© 2018 The Japan Mendel Society Cytologia 83(3): 337–342 Electron Microscopy and Structome Analysis of Unique Amorphous Bacteria from the Deep Sea in Japan Masashi Yamaguchi1*§, Hiroyuki Yamada2§, Katsuyuki Uematsu3, Yusuke Horinouchi4 and Hiroji Chibana1 1 Medical Mycology Research Center, Chiba University, 1–8–1 Inohana, Chuo-ku, Chiba 260–8673, Japan 2 Department of Mycobacterium Reference and Research, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo 204–8533, Japan 3 Marine Works Japan, Ltd., 3–54–1 Oppamahigashi, Yokosuka, Kanagawa 237–0063, Japan 4 Marine Biosystems Research Center, Chiba University, Kamogawa, Chiba 299–5502, Japan Received April 24, 2018; accepted May 20, 2018 Summary Structome analysis, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, is a useful tool for identification of unknown microorganisms that cannot be cultured. In 2012, we discovered a unique microorganism with a cell structure intermediate between those of prokaryotes and eukaryotes from the deep sea off the coast of Japan and named it Parakaryon myojinensis. We also reported another unique bacterium found in the same place that we named as Myojin spiral bacteria. Here, we report the third unique bacteria we discovered by structome analysis and 3D reconstruction using serial ultrathin sectioning of freeze-substituted specimens from the same place. The bacteria showed elongated flattened cell bodies with uneven surfaces. The cells consisted of outer amorphous materials, cell wall, cytoplasmic membrane, ribosomes, fibrous materials, and vacuoles. They had a total length of 1.82±0.40 µm, a total volume of 0.37±0.09 µm3, and had 1150±370 ribosomes within a cell; the density of the ribosomes in the cytoplasm was 312±41 per 0.1fL. Each bacterium showed different shapes but appears to belong to a single species because they have similar size and volume, have similar internal structure, inhabit a confined area, and have similar ribosome density in the cy- toplasm. We named it the ‘Myojin amorphous bacteria’ after the location of discovery and its morphology. This is the first report on the existence of amorphous bacteria. Key words 3D reconstruction, Amorphous bacterium, Deep sea, Freeze-substitution fixation, Serial ultrathin sectioning, Structome. In 2012, we discovered a unique microorganism that 3D reconstruction of freeze-substituted specimens. This has cellular structures intermediate between proKaryotes microorganism is unique since individuals of this spe- and euKaryotes from the deep sea off the coast of Japan cies showed different shapes. We named it the ‘Myojin and named it P. myojinensis after the location of discov- amorphous bacteria’ (MAB) after the location of discov- ery and its intermediate morphology (Yamaguchi et al. ery and morphology. 2012). From our observations using ultrathin sections of freeze-substituted specimens with electron microscopy, Materials and methods it became apparent that there are many other strange mi- croorganisms in the deep sea (Yamaguchi and Worman Sample collection, specimen preparation, and elemen- 2014, Yamaguchi 2015). We reported the second unique tary analysis microorganism from the same place and named it Myo- Samples were collected from hydrothermal vents at jin spiral bacteria (MSB) (Yamaguchi et al. 2016). the Myojin Knoll (32°08.0′N, 139°51.0′E) off the coast Here, we report the third unique microorganism from of Japan at a depth of 1240 m in May 2010 (Yamaguchi the same place based on structome analysis [‘structome’ et al. 2012). Small invertebrates, such as Polychaetes, was defined as the ‘quantitative and three-dimensional and their associated microorganisms were collected and structural information of a whole cell at electron micro- fixed with 2.5% glutaraldehyde. They were brought to scopic level’ (Yamaguchi 2006, Yamaguchi et al. 2011a)] the laboratory at Chiba University, snap-frozen, freeze- using serial ultrathin sectioning electron microscopy and substituted (Yamaguchi et al. 2011b, Yamaguchi 2013), and embedded in an epoxy resin. Serial ultrathin sec- * Corresponding author, e-mail: [email protected] tions of 70-nm thicKness were cut, picKed up on slit grids § These authors contributed equally to this worK. (Yamaguchi et al. 2009, Yamaguchi and Chibana 2018), DOI: 10.1508/cytologia.83.337 With supplementary data of figures (Figs. S1–S7) and movies stained with uranyl acetate and lead citrate (Yamaguchi (Movies 1–10). et al. 2005), and observed in a JEM-1400 electron mi- 338 M. Yamaguchi et al. Cytologia 83(3) croscope (JEOL, ToKyo, Japan) at 6000 to 25000 nomi- brane, inner membrane, ribosomes, fibrous materials, nal magnifications. and vacuoles. The outer and inner membranes consisted Elementary analysis was undertaKen on an ultrathin of three leaflets (Fig. 2); the outer leaflet was electron section after staining with uranyl acetate and lead citrate dense, the middle leaflet was electron transparent and at 200 KV using the EDAX EDS system (EDAX Inc., the inner leaflet was electron dense. The outer and inner Mahwah, NJ, U.S.A.) in a Tecnai 20 transmission elec- membrane thicKness measured 12.0±1.1 nm (22 mea- tron microscope (FEI, Hillsboro, OR, U.S.A.). surements) and 11.8±1.6 nm (22 measurements) respec- tively. The space between the outer membrane and inner Structome analysis of cells membrane measured 6.8±1.6 nm (15 measurements). Structome analysis of the amorphous bacteria using serial sections was undertaKen on 10 individuals using Structome analysis of the MAB micrographs of 15 to 35 complete serial sections for Structome analysis of the MAB was performed by each individual. Image analyses were performed using examining complete serial sections of 10 cells (Fig. Fiji (Image J, http://imagej.nih.gov/ij/) software (Kremer 3, S1–S6) and constructing 3D movie files of 10 cells et al. 1996, Schindelin et al. 2012). Briefly, cell length (Movies S1–S10). Table 1 shows the results of struc- was calculated by multiplying the number by 70 nm tome analysis of 70-nm-thick serial sections of Cell 1 to (thicKness of each section). The cross-sectional area of Cell 10. The cell length was 1.82±0.40 µm; the surface each cell was determined using the ‘Measure’ command area was 3.42±0.73 µm2; the volume of the cells was in the ‘Analyze’ menu of ImageJ/Fiji by tracing the cell 0.37±0.09 µm3; they had 1150±370 ribosomes in a cell, wall using the polygonal selection menu in the ImageJ and the density of the ribosomes in the cytoplasm was window and converting the area result above into µm2 312±41 per 0.1fL. by multiplying the square of the ratio of scale (nm) on the image. The surface area (µm2) of each cell was cal- Individual shapes of the MAB culated as the cumulative area of a trapezium of the cell The individual shapes of MAB were analyzed by in each section using the formula for calculating the area constructing 3D movie files of 10 cells from complete of a trapezoid, where the perimeter of the cell walls in a serial sections (Fig. 4, Movies S1–S10). It was found that given section and the previous section were used as the the bacteria showed elongated flattened cell bodies with upper base and lower base, respectively, and the section uneven surfaces, but each individual cell has a unique thicKness (70 nm) was used as the height. The volume shape. Strangely, all the bacteria examined were oriented (f L =µm3) of each cell was calculated as the cumulative in the same direction. volume of cell segments having the cell’s cross-sectional area as the base and the section thicKness (70 nm) as the Discussion height. Three-dimensional reconstruction of each cell was performed with TraKEM2 menu of ImageJ accord- Preservation of cell structure in natural state ing to TraKEM2 tutorials (https://imagej.net/TraKEM2_ It is essential to observe cell structures in a natural tutorials). state at a high magnification to perform structome analy- Ribosomes, recognized as electron dense particles sis of microorganisms. Since conventional chemical with 20 nm diameters in the cytoplasm of the cell cross- fixation often leads to the destruction of cell structures, section in each serial ultrathin section, were enumerated rapid freeze-freeze substitution method should be em- using the ‘Multi-point Tool’ in ImageJ/Fiji (Schindelin ployed for structome analysis (Yamaguchi et al. 2011b). et al. 2012). The total number of ribosomes in each cell Although samples from the deep sea must be fixed with and the number of ribosomes per 0.1 fL of cytoplasm glutaraldehyde aboard the ship (since rapid freezing can- were calculated based on the volume of each cell deter- not be performed aboard the ship), good preservation mined as described above. of cell structure can be obtained by employing rapid freezing for the samples fixed with glutaraldehyde (Ya- Results maguchi et al. 2011b). Thus, the micrographs of MAB in the present study appear to show good ultrastructural The cell structure of the MAB preservation of cell structures appropriate for structome The cell structure was examined by serial ultrathin analysis. sectioning. Figure 1 shows a low magnification image of one of the serial sections. The MAB was found ag- Features of the MAB gregated within a confined space. Each MAB individual Each MAB individual cell was found to have a dif- cell showed a different shape, although their sizes and ferent shape. This is unique since most bacteria form appearances were similar. Figure 2 shows a high magni- spheres, cylinders, or spirals, and show essentially fication image of one ultrathin section of the MAB. They the same morphology if they belong to the same spe- consisted of outer amorphous materials, outer mem- cies. The MAB have outer and inner membranes; each 2018 Amorphous Bacteria from the Deep Sea 339 Fig.