Biochemical Analysis of Whole <i>Spiroplasma</i> Cells.

<p>Total dry mass of analyzed cells was 1,022 µg. Individual values are given as absolute weights (in µg) within this total amount, as well as in percent of the total dry mass. <i>n</i> = 3 analyses per sample.</p>1<p>Total protein, lipid, and carbohydrate content are the sum of all subtypes.</p>2<p>Unconjugated protein is defined as pure protein or peptide free of detectable carbohydrate or lipid; it is derived by subtracting the sum of lipoprotein and glycoprotein from total protein.</p>3<p>Free lipid is defined as lipids with no detectable carbohydrate or protein; it is derived by subtracting the sum of lipid subtypes from total lipid.</p>4<p>Free carbohydrate is defined as carbohydrates with no detectable lipid or protein.</p

Geometric Parameters of <i>Spiroplasma</i> Cells.

<p>Geometric parameters of <i>Spiroplasma</i> cells were measured directly from high-intensity, dark-field light microscopy and cryoelectron microscopy. Using the helical symmetry of the cell, parameters were extrapolated to entire cells. STEM mass data, obtained per unit length or area, were similarly extrapolated to whole cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone-0087921-g001" target="_blank"><b>Fig. 1</b></a> illustrates diagramatically several of these parameters.</p

STEM dark field image of freeze-dried, intact <i>Spiroplasma</i> cells.

<p>A thick carbon lace (C) supports a thin (∼3 nm) carbon film. Cells (S) and TMV particles (T), the latter used for mass calibration, are scattered on the carbon support. The cells are polar and have distinct tapered (left edge) or round ends (not seen). Older cells are known to vesiculate, as indicated by the asterisk. Note the difference in mass, as reflected by differences in image brightness, between the straight, uniform tubular cell segments (dimmer) and the heavier (brighter) inflection points of the collapsed coils. Only the straight, uniform segments were used for mass measurements. Scale bar = 200 nm.</p

Estimating the cell membrane thickness.

<p><b>[A]</b> The membrane of cells freeze-substituted in acetone/uranyl acetate (UA, red) is not contrasted, whereas the cytoskeleton is highly contrasted, so that its interface (red line) with the cytoplasm (gray) is sharply resolved. In cells freeze-substituted in acetone/osmium tetroxide (OS, blue) or acetone/glutaraldehyde (GA, green), the membranes themselves (light blue ring) are highly contrasted. The difference (<i>Δd</i>/2) in projected diameters of circular cross-sections (arrows) of differentially stained cells is a good estimate of membrane thickness, <i>t_memb</i>. <b>[B]</b> Histograms of diameters of circular cross-sections of cells freeze-substituted in uranyl acetate (UA, red), osmium tetroxide (OS, blue) and glutaraldehyde (GA, green) are shown. The average diameters of the GA and OS specimens are very similar – 504±53 and 511±63 pixels (SD), respectively – whereas that of UA – 403±40 – is smaller. The difference (<i>Δd</i>) indicated by the double-headed arrow implies that the membrane thickness, <i>t_memb</i>, is ∼ 0.2<i>d</i>/2. The pixel size is 0.39 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone-0087921-g001" target="_blank"><b>Fig. 1B.</b></a></p

STEM, dark-field image of collapsed <i>Spiroplasma</i> vesicles on a thin carbon film.

<p><b>[A]</b> Completely collapsed, flat vesicles are marked (V). Only uniform areas of these vesicles were used for mass-per-area measurements. Many small, tightly aggregated vesicles are scattered on the carbon film. TMV particles (T) were used as mass standards. Scale bar = 100 nm. <b>Inset:</b> Vitrified membrane vesicles and a vitrified cell, suspended over a hole in a carbon film, are shown for comparison with the freeze-dried specimen<b>.</b> Scale bar = 0.4 µm.</p

Hydrodynamic studies of a <i>Spiroplasma</i> cell population.

<p><b>[A]</b> Dynamic light scattering of live <i>Spiroplasma</i> cells in isotonic PBS. Shown is the autocorrelation g⁽²⁾-1 function acquired at 90° (crosses) overlaid with the best-fit single species fit (solid line). The data estimate an average diffusion constant of 6.1×10⁻⁹ cm²/sec. <b>[B]</b> Evolution of concentration profiles across the Spiroplasma sample in PBS at various times after the start of centrifugation at 3,000 rpm. <b>[C]</b> Sedimentation coefficient distributions calculated by least-squares modeling of the concentration distributions in <b>[B]</b> by superposition of Lamm equation solutions of non-diffusing species, <i>ls-g*(s)</i>. Shown are the distributions obtained at <i>Spiroplasma</i> concentrations of 0.015 mg/ml (dash-dotted line), 0.15 mg/ml (solid line), 1.5 mg/ml (dashed line) and buffer (dotted line).</p

A histogram of the mass-per-length distribution for <i>Spiroplasma</i> cytoskeletal ribbons.

<p>A Gaussian fit (r² = 0.932) is superimposed on the histogram suggesting a normal distribution. The average linear mass density is 189±70 kDa/nm (SD).</p

Histogram of the mass-per-area distribution for a collapsed, flat population of <i>Spiroplasma</i> membrane vesicles.

<p>A Gaussian fit (r² = 0.97) is superimposed on the histogram suggesting a normal distribution. The average mass density per unit area of membrane is 4.67±0.70 kDa/nm² (SD).</p