Histogram of the mass-per-length distribution in MDa/nm for a <i>Spiroplasma</i> cell population.

<p>The Gaussian (r² = 0.97) fit superimposed on the histogram implies a normal distribution. The average mass is 3.74±0.68 (SD) MDa/nm. The mass of an average cell is 22.2 GDa.</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

Cell geometry and measurble parameters.

<p><b>[A]</b> Schematic diagram showing a right-handed, 1.5 turn helical tube with dimensions similar to an average <i>Spiroplasma</i> cell. LM and EM subscripts indicate parameters measured directly by light- and electron microscopy, respectively. one helical repeat (<i>P</i>; vertical black arrow) and corresponding helical centerline, <i>L_C–</i>black line, are marked. The basic tube diameter (<i>d;</i> grey horizontal arrow), tube centerline diameter (<i>D;</i>black horizontal arrow), shortest line, <i>L_S</i>–blue line–and corresponding inner tube diameter (<i>D-d;</i>blue arrow) and outer coiled-tube diameter (<i>D+d;</i>red arrow) are labeled. The center of the cytoskeletal ribbon, <i>L_R</i>, (magenta) follows the geometrically shortest helical line (blue). See <b>Eqs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921.e002" target="_blank">2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921.e027" target="_blank">17</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921.e029" target="_blank">18</a></b> for analytical details <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921-Trachtenberg4" target="_blank">[14]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone-0087921-t001" target="_blank"><b>Table 1</b></a> for actual dimensions. <b>[B] </b><i>Spiroplasma</i> cell compartments and measured STEM parameters are illustrated. The major mass compartment is a membrane tube of diameter d_cell and thickness <i>t_memb</i> (yellow) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone-0087921-g007" target="_blank"><b>Fig. 7</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone-0087921-t001" target="_blank"><b>Table 1</b></a>) to which a seven-fibered cytoskeletal ribbon (red) is attached. Together these form the dynamic cell envelope. The following parameters were determined on freeze-dried preparations: (i) the mass-per-length of whole, intact cells (<i>m_cell</i>); (ii) the mass-per-area of patches of defined areas of empty membrane vesicles (<i>µ_memb</i>); and (iii) the mass-per-length of isolated cytoskeletal ribbons (<i>m_R</i>) and their component single fibrils (<i>m_fibril</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921-Trachtenberg5" target="_blank">[16]</a>. Ribosome counts, <i>n_ribosome</i>, per unit area of thin sections were used to estimate the number of ribosomes per cell (<b>Eqs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921.e033" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone.0087921.e042" target="_blank">24</a></b>).</p

Gene expression.

<p>2D gels of cytoplasm <b>[A]</b>, membranes <b>[B]</b> and whole cells <b>[C]</b>. Gels were aligned and scaled using 20 common spots on gel [<b>A</b>] as a reference. About 600 spots can be counted. The cytoplasm comprises about 25% of the mass. Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087921#pone-0087921-t002" target="_blank"><b>Table 2</b></a><b>,</b> which presents quantitative chemical analysis of unmodified proteins as well as the post-translationally modified glycoproteins and lipoproteins.</p

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

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

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

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