Figure 1.
(A) Iron tolerance affected by ccc1Δ, ferritin, and Fe2+/Fe3+. Culture of 2.0 OD600 was diluted 102, 103, 104, and 105-fold in water, spotted by 10 ul from left to right for each strain, and grown on indicated plates. Empty, single-copy empty plasmid-harboring strain; Ferritin, single-copy ferritin gene set plasmid-harboring strain; Multi-empty, multi-copy empty plasmid-harboring strain; Multi-ferritin, multi-copy ferritin gene set plasmid-harboring strain. (B) Cell attraction towards magnet. Cell suspensions were placed over magnets (4×4 gray circles) and a picture taken at indicated times. Corresponding video is Video S1. (C) SQUID magnetization curve. Induced specific magnetic moment was measured as a function of applied magnetic field. Lines for the four strains without ferric citrate supplementation (No iron supplement) are almost identical and overlapped; mean ± s.d. (n = 3).
Table 1.
Mass magnetic susceptibility of ferric citrate–supplemented cells.
Figure 2.
Electron micrographs of magnetized cell.
Normally grown cells (A–D) and iron-supplemented cells (E–T) of wild-type (A, E, I, M, and Q), ferritin-expressor (B, F, J, N, and R), ccc1Δ (C, G, K, O, and S), and ccc1Δ ferritin-expressor (D, H, L, P, and T) are shown. (M–P) 3× magnification of (I–L), respectively. Unstained sections are shown in (Q–T). Arrows indicate electron-dense particles. Scale bar, 500 nm.
Figure 3.
EDS elemental analysis for magnetized cell.
Wild type (A) and ccc1Δ (B) are analyzed for counter image of dark-field electron micrograph (EM), iron (Fe), nitrogen (N), phosphorous (P), and oxygen (O). Arrows indicate electron-dense deposits. Two-fold magnifications are shown on the bottom-right. EDS spectra are shown for cytoplasmic region of wild type (C) and ccc1Δ (D) and electro-dense particles of wild type (E) and ccc1Δ (F), respectively. Vertical axis is for counts and horizontal axis is for energy (keV). Note copper peaks are from EM grid. Unassigned peaks are presumably from uranium used for staining. Relative element counts of iron (G) and phosphorous (H) versus nitrogen are calculated for sectioned whole cell region of normally grown wild type, iron-supplemented (+iron) wild type, and ccc1Δ.
Figure 4.
Magnetizable column entrapment.
(A) Wild type cells grown in normal medium (Normal) and in 20 mM ferric citrate (iron supplemented) were applied to magnetizable column, and fractions of flow-through (Flow), wash-out (Wash), and trapped cells released after magnet detachment (Released) were collected and monitored by OD600. (B) Magnetic entrapment efficiency of the four strains. Percentages of trapped cells were calculated; mean ± s.d. (n = 3).
Figure 5.
Effect of TCO89 on magnetization.
(A) Reduced magnetic attraction of tco89Δ. Cell suspensions were adjusted to 0.5 OD600, attached to magnet at half of the bottom, and let stand for 30 min. (B–E) Magnetizable column entrapments were performed as in Figure 3B. (B) TOR pathway gene knockouts. (C) Effect of TCO89 copy number. Strains harboring indicated plasmids were grown in selection medium to keep plasmid. (D) Induction of TCO89 expression under galactose inducible GAL1 promoter. tco89Δ cells harboring indicated plasmid were pre-cultured in raffinose medium containing 10 mM ferric citrate and transferred to raffinose (–) or galactose (+) medium containing 10 mM ferric citrate. (E) Genetic interaction between CCC1 and TCO89. ccc1Δ and ccc1Δtco89Δ double knockouts were grown in synthetic complete medium containing 10 mM ferric citrate.
Figure 6.
Effects of TORC1 activity on magnetization.
(A)Wild type harboring empty plasmid (Wild type), harboring multi-copy TCO89 plasmid (Multi-TCO89), and tco89Δ harboring empty plasmid (tco89Δ) were grown in selection medium containing 10 mM ferric citrate and 10 nM (red column), 20 nM (green column) rapamycin, or 0.2% dimethyl sulfoxide (DMSO) (blue column) as control. (B) Wild type (blue column) and tco89Δ (red column) are pre-cultured in synthetic complete medium and grown in indicated media containing 10 mM ferric citrate. Indications of media are synthetic complete medium containing 2% or 3% glucose (2% Glu, 3%Glu), 2- or 3-fold concentration of amino acid supplements (2×N, 3×N), or rich medium containing 2%, 3%, or 4% glucose (2% Glu, 3%Glu, and 4%Glu), respectively. Magnetization was measured as in Figure 3B.
Figure 7.
(A) Dose-dependent effect of TCO89 on cell redox state. Culture of 2.0 OD600 was diluted 102, 103, 104, and 105-fold in water, spotted by 10 ul from left to right for each strain, and grown on indicated plates for 2 d, and colony staining by methylene blue was observed. Bluer color indicates more oxidized. (B) Cellular NADP measurement. NADP+ (red column) and NADPH (blue column) were extracted from cells grown in liquid medium, measured, normalized by cell number, and plotted; mean ± s.d. (n = 3).