days at 37°C, and then cell-free supernatants were collected and consecutively filtered through 50-, 30-, 10-, and 3-kDa cellulose filters. For each fraction, the amount of HGA-melanin was determined by measuring the OD₄₀₀ of the sample (A), the level of total associated iron was determined by the ferrozine assay in the presence of the reducing agent vitamin C (B), and the amount of associated ferrous iron was ascertained by the ferrozine assay in the absence of vitamin C (C). The data are the means and standard deviations obtained from triplicate samples. In panel A, all wild-type fractions contained more pigment than did the corresponding mutant samples (***, P < 0.001), with the exception of the 30- to 50-kDa fraction, which was devoid of melanin in both cases. In panels B and C, the >50-kDa, 10- to 30-kDa, and 3- to 10-kDa fractions of the wild type contained more iron than did the corresponding fractions of the mutant (***, P < 0.001), whereas the <3-kDa fraction of the mutant had more total iron than that of the wild type (***, P < 0.001).