Structome analysis is defined as the quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level. High resolution 3D ultrastructural microscopy of entire cells is required to obtain quantitative insights into the subcellular localization, geometric arrangement and distribution of cellular components – fundamental determinants of cellular function.
This is the first report of Escherichia coli structome analysis with direct measurement of the cell profiles on the serial ultrathin sections. In this report, ribosome number and density are compared between already structome analyzed species and discussed about the correlation with doubling time.
Fig. 1. Serial sections of Escherichia coli. The numbers indicate the sequence relative to the first section. The figure shows 12 complete sections. This cell was found to contain 21,700 ribosomes. (Modified from Yamada et al. Microscopy 66, 283– 294, 2017)
In series of studies by researchers Drs. Masashi Yamaguchi and Hiroyuki Yamada, the structome analysis has been performed in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, Myojin amorphous bacteria, and Mycolicibacterium smegmatis. Information obtained from these structome analysis revealed the presence of ribosome density in the organism’s cytoplasm using transmission electron micrographs of the exquisitely preserved cell structure which were processed through rapid cryofixation and freeze substitution.
Surprisingly, there are only a few studies reporting profile data of E. coli cells. In these studies, cell profiles and E. coli cells ribosome enumeration were measured by phase contrast microscopy, fluorescent microscopy, super-resolution imaging and transmission electron microscope. However, the volumes of the cells were not measured but calculated using formula based on the diameter and the length of the cells and the number of ribosomes were calculated utilizing the quantity of total RNA and transfer RNA in the cells.
Nevertheless, in another study, fluorescent-labeled ribosomes were numbered using super-resolution imaging. Anyhow through these techniques the precise ribosome number of ribosomes in the live E. coli cells were not discussed because live cells and dead cells were not able to be differentiated.
On the other hand, structome analysis can examine whole cell body in serial ultrathin sections by observation with transmission electron microscope. Then, also the profile of the cell outlined by outer membrane or plasma membrane can be observed and the ribosomes as electron dense particles with diameter of some 20 nm (Cover Page) can be visualized. Therefore, the ribosome number in a single live cell and the ribosome density per unit volume of the cytoplasm can be provided. Finally, in structome analysis system by Drs. Yamada and Yamaguchi, it is feasible to compare the ribosome number or density per cell between already examined species.
This is the first report of E. coli structome analysis based on examination of serial ultrathin sections (Fig. 1) and the third report in prokaryotic cells, following M. tuberculosis and Myojin spiral bacteria. In this research article, the profiles of E. coli cells based on the structome analysis obtained from serial ultrathin section examinations are discussed. Based on the comparison of one-dimensional data of E. coli cells obtained from phase contrast microscopy, scanning electron microscopy and structome analysis, it is observed that the diameter and length of E. coli cells obtained from scanning electron microscopy examination were significantly smaller than phase contrast microscopy and structome analysis. Therefore, it is suggested that scanning electron microscopy examination may be inadequate for exact measuring of bacterial cell profiling.
This study revealed that E. coli cells have the highest ribosome density of all microorganisms subjected to structome analysis. Here, authors showed that E. coli cells have average total ribosome number and ribosome density per cytoplasm as 26 100 ± 4020 and 2840 ± 120, respectively (Fig. 2).
Based on the results from the previous and the present structome analysis, it is revealed that ribosome density is quite unique to each species with small deviation whereas according to the volume of the cytoplasm total number of ribosomes varies from cell to cell in the same species. Hence, authors conclude that cytoplasmic ribosome density in the single cell can be used as species-specific unique determinant of microorganisms, giving the information on their growth rate (Fig. 3). Therefore, it is clear that structome analysis is unique and useful approach for study of microorganism.
Fig. 2. Volume of cytoplasm in 9 E. coli cells varies from cell to cell depending on their cell length with constant cell diameter. Likewise, total ribosome number in the cytoplasm varies from cell to cell. However, ribosome density in each cell is extremely constant (2,840 ± 120 / 0.1 fl cytoplasm). The ribosome density can be used as a species-specific parameter. (Modified from Yamada et al. Microscopy 66, 283–294, 2017)
Fig. 3. Correlation curve between ribosome density per 0.1 fl cytoplasm and doubling time (min). Correlation curve was drawn based on known doubling time and ribosome densities per 0.1 fl cytoplasm enumerated in previous structome analysis of E. coli, M. tuberculosis, and S. cereviciae. Number in parenthesis indicates ribosome density per 0.1 fl cytoplasm (left) and doubling time (min) (right). Underlined doubling time (min) in species with unknown doubling time, Myojin spiral bacteria, Myojin amorphous bacteria, E. dermatitidis, and M. smegmatis were calculated based on the formula y = 4998.5e−0.002x. (Reprinted from Yamada et al. Front. Microbiol. 9:1992. doi: 10.3389/fmicb.2018.01992)
This work is partly supported by both a Health Science Research Grant (H24-SHINKO-IPPAN-011) from the Ministry of Health, Labour and Welfare of Japan and Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development, AMED.
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About:
Hiroyuki Yamada, Ph.D.,
Senior Researcher,Department of Mycobacterium Reference and Research, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association.
Dr. Hiroyuki Yamada was born in Chiba prefecture, Japan, in 1959. He graduated from Toho University in 1982. Dr. Yamada was awarded Ph. D from Toho University in 2004, based on his extensive research studies on the cytokine expression patterns in experimental murine tuberculosis.
In 1983, Dr. Yamada started his career with the Department of Anatomy and Pathology, the Research Institute of Tuberculosis, JATA. In his tenure with this institute, he was involved with the studies on the carcinogenicity of diesel exhaust particle, and mineral fibers containing asbestos, with analytical electron microscopy. Later, in 2005, Dr. Yamada moved to the Department of Mycobacterium Reference and Research. Here, he developed artificial sputum, based on cultured human cells, formalin-fixed tubercle bacilli, and polyacrylamide, which can prepare completely negative grade as well as 3+ grade. This was used for training and quality assurance of laboratory microscopists.
Recently, Dr. Yamada has studied and reported Structome Analysis of several bacterial cells such as Mycobacterium tuberculosis (2015), Escherichia coli (2017), and Mycolicbacterium smegmatis (2018), based on Dr. Masashi Yamaguchi’s research studies. On the basis of his extensive studies and analysis, Dr. Yamada has put forward a research proposal about the universal and species-specific close correlation between the growth rate and the ribosome density per unit volume of bacterial cytoplasm.
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