Fundamental Cell Morphologies Examined With Cryo-TEM of the Species in the Novel Five Genera Robustly Correlate With New Classification in Family Mycobacteriaceae

Abstract

A recent study proposed the novel classification of the family Mycobacteriaceae based on the genome analysis of core proteins in 150 Mycobacterium species. The results from these analyses supported the existence of five distinct monophyletic groups within the genus Mycobacterium . That is, Mycobacterium has been divided into two novel genera for rapid grower Mycobacteroides and Mycolicibacterium , and into three genera for slow grower Mycolicibacter , Mycolicibacillus , and an emended genus Mycobacterium , which include all the major human pathogens. Here, cryo-TEM examinations of 1,816 cells of 31 species (34 strains) belonging to the five novel genera were performed. The fundamental morphological properties of every single cell, such as cell diameter, cell length, cell perimeter, cell circularity, and aspect ratio were measured and compared between these genera. In 50 comparisons on the five parameters between any two genera, only five comparisons showed “non-significant” differences. That is, there are non-significant differences between slow grower genus Mycolicibacillus and genus Mycobacterium in average cell diameter ( p = 0.15), between rapid grower genus Mycobacteroides and slow grower genus Mycobacterium in average cell length ( p > 0.24), between genus Mycobacteroides and genus Mycobacterium ( p > 0.68) and between genus Mycolicibacter and genus Mycolicibacillus ( p > 0.11) in average cell perimeter, and between genus Mycolicibacterium and genus Mycobacterium in circularity ( p > 0.73). The other 45 comparisons showed significant differences between the genera. Genus Mycobacteroides showed the longest average cell diameter, whereas the genus Mycolicibacter showed the shortest average diameter. Genus Mycolicibacterium showed the most extended average cell length, perimeter, and aspect ratio, whereas the genus Mycolicibacillus showed the shortest average cell length, perimeter, and aspect ratio. Genus Mycolicibacillus showed the highest average cell circularity, whereas genus Mycobacterium showed the lowest average cell circularity. These fundamental morphological data strongly support the new classification in the family Mycobacteriaceae , and this classification is rational and effective in the study of the members of the family Mycobacteriaceae . Because both the genus ...

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Electron microscopy analysis of soft materials with freeze‐fracture techniques

“…TEM is also used for observing the structure of tissues or cellular units in animal organs. A cryogenic TEM (cryo‐TEM) through rapid freezing is mainly used to identify the microstructure of the tissue constituting the surface 15,31,65,66 . However, as a method of obtaining and observing the slices, the analysis of the internal structure is limited.…”

“…Structural analysis of nano‐ and micro‐materials is essential in various material sciences such as polymers, 1–4 energy materials, 5,6 semiconducting materials, 7–9 and biomaterials 10–16 . Depending on the size and properties of the material, sophisticated pretreatment, and observation techniques are essentially required.…”

“…Above all, it is impossible to directly analyze the deep internal structures surrounded by solvents or membrane layers with SEM or TEM. Various studies have been proposed to improve these analytical limitations, such as the cryo‐method 15,19,29–33 and cryo‐ultra‐microtomy, 34 and freeze‐fracture technique 12,29,35–41 …”

“…Structural analysis of nano-and micro-materials is essential in various material sciences such as polymers, [1][2][3][4] energy materials, 5,6 semiconducting materials, [7][8][9] and biomaterials. [10][11][12][13][14][15][16] Depending on the size and properties of the material, sophisticated pretreatment, and observation techniques are essentially required. Scanning electron microscopy (SEM), 2,17,18 transmission electron microscopy (TEM), 11,[19][20][21][22] atomic force microscopy (AFM), [23][24][25] and x-ray diffraction (XRD) [26][27][28] are generally used to analyze structures that have nanometer scales.…”