Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination

Abstract

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

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

“…Therefore, the average length, perimeter, and aspect ratio of each species may increase, and the average circularity may decrease according to future investigations. Finally, as we have proposed in our previous report, which undertook a structome analysis in MSG (Yamada et al, 2018), MSG should be interpreted as a completely distinct species from MTB. M. bovis BCG strains or MTB auxotroph strains are more appropriate to use as surrogate species or strains, which can be manipulated in the biosafety level 2 facility (Vilcheze et al, 2018).…”

“…The microscope was operated at 300 kV (JEM-3100, JEOL, Tokyo, Japan), 200 kV (JEM-2200FS and JEM-2100Plus, JEOL, Tokyo, Japan) or 120 kV (JEM-1230 and JEM-2100Plus, JEOL, Tokyo, Japan) acceleration voltages, and the samples on the grids were cooled during examination with liquid nitrogen in the single tilt nitrogen cryotransfer holder described above at −175.9 to −177.8 • C. Raw images of the intact cells were recorded at the magnification of ×6,000, ×8,000, or ×10,000 according to the length of a single cell on a 4K × 4K charge-coupling device (CCD) sensor (F415, TVPIS, Germany) with JEM-3100 and JEM-2200FS, on 1K × 1K CCD digital camera system (OSIS MegaView G2, Olympus, Tokyo, Japan) with JEM-1230, or 2K × 2K high sensitivity CMOS camera system (JEOL, Tokyo, Japan) with JEM-2100Plus. Damaged or bent cells and the cells embedded in the thicker ice were not examined because the exact cell morphology could not be obtained (Yamada et al, 2012(Yamada et al, , 2018.…”

“…Our previous studies reported on a structome analysis of MTB and Mycolicibacterium smegmatis (MSG, basonym Mycobacterium smegmatis), which revealed that non-pathogenic MSG is entirely distinct from virulent MTB (Yamada et al, 2015(Yamada et al, , 2018. However, MSG has been often used as an alternative to MTB, but MSG is a distinct species in a genus different from MTB (Yamada et al, 2018). The present study was based on the novel classification and comparison of fundamental cell morphology data obtained from cryo-TEM examination between the five genera.…”

PE_PGRS31-S100A9 Interaction Promotes Mycobacterial Survival in Macrophages Through the Regulation of NF-κB-TNF-α Signaling and Arachidonic Acid Metabolism

“…Ms is often used as an alternative for M. tb in experimental TB, because Ms offers some technical benefits, such as a shorter generation time and negligible risk to laboratory workers compared to M. tb. Several phenotypes, including total cytoplasmic ribosome number, antigenicity, acid-fastness, and the mechanism of drug resistance in Ms are also different to those in M. tb (Yamada et al, 2018). Approximately 30% of M. tb proteins lack conserved orthologs in Ms compared to 3% being absent in BCG (Altaf et al, 2010).…”

“…Ms is a rapidly growing bacterium (Gupta et al, 2018;Yamada et al, 2018;Nikitushkin et al, 2020) that is frequently used as a substitute for M. tb in studies (Doddam et al, 2019;Kaur and Kaur, 2019;Maslov et al, 2019;Wang et al, 2019). The full-length Rv1768 gene (1768 bp) was cloned into the mycobacterial shuttle vector pMV261 and transformed into Ms to construct a recombinant Ms expressing Rv1768 [Ms (pMV-1768)] (Supplementary Figures S1F,G).…”