Figure 2. In vitro metal competition.

In vitro growth measurements of S. pneumoniae wild-type (D39) and ΔpsaA. (A) Bacteria were grown in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 1000∶1, 250∶1, 100∶1, 50∶1 10∶1, 1∶1, and C+Y with 1 µM MnSO₄, respectively. Data are mean (± SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (B) Bacteria were grown in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 100∶1, 250∶1, 100∶100, 250∶250, and C+Y+1 µM MnSO₄ supplementation, respectively. Data are means (±SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (C) Bacteria were grown in C+Y medium supplemented with 1 µM MnSO₄ until an A ₆₀₀ of 0.3 was reached. Cells were washed in C+Y medium and then inoculated to an A ₆₀₀ of 0.2 in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 100∶1, 300∶1, and 1000∶1. Data are means (±SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (D) In vitro growth measurements of the ΔpsaA mutant. Bacteria were grown in C+Y medium consisting of the following Zn(II):Mn(II) ratios (in µM): 100∶1, 10∶1, and C+Y+1 µM Mn supplementation, respectively. The wild-type S. pneumoniae D39 grown in C+Y+1 µM Mn supplementation is shown for reference. Data are means (±SEM) A ₆₀₀ measurements from seven independent biological experiments (n = 7). (E) S. pneumoniae intracellular Zn(II) accumulation determined by ICPMS. Data are mean (± SEM) ng Zn(II)/g cell measurements from duplicate measurements of 2 independent biological experiments. (F) Intracellular Mn(II) accumulation determined by ICPMS. Data are mean (± SEM) ng Mn(II)/g cell measurements from duplicate measurements 2 independent biological experiments. The statistical significance of the differences in concentrations was determined by a two-tailed unpaired t-test. P-values of <0.05, <0.005 and <0.0005 are denoted by *, ** or ***, respectively.