There are only two kinds of organisms on the Earth: prokaryotes and eukaryotes. Eukaryotes are thought to have developed from prokaryotic predecessors; however, the large differences in their cellular structures results in equally large questions of how the process might have occurred. One way to address this question is to find an organism intermediate between prokaryotes and eukaryotes. Dr. Masashi Yamaguchi payed attention to the deep sea to find such organism, because the deep sea exhibits the extreme environmental stability that allows for survival of morphologically stable organisms over long periods of time, such as the coelacanth fish, which has been surviving with little morphological change for 400 million years in the deep sea.
In 2010, Dr. Yamaguchi and his research team left Yokosuka harbor on the vessel Natsushima heading to the Myojin Knoll, which is located about 100 km south of Hachijo Island off the coast of Japan. Samples were collected from hydrothermal vents at a depth of 1200 m using a remotely operated vehicle, Hyper-Dolphin (Fig. 1). There was a dense population of larger creatures near the hydrothermal vents (Cover Page). They collected small invertebrates, such as scale worms (Fig. 2a), and their associated microorganisms. Most of the collected creatures were alive when they were lifted onto the deck of the ship. The specimens were fixed with 2.5% glutaraldehyde in sea water, kept on ice, and transported to the laboratory in Chiba University.
Fig. 1. The remotely operated vehicle, Hyper-Dolphin (arrow). (Reprinted from Yamaguchi and Worman, Jpn J Protozool, 47, 29-48, 2014)
Fig. 2. a. A scale worm that they collected from deep sea. b. A transverse section of a scale worm chaeta and the many associated microorganisms on the surface of the chaeta. (Reprinted from Yamaguchi and Worman, Jpn J Protozool, 47, 29-48, 2014)
Many microorganisms were found associated with the chaetae of scale worms (Fig. 2b). Although the ultrastructure of microorganisms cannot be observed by light microscopy (Fig. 2b), they were clearly observed by electron microscopy (Fig. 3). Through the initial observations shown in Fig. 3a-c, Dr. Yamaguchi noticed that it is necessary to observe deep-sea microorganisms in three-dimensions to truly understand the whole cell structure. He also noticed that the ultrastructure of microorganisms is heavily damaged by conventional chemical fixation (Fig. 3a-c) and thus there was a need to develop methods for observing intact morphology at high resolutions.
He thought rapid-freeze freeze-substitution even after glutaraldehyde fixation would give a significantly better preservation of the ultrastructure of deep-sea specimens because he previously had good results from freeze-substitution of glutaraldehyde-fixed yeast cells. The chaetae with associated microorganisms were cut from glutaraldehyde-fixed scale worms and sandwiched between two copper discs. They were snap-frozen by being plunged into melting propane kept in liquid nitrogen. The specimens were freeze-substituted in acetone containing 2% osmium tetroxide at –80°C for 2 to 6 days and embedded in epoxy resin (Dr. Yamaguchi refers to this new method as chemical fixation/freeze-substitution method, as opposed the conventional chemical fixation method). Ultrathin sections were stained with uranyl acetate and lead citrate, covered with Super support film, and observed in a JEM-1400 electron microscope.
Figure 3 clearly shows improvement of cell structure preservation of deep-sea microorganisms by this new method. Fig. 3a-c shows electron microscopic images of microorganisms prepared by conventional chemical fixation. They appeared distorted in shape (Fig. 3b), membranes were not smooth (Fig. 3b), and the contents of the cytoplasm seem to be artificially extracted (Fig. 3a-c). Fig. 3d-f shows electron microscopic images of similar microorganisms prepared by chemical fixation/freeze substitution method. They appeared natural in shape (Fig. 3e), membranes were smooth (Fig. 3e), and the contents of the cytoplasm were well preserved (Fig. 3d-f).
Fig. 3. Development of a specimen preparation method for deep-sea microorganisms. a, b, c Ultrathin sections with conventional chemical fixation. Note that membranes are not smooth and cytoplasmic structures appear to be distorted and extracted. d, e, f Ultrathin sections prepared using freeze-substitution after glutaraldehyde fixation. Note that membranes are very smooth, cytoplasm is filled with electron-dense components and vacuoles (d) and the cell (e) are nearly spherical showing natural forms and high-resolution images. C, cytoplasm; CW, cell wall; M, membrane; OM, outer membrane; V, vacuole (Reprinted from Yamaguchi et al., J Electron Microsc, 60, 283-287, 2011).
With this new method of fixation and serial ultrathin sectioning, structome analysis was undertaken on several microorganisms from the deep sea that led to the discovery of new microorganisms. Parakaryon myojinensis was the first unusual microorganism that showed cellular structure intermediate between those of prokaryotes and eukaryotes, and will be discussed in detail in next section.
The Myojin spiral bacteria was the second unique microorganism found from the same place by high-voltage electron microscopy tomography and structome analysis using serial ultrathin sectioning of freeze-substituted specimens. (Fig. 4). The bacteria had a total length of 1.77 ± 0.48 µm and a total diameter of 0.45 ± 0.05 µm, and showed either clockwise or anti clockwise spiral. The cells had a cell surface membrane, thick fibrous layer, ribosomes and inner fibrous structures. They had no flagella. The bacteria had 322 ± 119 ribosomes per cell. This ribosome number is only 1.2% of that of Escherichia coli and may reflect a very slow growth rate of this organism in the deep sea.
Fig. 4. 3D images of Myojin spiral bacteria observed by high-voltage electron microscopy tomography. a, b one bacterium; c another bacterium.
The Myojin amorphous bacteria was the third unique microorganism discovered from the same place by structome analysis and 3D reconstruction. 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 1,150 ± 370 ribosomes within a cell; the density of the ribosomes in the cytoplasm was 312 ± 41/0.1μm3. 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 cytoplasm. The bacteria are unique because each individual cell has a different shape, since most bacteria form spheres, cylinders, or spirals and show essentially the same morphology if they belong to the same species.
There are a few studies of newly discovered deep sea microorganisms that were characterized morphologically, genetically and/or biochemically after being cultured in the lab. However, culturing practices are always biased towards certain types of microorganisms with particular tolerances. Considering the fact that standard methods fail to successfully culture most (99 %) microbes, most organisms are overlooked by these methods. Their strategy of direct observation of individual microorganisms is time- and labor-intensive but has the advantage of sampling deep-sea microorganisms without bias toward organisms able to thrive in particular culturing conditions.
The deep sea is an extremely stable environment in which there might be very little selective pressure for change and low levels of competition, leading to still surviving ‘living fossils’ that may retain features long absent from more typical lineages in more ‘normal’ environments. The deep sea is ‘a museum of living ancient microorganisms’.
For more information, read articles:
- Yamaguchi M: An electron microscopic study of microorganisms: from influenza virus to deep-sea microorganisms. JSM Mycotoxins 65: 81-99, 2015.
- Yamaguchi M, Namiki Y, Okada H, Uematsu K, Tame A, Maruyama T, Kozuka Y: Improved preservation of fine structure of deep-sea microorganisms by freeze-substitution after glutaraldehyde fixation. J Electron Microsc. 60: 283-287, 2011.
- Yamaguchi M, Yamada H, Higuchi K, Yamamoto Y, Arai S, Murata K, Mori Y, Furukawa H, Uddin MS, Chibana H: High-voltage electron microscopy tomography and structome analysis of unique spiral bacteria from the deep sea. Microscopy 65: 363-369, 2016.
- Yamaguchi M, Yamada H, Uematsu K, Horinouchi Y, Chibana H: Electron microscopy and structome analysis of unique amorphous bacteria from the deep sea in Japan. Cytologia 83: 337-342, 2018.