Article Figures & Data
Figures
- FIG. 1.
Electromobility of shed GXM from JEC21 cells changes over time in rich medium. All samples were heated (15 min at 70°C) to denature enzymes, centrifuged (16,000 × g for 3 min) to separate supernatant and cells, and stored at 4°C. Samples (15 μl) of culture supernatants were mixed with 6× DNA loading dye (4), loaded on a 0.6% certified megabase agarose (Bio-Rad) gel, and subjected to electrophoresis (15 h at 25 V) in 0.5× TBE (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA, pH 8.3). Samples containing more GXM (later time points) were diluted in distilled water based on a pilot gel, as sample normalization based on enzyme-linked immunosorbent assay determination of GXM concentration (23) did not yield equal blot intensity. (This may reflect changes over time in GXM antibody reactivity [7, 11, 18] and/or transfer efficiency.) The gel was transferred onto a positively charged nylon membrane and immunoblotted with 1 μg/ml anti-GXM antibody 3C2 as described in the text. Initial sample heating and dilution in distilled water did not alter electrophoretic migration (data not shown). St, starter culture (at 1.6 × 107 cells/ml); arrowhead, well position; arrow, direction of migration; other numbers, sampling time (hours) after the start of the experiment.
- FIG. 2.
GXM shed from cells grown under capsule-inducing conditions displays electrophoretic mobility that is lower than that for cells grown in rich medium. Cultures of JEC21 cells were grown overnight and then washed and diluted to 106 cells/ml for continuous growth as indicated, followed by analysis of GXM as described for Fig. 1 (lanes rotated 90 degrees counterclockwise). Top lane, cells grown in YPD for 75 h, to a final concentration of 4 × 108 cells/ml; bottom lane, cells grown in DMEM (Sigma catalog no. D5796) in the presence of 5% CO2 at 30°C for 14 h, to a final concentration of 1.6 × 106 cells/ml. Note that the growth conditions for the YPD sample, 75 h of continuous growth, correspond to those that yielded the slowest-migrating material in Fig. 1.
- FIG. 3.
Capsule size correlates with the electromobility of shed GXM. Capsule-induced JEC21 cells were washed and resuspended in YPD, and samples were taken at 4, 8, and 24 h after the medium change. (A) Two representative micrographs of cells at each time point, imaged as described in Table 1; scale bar, 10 μm. (B) Capsule thickness of 50 cells was measured as in Table 1, with frequency plotted as a histogram (bin width equals 1 μm with the bin maxima indicated on the abscissa, in decreasing order). (C) Electromobility of shed GXM, analyzed as described for Fig. 1. Ind, original induced sample; 4, 8, and 24 indicate hours after medium change.
Tables
- TABLE 1.
Cell density and capsule thickness in continuous and diluted cultures
Hour Continuous culture Diluted culture Cell densitya Capsule thicknessb Cell density Capsule thickness 11 9.1 × 107 0.80 ± 0.03 9.1 × 107 0.80 ± 0.03 24 1.5 × 108 1.35 ± 0.06 8.6 × 107 0.90 ± 0.02 35 1.8 × 108 1.37 ± 0.07 6.4 × 107 0.82 ± 0.03 48 2.1 × 108 1.60 ± 0.19 1.0 × 108 1.02 ± 0.05 75 4.3 × 108 1.54 ± 0.08 1.7 × 108 0.93 ± 0.02 ↵ a Cell density at the indicated time of growth (cells/ml), determined by counting cells on a hemocytometer.
↵ b Capsule thickness of India-ink-stained cells, measured using an Axioskop 2 fluorescence microscope (Carl Zeiss). The averages ± standard errors of the means (μm) of 50 cells for each time point are tabulated.
