Figure Legends
Fig. 1. Onset of iron oxidation is influenced strongly by initial cell density and presence of sulfur. (A) The ferrous iron oxidation (gray, unfilled) and pH (light blue, unfilled) in the absence of c-sulfur, and the effect of adding 10 g/L c-sulfur to AFM1 medium on ferrous iron consumption (black, filled) and pH (blue, filled) is shown. Squares, diamonds, and triangles indicate the three experimental replicates. The initial cell density was OD600 of 0.05. (B) shows the same data with an initial cell density of OD600 of 0.005. The culture highlighted in the red line is shown in (D). (C) Time course data of ferrous iron consumption in AFM1 medium with varying concentrations of iron (72 mM, orange squares; 1 mM, gray triangles; 100 µM blue diamonds; no iron, yellow crosses; no iron and abiotic, green circles) and 10 g/L c-sulfur. The initial cell density was OD600 of 0.001 for biotic cultures. After consumption of the ferrous iron, sulfur oxidation continues until pH 1.3 where ferric iron reduction activity can be measured through an increase in ferrous iron concentration. (D) Images showing the changes in the appearance of c-sulfur and medium over time. The data displayed for (A, B) are all measurements taken from three independent cultures for each condition. Data for (C) represent mean measurements taken from three independent cultures, and the error bars represent the SD. Many error bars are smaller than the size of the symbols.
Fig. 2. The dispersion of sulfur affects bioavailability changes substrate utilization behavior in A. ferrooxidans . (A) Time course data of ferrous iron consumption (black) and pH (blue) in AFM1 medium with 1 g/L c-sulfur (squares) or d-sulfur (diamonds). (B) Time course data showing the effect of d-sulfur concentration on ferrous iron oxidation (black, top) and pH (blue, bottom) in AFM1 medium with 10 g/L (squares), 5 g/L (diamonds), 2.5 g/L (triangles), 2 g/L (crosses), or 1 g/L (circles; same data as diamonds in (A). (C) Time course data demonstrating that growth in AFM1 medium with lig-sulfur displays similar behavior to d-sulfur using a concentration of 10 g/L (squares) or 1 g/L (circles). An initial cell density, OD600 of 0.005, of A. ferrooxidanswas used for all cultures. For (A, B, C), data represent mean measurements taken from three independent cultures, and the error bars represent the SD. Many error bars are smaller than the size of the symbols.
Fig. 3. Dispersed sulfur enables rapid acidification of media to induce ferric iron reduction. Time course data demonstrating ferric iron reduction with 10 g/L d-sulfur in AFM1 medium containing 72 mM ferric iron. The abiotic condition is shown in black and the biotic condition is shown in green with the change in pH (squares) and ferrous iron (circles) shown. The initial cell density was OD600of 0.005 for biotic cultures. Data represent means using measurements taken from three independent cultures, and the error bars represent the SD. Many error bars are smaller than the size of the symbols.
Fig. 4. Selective pressure from kanamycin is maintained during growth on lig-sulfur from a high pH with small amounts of iron. (A, B, C, D) Histograms of GFP fluorescence over three passages (green, AF-GFP control; pale blue, 1st; sky blue, 2nd; blue, 3rd) in various media using an initial cell composition of AF-GFP cells and WT in a 1:100 ratio and initial cell density of OD600 of 0.005. After each passage, cells were diluted hundred-fold into fresh medium for the next passage. (A) Passages in AFM1 medium. (B) Passages in AFM1 medium with kanamycin. (C) Passages in LSM4 medium (initial pH 1.8) with kanamycin. (D) Passages in LSM4 medium (initial pH 5.0) with kanamycin. 250 µg/mL kanamycin sulfate were used for (B, C, D). The data shown represents all gated events.
Fig. 5. Average MFI values for thrice passaged mixtures of WT and AF-GFP against pure cultures of AF-GFP. The MFIs of GFP fluorescence were averaged from the histograms shown in Fig. 4. Data represent means using measurements taken from three histograms, and the error bars represent the SD.
Fig. 6. The population behavior of A. ferrooxidans when provided ferrous iron and/or sulfur for metabolic energy can be described by knowing the pH, percentage of planktonic cells in relation to the total population, and the hydrophobicity of the sulfur. The larger markers correspond to relatively higher activity per cell and the smaller markers correspond to relatively lower activity per cell in each of the given regions. Marker Z is unshaded as no growth was observed with the reported conditions. (A-J) Markers refer to conditions described in this study. (V-Z) Markers refer to conditions estimated from methods and data provided in other studies. (A) AFM1 medium with initial OD600 of 0.05 at inoculation (Fig. 1A). (B) AFM1 medium with initial OD600 of 0.005 at inoculation (Fig. 1B). (C) AFM1 medium and 10 g/L c-sulfur with initial OD600 of 0.05 at inoculation (Fig. 1A). (D) AFM1 medium and 0.75 g/L with cell lysate sulfur with initial OD600of 0.005 at inoculation (Fig. S5). (E) AFM1 medium and 1 g/L d-sulfur or lig-sulfur with OD600 of initial 0.005 at inoculation (Fig. 2B, 2D). (F) AFM1 medium and 10 g/L c-sulfur with initial OD600 of 0.005 at inoculation (Fig. 1B). (G) AFM1 medium and 10 g/L c-sulfur with initial OD600 of 0.05 at late exponential phase (Fig. 1A). (H) AFM1 medium and 10 g/L d-sulfur with initial OD600 of 0.005 at late exponential phase (Fig. 3B). (I) AFM1 medium and 10 g/L c-sulfur with OD600 of 0.005 at late exponential phase (Fig. 1B). (J) LSM4 medium at initial pH 5.0 with initial OD600 of 0.005 at inoculation. (V) 5 mM ferric iron and 5 g/L elemental sulfur initially ((Johnson et al., 2017); from Fig. 1). (W) 5 mM ferric iron and 5 g/L elemental sulfur at late exponential phase ((Johnson et al., 2017); from Fig. 1). (X) 9K-Fe+S medium (10 g/L elemental sulfur) with cells domesticated in 9K-Fe medium at inoculation ((Zhang, Yang, Liu, & Qiu, 2013); from Fig. 5). (Y) Fe(II) + S0 medium (10 g/L elemental sulfur) sub-cultured in the presence of iron and sulfur at inoculation ((Ponce et al., 2012); from Fig. 2). (Z) ATCC medium 2039 adjusted to pH 1.0 at inoculation ((Chao et al., 2008); from Fig. 2).