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© 2018 The Japan Mendel Society Cytologia 83(3): 337–342

Electron Microscopy and Structome Analysis of Unique Amorphous from the in Japan

Masashi Yamaguchi1*§, Hiroyuki Yamada2§, Katsuyuki Uematsu3, Yusuke Horinouchi4 and Hiroji Chibana1

1 Medical 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 at the electron microscopic level, is a useful tool for identification of unknown that cannot be cultured. In 2012, we discovered a unique with a cell intermediate between those of and 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, , 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 was 312±41 per 0.1fL. Each bacterium showed different shapes but appears to belong to a single because they have similar size and volume, have similar internal structure, inhabit a confined area, and have similar density in the cy- toplasm. We named it the ‘Myojin amorphous bacteria’ after the location of discovery and its . 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 intermediate between prokaryotes microorganism is unique since 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 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 (EDAX Inc., the inner leaflet was electron dense. The outer and inner Mahwah, NJ, U.S.A.) in a Tecnai 20 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 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 . 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 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. 1. Low magnification view of an ultrathin section of the freeze-substituted MAB. There are 17 MAB in this field (arrows). The numbers indicate the cell number and the section numbers of the cell. For example, 1-11 shows the eleventh section of cell 1. Electron-dense inorganic materials (E) were found to contain iron (Fig. S7).

Fig. 2. High magnification of MAB. The bacterium consists of outer amorphous materials (A), outer membrane (OM), inner membrane (IM), ribosomes (R), fibrous materials (F), and vacuoles (V). 340 M. Yamaguchi et al. Cytologia 83(3)

Fig. 3. Complete serial sections of Cell 7. The numbers indicate the section number.

Table 1. Structome of the MAB using 70-nm-thick serial sections.

Number of Cell length Surface area Cell volume Total ribosome Ribosome density Number Cell sections (µm) (µm2) (µm3) number per 0.1 fL cytoplasm

1 15 1.05 2.13 0.21 500 236 2 23 1.61 4.04 0.44 1110 252 3 24 1.68 3.13 0.31 1050 338 4 24 1.68 3.28 0.34 1090 323 5 24 1.68 3.49 0.34 1000 295 6 24 1.68 3.49 0.34 1080 315 7 29 2.03 3.65 0.33 990 304 8 29 2.03 3.88 0.39 1340 345 9 33 2.31 2.50 0.55 1980 361 10 35 2.45 4.64 0.42 1360 348 Average 26 1.82 3.42 0.37 1150 312 SD 5.7 0.4 0.73 0.09 370 41 Minimum 15 1.05 2.13 0.21 500 236 Maximum 35 2.45 4.64 0.55 1980 361 showing a three-layer structure. This double membrane than M. tuberculosis and MSB. E. coli grows rapidly and structure is similar to the cell wall structure of Gram- divides every 20 min. It has 26100 ribosomes in a cell negative bacteria (Madigan et al. 2015). with a density of 2840 ribosomes per 0.1fL on average (Table 2). M. tuberculosis grows slowly and divides only Comparison of the structomes of MAB with those of every 20 h (Yamada et al. 2015). It has 1670 ribosomes , Mycobacterium tuberculosis, and MSB in a cell with a density of 717 ribosomes per 0.1fL on Structome data of the MAB were compared with average (Table 2). The total number of ribosomes in the those of E. coli, M. tuberculosis, and MSB (Tab. 2). The MAB was only 4.4% of E. coli, 68.9% of M. tuberculo- volume of the MAB was 32% of E. coli, 128% of M. sis, and 357% of MSB. The density of the ribosomes was tuberculosis, and 206% of MSB. Thus, the MAB appear 11.0% of E. coli, 43.5% of M. tuberculosis, and 144% to be a small organism compared to E. coli, but bigger of MSB. Since the number of ribosomes (and ribosome 2018 Amorphous Bacteria from the Deep Sea 341

Fig. 4. 3D reconstructions of Cell 1 to Cell 10. a, front view; b, side view. Note each bacterium has a different shape.

Table 2. Comparison of the structomes between the MAB and other bacteria.

Cell volume Ribosome number within the cell Ribosome density Species Reference (µm3, average) (Average) (Number per 0.1fL, average)

E. coli 1.16 26100 2840 Yamada et al. (2017) M. tuberculosis 0.29 1670 717 Yamada et al. (2015) MSB 0.18 322 216 Yamaguchi et al. (2016) MAB 0.37 1150 312 Present study Proportion to E. coli 32% 4.4% 11.0% Proportion to M. tuberculosis 128% 68.9% 43.5% Proportion to MSB 206% 357% 144% density) appears to reflect the growth rate of each - research for establishing a method to analyze ism (Yamada et al. 2017), the growth of the MAB may of the individual microorganisms observed on ultrathin be slower than E. coli and M. tuberculosis. The longer sections. doubling of the deep-sea microorganisms can be expected because they live at low temperature (2–4°C) Do MAB change shapes? and in environments offering low . If the MAB are flexible, they may change shapes with time like mycoplasma (Maniloff and Morowitz 1972). Do the MAB belong to a single species? The mycoplasma has no cell walls and is enclosed by In the world of organisms, individuals belonging to only a single . On the other hand, the the same species exhibit the same shape. This is true not MAB have an external amorphous material, a cell wall, only in and but also microorganisms. In and a cytoplasmic membrane; similar cell surface struc- the present study, individual cells of MAB show differ- ture to Gram-negative bacteria. Although we cannot ent shapes. Do the MAB belong to a single species? We determine if the MAB are rigid or flexible, we think they think they belong to a single species for the following are rigid and does not change shape with time. reasons. 1) Individual cells of MAB show similar size and volume (Table 1). 2) They show similar internal of MAB structure (Figs. 1–3, S1–S6). 3) They inhabit a confined Most rod-shaped bacteria proliferate by binary fis- area. 4) They show similar ribosome density in the cy- sion. They have similar size, shape, and structures along toplasm of the cell (this characteristic strongly suggests the cell . However, the MAB have different sizes that they belong to the same species (Yamada et al. and shapes along the cell body because cross sections 2017). analysis of the individual MAB cells of each bacterium showed different shapes. How do the observed in electron microscopy should be performed to MAB proliferate? It may not be easy to control cell divi- confirm that they have the same genome and to establish sion under such situation. If the MAB are flexible, they their taxonomic position. Our team is now conducting may divide by fission like mycoplasma, showing coccoid 342 M. Yamaguchi et al. Cytologia 83(3) cells connected by a membrane tubule (Maniloff and precise cell profiles and the ribosome density. Microscopy 66: Morowitz 1972). However, we did not observe such im- 283–294. Yamaguchi, M. 2006. Structome of Exophiala cells determined ages in our samples in the present study nor cell images by freeze-substitution and serial ultrathin sectioning electron that appear to be in cell division. The division mecha- microscopy. Curr. Trends Microbiol. 2: 1–12. nism of the MAB remains to be answered in the future. Yamaguchi, M. 2013. Ultrathin sectioning of the Myojin parakaryote discovered in the deep sea. Cytologia 78: 333–334. Acknowledgements Yamaguchi, M. 2015. An electron microscopic study of microorgan- isms: from influenza to deep-sea microorganisms. Myco- toxins 65: 81–99. We thank John and Sumire Eckstein for their criti- Yamaguchi, M. and Chibana, H. 2018. A method for obtaining se- cal reading of the manuscript. We also thank the crew rial ultrathin sections of microorganisms in transmission electron of the research vessel Natsushima and the staff of Japan microscopy. J. Vis. Exp. 131: e56235. 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