Structome Analysis of Virulent <i>Mycobacterium tuberculosis</i>, Which Survives with Only 700 Ribosomes per 0.1 fl of Cytoplasm

<div><p>We previously reported the exquisite preservation of the ultrastructures of virulent <i>Mycobacterium tuberculosis</i> cells processed through cryofixation and rapid freeze substitution. Here, we report the “structome” analysis (i.e., the quantitative three-dimensional structural analysis of a whole cell at the electron microscopic level) of virulent <i>M. tuberculosis</i> using serial ultrathin sections prepared after cryofixation and rapid freeze substitution and analyzed by transmission electron microscopy. Five <i>M. tuberculosis</i> cells, which were contained in the serial ultrathin cross sections encompassing from one end to the other, were cut into 24, 36, 69, 55, and 63 serial ultrathin sections, respectively. On average, the cells were 2.71 ± 1.05 μm in length, and the average diameter of the cell was 0.345 ± 0.029 μm. The outer membrane and plasma membrane surface areas were 3.04 ± 1.33 μm² and 2.67 ± 1.19 μm², respectively. The cell, outer membrane, periplasm, plasma membrane, and cytoplasm volumes were 0.293 ± 0.113 fl (= μm³), 0.006 ± 0.003 fl, 0.060 ± 0.021 fl, 0.019 ± 0.008 fl, and 0.210 ± 0.091 fl, respectively. The average total ribosome number was 1,672 ± 568, and the ribosome density was 716.5 ± 171.4/0.1 fl. This is the first report of a structome analysis of <i>M. tuberculosis</i> cells prepared as serial ultrathin sections following cryofixation and rapid freeze substitution and examined by transmission electron microscopy. These data are based on the direct measurement and enumeration of exquisitely preserved single-cell structures in transmission electron microscopy images rather than calculations or assumptions from indirect biochemical or molecular biological data. In addition, these data may explain the slow growth of <i>M. tuberculosis</i> and enhance understanding of the structural properties related to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance, particularly in regard to the ratio of target to drug concentrations.</p></div

Three-dimensional profiles of <i>M</i>. <i>tuberculosis</i> cells.

<p>Three-dimensional profiles of <i>M</i>. <i>tuberculosis</i> cells.</p

One- and two-dimensional properties of five <i>M</i>. <i>tuberculosis</i> cells obtained from serial ultrathin section TEM examination.

<p>The size of each schema correlates with the cell’s relative width and length. The diameter, length, aspect ratio, outer membrane (OM) surface area, and plasma membrane (PM) surface area of each of five cells are indicated.</p

TEM images of five cross-sectioned <i>M</i>. <i>tuberculosis</i> cells.

<p>Cells were cut in the middle. The outer membrane (OM), periplasm (asterisk), plasma membrane (PM), and ribosomes (R) are visible as shown in the bottom right panel (enlarged image of cell 3). The cytoplasm of cell 2 appeared to have degraded, as evidenced by its dark color and fewer ribosomes. Cell 3 can be seen to the upper left of cell 2. Bar: 100 nm.</p

One-dimensional profiles of <i>M</i>. <i>tuberculosis</i> cells.

<p>One-dimensional profiles of <i>M</i>. <i>tuberculosis</i> cells.</p

Pie graphs showing the volumes of the cell compartments for each of the five <i>M</i>. <i>tuberculosis</i> cells examined.

<p>The size of each pie correlates with the cell’s relative volume. The whole-cell volume, proportion of the volumes of the cell compartments, total number of ribosomes, and ribosome density for each of five cells are indicated.</p