Jpn. J. Protozool. Vol. 47, No. 1, 2. (2014)

29

Review

Deep-sea microorganisms and the origin of the
eukaryotic cell
Masashi YAMAGUCHI1* and Cedric O’Driscoll WORMAN2
1

Medical Mycology Research Center, Chiba University, Chiba 260-8673, Japan
Department of Biology, Francis Marion University, Florence, SC 29502, USA

2

SUMMARY
Living cells are deeply divided into two enormously divergent levels of complexity: prokaryotic
and eukaryotic. Eukaryotes are thought to have developed from prokaryotic predecessors; however thelarge differences in their cellular structures results in equally large questions of how the process might
have occurred. In 2012, in the deep-sea off the coast of Japan, we discovered a unique microorganism
appearing to have cellular features intermediate between prokaryotes and eukaryotes. The organism, the
Myojin parakaryote (tentatively named by Yamaguchi et al., 2012), was two orders of magnitude larger
than a typical bacterium and had a large “nucleoid”, consisting of naked DNA fibers, surrounded by asingle layered “nucleoid membrane”, and bacteria-like “endosymbionts”, but it lacked mitochondria.
This organism exemplifies a potential evolutionary path between prokaryotes and eukaryotes, andstrongly supports the endosymbiotic theory for the origin of mitochondria and the karyogenetic
hypothesis for the origin of the nucleus. In this review, we describe how the Myojin parakaryote wasdiscovered, the features of this organism, the significance of the discovery, and perspectives on future
research.
Key words: Prokaryotes, Parakaryotes, Mitochondria, Nucleus, Symbiosis, Electron microscopy, Freezesubstitution, Serial ultrathin sectioning, 3D reconstruction, Structome

INTRODUCTION
There are only two known ways of being aliving cell: the prokaryotic and the eukaryotic. Prokaryotes include the Bacteria and Archaea. Prokar*Corresponding author
Tel: +81-43-222-7171/Fax: +81-43-226-2486
E-mail: yama@faculty.chiba-u.jp
Received: 28 February 2014; Accepted: 05 June 2014.

yotic cells are generally only a few micrometers in
size, have simple cellular structures including cytoplasm with a fibrous nucleoid, ribosomes, aplasma membrane, and a cell wall. Eukaryotic
cells are much more complex and include both
single- and multi-cellular organisms; e.g., animals,
plants, fungi, and protists. Both groups,
particularly the eukaryotes, have wide cellular size
ranges, however eukaryotic cells typically have

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Deep-sea microorganisms and evolution

nearly 10,000 times the volume of prokaryotic
cells. Eukaryotic cells also have a nucleus
enclosed by a double membrane and show complex membranous cellular structures: endoplasmic
reticula, Golgi apparatuses, peroxisomes, lysosomes, endosomes, and various sizes and types of
vacuoles. Additionally, eukaryotic cells have either
one or both of two distinct types of organelles that
contain their own DNA: mitochondria and chloroplasts. Eukaryotic cells also have various types ofcytoskeletal structures: centrioles, microtubules
and microfilaments (Fig. 1) (Stanier and van Niel,
1962; Mayr, 1998; de Duve, 1996).
Eukaryotes are thought to have evolved fromprokaryotes, however, until recently, there were no
known examples of intermediate forms between
the vastly different prokaryotic and eukaryotic
levels of organization (de Duve, 1996; Doolittle,
1998). In fact, the differences in cellular structure
between prokaryotes and eukaryotes are soseemingly insurmountable that the problem of how
eukaryotes could have evolved from prokaryotes is
one of the greatest puzzles in biology (Koonin,2010). One way to address this question is to findan organism with intermediate organization andexamine its ultrastructure, DNA, and molecularmachinery in detail. The deep-sea is one of themost likely environments to find such an organism
because it exhibits the extreme environmental
stability that allows for the 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.
There are two major hypotheses regarding
the origin of eukaryotes (Dodson, 1979; Doolittle,1980). In the endosymbiotic theory, a larger,
amoeboid, heterotrophic, anaerobic prokaryote
engulfed smaller aerobic prokaryotes, some ofwhich stabilized as endosymbionts and became
integrated into the host cell as mitochondria rather
than be digested directly as a food source(Margulis, 1970; Whatley et al., 1979; Corsaro etFig. 1. Schematic diagrams of a prokaryotic cell (colon
bacillus, a) and a eukaryotic cell (rat pancreas, b). C, centrioles; CW, cell wall; G, Golgi apparatus; GG, glycogengranules; L, lysosomes; LD, lipid droplets; M, mitochondria; Mf, microfilaments; Mt, microtubules; N, nucleus;
NM, nuclear membrane; NP, nuclear pores; Nu, nucleolus;
PM, plasma membrane; R, ribosomes; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum;SG, secretion granules (From Yamaguchi (2013) withpermission).

al., 1999). In the autogenesis theory, the structuresand functions of eukaryotic cells developed
gradually from simple precursors in prokaryotic
cells (Raff and Mahler, 1972; Nakamura and Hase,
1990). Significant debate about how eukaryotes
originated continues in the present day (Kutscheraand Niklas, 2005; Zimmer, 2009).
In 2012, in the deep-sea off the coast ofJapan, we found a unique microorganism whichappears to have an intermediate cellular structure
between those of prokaryotes and eukaryotes(Yamaguchi et al., 2012). This organism wasdescribed using freeze-substitution electron
microscopy and structome analysis (Structome isdefined as the “quantitative and three-dimensional
structural information of a whole cell at theelectron microscopic level” (Yamaguchi, 2006;

Jpn. J. Protozool. Vol. 47, No. 1, 2. (2014)31

Yamaguchi et al., 2011a)). The organism wasnamed the “Myojin parakaryote” with thescientific name of Parakaryon myojinensis (“nextto (eu)karyote from Myojin”) after the discovery
location and its intermediate morphology between
the prokaryotes and the eukaryotes (Yamaguchi etal., 2012). In this review, we describe how the
Myojin parakaryote was discovered, the features ofthis organism, the significance of the discovery,
and perspectives on future research.
SAMPLE COLLECTION

Fig. 2. The remotely operated vehicle, Hyper-Dolphin(arrow).

In May 2010, we left Yokosuka harbor on
the research vessel Natsushima heading to the
Myojin Knoll (32°08.0’N, 139°51.0’E), which islocated about 100 km south of Hachijo Island off
the coast of Japan. Samples were collected fromhydrothermal vents at a depth of 1,240 m using a
remotely operated vehicle, Hyper-Dolphin (Fig. 2).
There was dense population of larger creatures

Fig. 3. The rich community of deep-sea organisms at a depth of 1,227.8 m on Myojin Knoll; including mussels (Mu)and crabs (C). Samples were collected with a slurp gun (suction sampler, S) or with a manipulator (M). Sample collection lasted 2 hours for each dive and was recorded on video. This picture was taken on the 15th of May, 2010.
Other information on time (10:01:55AM), dive number (1,126), direction (176.2), height from the sea bottom (0.5 m),
type of main camera (HD), sub-camera (CCD), depth of the machine (1,222.1 m), salinity (34.337 PSU), dissolved
oxygen concentration (1.4 mL/L), and temperature (4.453°C) was recorded. P. myojinensis was collected in this dive.

32

Deep-sea microorganisms and evolution(Fig. 4). The specimens were fixed with 2.5% glutaraldehyde in sea water, kept on ice, andtransported to the laboratory in Chiba University.
ELECTRON MICROSCOPIC OBSERVATION OF MICROORGANISMS BY CONVENTIONAL CHEMICAL FIXATION

Fig. 4. A scale worm (Polynoidae) that we collected fromdeep sea and its many chaetae.

(e.g., crabs and mussels) near the hydrothermal
vents (Fig. 3). We collected small invertebrates,
such as Polychaetes, and their associated microorganisms. Most of the collected creatures werealive when they were lifted onto the deck of theship. The animals were photographed directly or
under a stereomicroscope to record whole morphological features and aid in species identification

The glutaraldehyde-fixed samples were postfixed with 1% osmium tetroxide, dehydrated in a
graded series of ethanol, and embedded in epoxy
resin. Ultrathin sections were cut to a thickness of
70 nm, stained with uranyl acetate and lead citrate,
covered with Super Support Film (Nisshin EM,
Tokyo, Japan), and observed in a JEM-1400
electron microscope (JEOL, Tokyo, Japan) at 100
kV.
Many microorganisms were found associatedwith the chaetae of scale worms (Polynoidae) (Fig.
5). Although the ultrastructure of microorganisms
cannot be observed by light microscopy, they were

Fig. 5. A transverse section of a scale worm chaeta and the many associated microorganisms on the surface of the chaeta.

Jpn. J. Protozool. Vol. 47, No. 1, 2. (2014)33

Fig. 8. An example of unusual lens-shaped deep-sea microorganisms with internal cell wall-like gull-shaped
structures. Note the structure of the cytoplasm was heavily
damaged by the conventional specimen preparation(arrow).
Fig. 6. An example of unusual deep-sea microorganisms
from Myojin Knoll with an internal cell wall-like gullshaped structure. There were 4 individuals side by side
(only 3 are shown here), suggesting division by binary
fission.

Fig. 7. An example of unusual deep-sea microorganisms
from Myojin Knoll with unique comma-shaped nuclei-likestructure (N). They seem to have no mitochondria.

clearly observed by electron microscopy. Fig. 6
shows spherical microorganisms with cell walllike structures inside the cells. They measure about
2.5 µm in diameter and are postulated to divide bybinary fission because there were 4 individuals
side by side. If the electron-lucent gull-shaped
structures are indeed internal cell walls, these bacteria are the first observed with such a unique morphology. Cell walls on known bacterial cells arealways external to the cell membrane, thus thisconfiguration must have divergent and unknown
advantages. This inner cell wall structure might bean example of the diverse and seemingly bizarre
adaptations that are found in extreme environments, such as the deep sea, but are currently
unknown on the surface of the Earth.
Fig. 7 shows another example of the poorlystudied microbe community in the deep sea. These
spherical microorganisms measure about 1.0 µm in
diameter. Like the organisms in Fig. 6, they may
divide by binary fission since there are 4 individuals side by side. These microbes have uniquecomma-shaped nuclei-like structure and lack mitochondria. Fig. 8 shows yet another example of thetypes of interesting life forms we are finding in the
deep sea. In this case, the microorganism is lensshaped with a gull-shaped cell wall-like structure
inside the cell very similar to that seen in Fig. 6.

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Deep-sea microorganisms and evolution

Fig. 9. Ultrathin sections of deep-sea microorganisms prepared using conventional chemical fixation (CF method). Samples were fixed with 2.5% glutaraldehyde aboard the ship, transferred to the laboratory, post-fixed with 1% osmium
tetroxide, and embedded in epoxy resin. Note that membranes are not smooth (OM in b) and cytoplasmic structuresappear to be distorted and extracted (a–c). C, cytoplasm; CW, cell wall; D, electron-dense components; L, electronlucent components; M, membrane; OM, outer membrane; V, vacuole (also for Fig. 10) (From Yamaguchi et al. (2011b)with permission).

Through the initial observations shown in
Figs. 6–8, we noticed that it is necessary toobserve deep-sea microorganisms in threedimensions to truly understand the whole cellstructure. We also noticed that the ultrastructure of
microorganisms is heavily damaged by conventional chemical fixation and thus there was a needto develop methods for observing intact morphology at high resolutions (Therefore, the above
descriptions, which are based only on one ultrathin
section of chemically fixed cells, must be considered preliminary until we examine the 3D ultrastructure of the cells with the more precise
methods elucidated below).

Jpn. J. Protozool. Vol. 47, No. 1, 2. (2014)35

Fig. 10. Ultrathin sections of deep-sea microorganisms prepared using freeze-substitution after glutaraldehyde fixation(CF-FS method). Samples were fixed with 2.5% glutaraldehyde aboard the ship, transferred to the laboratory, cryofixedwith melting propane, freeze-substituted in acetone containing 2% osmium tetroxide at −80°C, and embedded in epoxy
resin. Ultrathin sections were cut to a thickness of 70 nm, stained with uranyl acetate and lead citrate, covered with Super
Support Film, and examined in an electron microscope. Note that membranes are very smooth (OM in b, M in c), cytoplasm is filled with electron-dense components and vacuoles (a–c), and the cell (b) and vacuoles (a) are nearly spherical,
showing natural forms and high-resolution images of ultrastructures in deep-sea microorganisms (From Yamaguchi et al.,(2011b) with permission).

DEVELOPMENT OF BETTER SPECIMENPREPARATION METHODSWe knew that rapid-freeze freezesubstitution fixation would preserve the natural
ultrastructure of our deep-sea microorganisms at

high resolution. However, the method cannot be
performed on a research vessel, because the time
for specimen preparation on board is limited andrefrigerants such as propane are too dangerous tobring into the vessel’s laboratory. We thought
rapid-freeze freeze-substitution even after glutar-

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Deep-sea microorganisms and evolution

aldehyde fixation would give a significantly better
preservation of the ultrastructure of deep-sea specimens because we previously had good resultsfrom freeze-substitution of glutaraldehyde-fixed
yeast cells (Yamaguchi et al., 2005).
The chaetae with associated microorganisms
were cut from glutaraldehyde-fixed scale worms,
rinsed with phosphate buffer (pH 7.2), 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 (Yamaguchi et al., 2011b) (We refer tothis new method as CF-FS (chemical fixationfreeze-substitution) method, as opposed the conventional chemical fixation method (CF method)).
Ultrathin sections were stained with uranyl acetate
and lead citrate, covered with Super Support Film,and observed in a JEM-1400 electron microscope
the same as with the conventional method.
Fig. 9a shows bacteria-like microorganisms,
0.3 to 0.5 µm diameter, prepared using CF method.

They appear to consist of a cell wall/outer cell
membrane and cytoplasm. Parts of the cytoplasm
in the center are often devoid of electron-dense
components, have fine filaments, and seem to have
had the contents artificially extracted. Fig. 10ashows bacteria-like microorganisms similar to
those in Fig. 9a, but prepared using CF-FS method.
They also appear to consist of a cell wall/outer cell
membrane and cytoplasm. The cytoplasm appears
to contain electron-dense components and anelectron-lucent vacuole, which is spherical inshape. CF-FS method preserves the natural morphology of the cell, including the vacuole, while
CF method disrupts fragile cellular structures andonly allows visualization of robust structures.
Fig. 9b shows a 2.2 to 2.5 µm diameter
microorganism prepared using CF method. It
appears to consist of an outer membrane and cytoplasm with several cellular compartments that are
surrounded by membranes but appear to be devoidof electron-dense materials inside, instead showing
scattered electron-opaque materials. Fig. 10bshows a microorganism similar to the one in Fig.

Fig. 11. An ultrathin section of Parakaryon myojinensis. Note the large irregular “nucleoid” (N) with single layer“nucleoid membrane” (NM), the presence of endosymbionts (E), and the absence of mitochondria. Also labeled are thecell wall (CW) and plasma membrane (PM) (From Yamaguchi et al. (2012) with permission).

Jpn. J. Protozool. Vol. 47, No. 1, 2. (2014)37

Fig. 12. Serial sections of P. myojinensis. The numbers at the lower left are sequential from the first section. 12 out of 67
sections. N, nucleoid.

9b, but prepared using CF-FS method. It appears
to consist of an outer membrane and a cytoplasm
which has several compartments surrounded bymembranes. The outer membrane has a verysmooth circumference consisting of an outerelectron-dense leaflet, a middle electron-lucent
leaflet, and an inner electron-dense leaflet. Thus,
CF-FS method prevents extraction of cytoplasm

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Deep-sea microorganisms and evolution

Fig. 13. The cellular components of P. myojinensis under high magnification. (a) and (b) The largest endosymbiont,
Endosymbiont 1 (E1), showing the endosymbiont nucleoid (EN) with DNA fibers (F), ribosomes (R), and the endosymbiont cell membrane (EM), as well as the cytomembranes (CM) and the phagosome space (PS) of the host. (c) The second largest endosymbiont (E2). (d) The smallest endosymbiont (E3). (e) A vacuole (V). (f) Small granular electrontransparent materials, which might be storage materials (S). (g) High magnification of the host “nucleoid” region (N)showing DNA fibers (F), the “nucleoid membrane” (NM), ribosomes (R), the cell wall (CW), and the plasma membrane(PM). (h) High magnification of the plasma membrane (PM). (i) The nucleoid (N) enclosed by the nucleoid membrane(NM) with a gap (G). Also apparent is Endosymbiont 1 (E1) surrounded by phagosome space (PS). A traced image of (i)is shown in Fig. 14g (From Yamaguchi et al. (2012) with permission).

components and preserves natural cell morphology.
Fig. 9c shows an apical part of a filamentous
multicellular microorganism with cell walls
prepared using CF method. The cytoplasm of eachcell contains several components, some of which
are electron-dense while others are electron-lucent.
Fig. 10c shows an apical part of a similar

filamentous multicellular microorganism preparedusing CF-FS method. The cytoplasm of each cell isfilled with similar components in both specimens,
however the morphology of each component appears clearly only in the CF-FS method specimen.
As these examples show, by applying rapidfreeze freeze-substitution after glutaraldehyde fixation (CF-FS method), it is possible to observe theJpn. J. Protozool. Vol. 47, No. 1, 2. (2014)39

Fig. 14. The three dimensional reconstruction of P. myojinensis. (a) The whole cell. (b) The nucleoid. (c) The cytomembrane system of the host cell. (d) The endosymbionts. (e) The distribution of vacuoles in the host cell. (f) The distribution of the small granulated electron-transparent materials in the host cell. (g) Trace image of Fig. 13i showing how thenucleoid region (N) was defined by the inner most cytomembrane (nucleoid membrane) (From Yamaguchi et al. (2012)
with permission).

ultrastructure of deep-sea microorganisms at highresolutions with minimal disturbance of theirnatural morphologies.
DISCOVERY OF A “MYOJIN PARAKARYOTE”
We prepared 420 specimen blocks by thisnew CF-FS method, made serial ultrathin sections
for all specimens, stained with uranyl acetate andlead citrate, and observed them with an electron
microscope. After one year of sectioning andobservation, we found a yeast-like microorganism
several microns in size with a cell wall (Fig. 11;
Yamaguchi et al., 2012).
However, this microorganism was found tolack a nucleus enclosed by a double membrane andmitochondria (Fig. 12). Instead, it had “endosymbionts” with bacteria-like morphology consisting of ribosomes and fibrous nucleoids but no cellwall (Fig. 13a, b). The “nucleoid” of the host cell
had a highly irregular shape and occupied most of
the host cytoplasm (Figs. 11 and 12). It consistedof fibrous material (DNA) and ribosomes (Fig.
13g). Interestingly, the “nucleoid” was different
from both the true nucleoids of prokaryotes andthe true nuclei of eukaryotes in that it was enclosed
by single-layer membrane, which we refer to as the“nucleoid membrane” (Figs. 11, 13g and 13i). The
nucleoid membrane was not a closed membrane
system but was interrupted by gaps (Fig. 13i)
through which the nucleoid region was connected40

Deep-sea microorganisms and evolution

Fig. 15. The volumetric proportions of the cell components in P. myojinensis (From Yamaguchi et al. (2012) withpermission).

to the cytoplasm, and was notably different fromthe nuclear envelopes of eukaryotic cells that are
made of closed double membranes.
The cell wall consisted of one layer and hada thickness of 80–120 nm (Fig. 13g). The plasma
membrane appeared to be a typical three leaflet
structure of electron-dense, electron-transparent,and electron-dense material (Fig. 13h) and had athickness of 19.4 ± 3.9 nm. The cell lacked
mitochondria, chloroplasts, a nucleolus, plastids,
Golgi apparatuses, peroxisomes, centrioles, spindle
pole bodies, and microtubules.
P. myojinensis was 10 µm in length and 3µm in diameter. By 3D reconstruction from the 67
complete serial sections and structome analysis,
we found that the four putative endosymbionts

Table 1. The structomes of P. myojinensis, S. cerevisiae, E. dermatitidis, and E. coli

Cell size (length × diameter)
Cell volume

Percent (%)
of whole
cell volume

Reference

Cell wall
Nucleoid or nucleus
Endosynbiont or mitochondria
VacuolesCytosol
Other components

Parakaryon
myojinensis

Saccharomyces
cerevisiae

Exophialadermatitidis

Escherichia coli

10.3 × 3.1 µm
52.6 µm3
25.6
40.8

3.9 × 3.2 µm
17.1 µm3
17.0
10.5

4.9 × 3.6 µm
36.0 µm3
21.8
7.3

Not reported
0.469 µm3

4.9

1.7

9.9

1.4
22.4
4.9
Yamaguchi et al.,
2012

5.8
64.0
1.0
Yamaguchi et al.,2011aThis table is modified from Table 2 of Yamaguchi et al. (2012).

6.2
47.5
7.3
Biswas et al.,2003

Not reported

Pilavtepe-Çelic
et al., 2008

Jpn. J. Protozool. Vol. 47, No. 1, 2. (2014)

apparent in the sectioned image in Fig. 11 (labeledE) were actually different parts of the one large
spiral endosymbiont (E1) (Fig. 14a and 14d) andthere were a total of three endosymbionts in thecell. The other two endosymbionts (E2 and E3;
Fig. 14d) were both rod-shaped and small, together
being only around one tenth the volume of thelarge endosymbiont.
The “nucleoid” of the host occupied 41% of
the cell volume (Fig. 15) and was surrounded by acomplicated cytomembrane system (Fig. 14c),
which occupied 1.7 times the area of the plasma
membrane. There were about 100 small vacuoles
in the cell (Fig. 14e), which occupied 1.4% of thecell volume (Fig. 15). The cell also contained
small granulated electron-transparent materials
that occupied 0.6% of the cell volume (Fig. 15)(Fig. 14f), and are typically considered to be storage materials (Biswas et al., 2003). The cytosol,
including the plasma membrane, cytomembranes,
and ribosomes, occupied 22% of the cell volume(Fig. 15).
MORPHOLOGY OF PARAKARYON
MYOJINENSIS
It is interesting to compare structomes for P.
myojinensis, a pair of eukaryotic yeasts(Saccharomyces cerevisiae and Exophiala
dermatitidis (black yeast)), and a prokaryote(Escherichia coli) (Table 1). P. myojinensis is
more than 100 times larger than E. coli, three times
larger than S. cerevisiae, and 1.5 times larger thanE. dermatitidis (Table 1). The size of prokaryotes
is typically confined to a few micrometers because
their metabolism is dependent on the diffusion ofmolecules. Exceptions to this general rule areallowed by special conditions and adaptations suchas extraordinarily large resource containing
vacuoles, which form the bulk of the cellularvolume in Thiomargarita namibiensis, or anutrient-rich environment, highly-folded cell
membrane (providing increased surface area for
diffusion), and polyploidy (multiple copies of agene enable the production of its product indifferent areas of the cell) of Epulopiscium
fishelsoni (Schulz and Jørgensen, 2001). Because
P. myojinensis exceeds the normal size forprokaryotes but lacks large vacuoles, a richenvironment, or a highly-folded cell membrane,
the organism likely has some kind of transportsystem within the cell; for example, cytoskeleton
molecules like actin. We do not know what kind of
intracellular transport system this organism has
and further study is necessary, however the sizeand complexity of the “nucleoid” and thecytomembrane system are suggestive of theirpotential involvement.
This “nucleoid” of P. myojinensis occupies
more than 40% of the cell volume, whereas the

Table 2. Features of Parakaryon myojinensis
1

Cell size

2

Nucleoid3

Nucleoid membrane

4

Endosymbionts

5

Other organelles

Much larger than ordinary prokaryotes; more than 100 times larger than E. coli; three times
larger than S. cerevisiae
Consists of prokaryote type DNA fibers and no nucleolus structure; very large and consistsof more than 40% of the cell volume
Single membrane surrounding the nucleoid; pierced with gaps
Similar ultrastructure to modern eubacteria consisting DNA fibers and ribosomes; lack cell
walls but enclosed by cell membranes
Cell wall, plasma membrane, complex cytomembrane systems, many vacuoles, small granular electron-transparent materials; none of the following: mitochondria, chloroplasts, plastids, Golgi apparatus, peroxisomes, centrioles, spindle pole body, microtubules

This table is modified from Table 3 of Yamaguchi et al. (2012).

41

42

Deep-sea microorganisms and evolution

nuclei of the yeasts occupy only 7–11% of the cellvolumes (Table 1). The endosymbionts in P.
myojinensis occupy about 5% of the cell volume, apercentage which is in between the mitochondrial
volumes in S. cerevisiae (2%) and E. dermatitidis(10%) (Table 1). The presence of a giant endosymbiont is reminiscent of the presence of a giant
mitochondrion in S. cerevisiae (Yamaguchi et al.,2011a). The endosymbionts themselves of P.
myojinensis might fuse or divide during the cell
cycle as do the mitochondria of yeast cells. Further
study is needed to clarify the nature of the symbiosis between the host and the endosymbionts.
Table 2 summarizes the features of P. myojinensis.
POSSIBLE
ORIGINSOF
MYOJINENSIS SPECIMEN

THE

P.

One possibility for the presence of putative“endosymbionts” in a larger cell is predatory orparasitic bacteria living within a prokaryotic host(Guerrero et al., 1986; Larkin et al., 1990). These
parasitic bacteria present as intact within the hostcells: they show dense cytoplasms, maintain their
original rod shapes, and have cell walls. However,in these cases, the cytoplasm of the host cellbecomes less dense and shows irregularmorphology and thus appears to be degraded by
the internal bacteria.
If P. myojinensis is a similar case of a hostwith parasites/predators, the specimen is a snapshot of a short-term predatory interaction. This
scenario is unlikely to be the case because of thefollowing three reasons.
First, unlike the reported bacterial parasitehost interactions mentioned above, P. myojinensis
contained multiple endosymbionts of varying
morphology (Fig. 14d). It is unlikely that multiple
bacteria of different species attacked a host at thesame time. Also, the endosymbionts in P.
myojinensis lack a cell wall and it is unlikely that avery small bacterium without a cell wall (E3 in
Fig. 14d) would be able to successfully attack aprokaryote that had a cell wall. Therefore theseendosymbionts cannot have recently entered thehost cell as independently living bacteria.
Second, the cytoplasms of both theendosymbionts and the host show intact cellular
structures, so no digestion in either host or endosymbionts seems to have occurred. Thus it appears
that both the host and the endosymbionts are ingood condition and the symbiosis is a long-term
interaction.
Third, if P. myojinensis is a snapshot of apredator-prey interaction, there must be dense
populations of hosts because bacterial predators
cannot survive for long between hosts. However,after 14 years, and more than 12,000 micrographsof microorganisms from the deep-sea off the coastof Japan, only one microorganism like P.
myojinensis was found. This suggests that P.
myojinensis lives at extremely low densities and istherefore unlikely to be an interaction between
predators/parasites and a host.
Instead, P. myojinensis seems to be a stablespecies that originated through an endosymbiotic
event in the past involving a larger prokaryote andsmaller bacteria as discussed by Margulis (1970).
Potentially, P. myojinensis could even be aconservative descendent of the transitional lineage
between prokaryotes and eukaryotes, however the
likelihood of that is low and depends a great deal
on how readily bacterial endosymbionts areincorporated into larger cells. At the time of theendosymbiotic event the ancestor of P. myojinensis
probably had no cell wall, otherwise it would havebeen unlikely to be able to engulf the bacteria in
the first place. P. myojinensis currently has acomplicated cytomembrane system and a phagosome-like organelle that contains endosymbionts
thus it seems likely that its ancestor had the ability
to engulf free-living bacteria. The cell wall of P.
myojinensis most likely formed at some point intime after the endosymbiotic process was complete. Also, the lack of cell walls in the endosymbionts of P. myojinensis is likely a derivedJpn. J. Protozool. Vol. 47, No. 1, 2. (2014)

characteristic, as they probably had cell walls likeother prokaryotes at the beginning of the endosymbiotic event. The cell walls of the endosymbionts
must have been lost during a long-term symbiosis
as they were no longer needed for protection andwould have likely interfered with the benefits ofsymbiosis.
It is fervently hoped that genetic andbiochemical work will be possible in the futurewhen another specimen of P. myojinensis is foundto enable estimates of its phylogenetic position andthe timing of its endosymbiotic event.
IMPLICATIONSFORTHEORIES
OF
MITOCHONDRIALAND
NUCLEARDEVELOPMENT
According to the endosymbiosis theory, theancestor of mitochondria is believed to be an αproteobacterium. There are several lines of evidence for this theory. 1) Mitochondria have theirown DNA (Nass, 1969). 2) They have a double
membrane structure (Nass, 1969). 3) They have
70S ribosomes that are similar to bacterial ribosomes and different from eukaryotic ribosomes(Nass, 1969). 4) They multiply within the cell bydivision (Kuroiwa et al., 1977). 5) The genome of
Rickttsia prowazekii (an α-proteobacterium) was
found to be similar to the mitochondrial genome(Anderson et al., 1998).
The relationship between P. myojinensis andits endosymbionts must be a beneficial one for it tohave lasted long enough for the endosymbionts tolose their cell walls and host to gain its cell wall. It
seems likely that the endosymbionts in P.
myojinensis are descendants of bacteria engulfed
by a larger prokaryote in the past, thus the micrographs of the present study may provide an example that shows another endosymbiotic event that
lends support to the hypothesis that the highlyderived mitochondria in eukaryotes could indeed
have evolved from bacteria.
The origin of the eukaryote nucleus is anoth-43

er mystery to which P. myojinensis may provide
clues. There are currently several hypotheses vying
for dominance (Lake and Rivera, 1994; Martin,
1999, 2005; Pennisi, 2004). The eukaryote nucleus
could have evolved gradually by the development
and elaboration of an inner cytomembrane system(Cavalier-Smith, 1988; Nakamura and Hase,
1990), however it could also have been engendered by fusion or symbiosis between multiple
prokaryotes (Hartman, 1984; Moreira and LópezGarcía, 1998; Horiike et al., 2001).
The “nucleoid” of P. myojinensis is not atrue nucleus because it does not contain chromatin(DNA associated with histone proteins), which is ahallmark of eukaryotic nuclear organization.
Instead the “nucleoid” contains naked DNA fibers,
much like the prokaryotic nucleoid. However, trueprokaryotic nucleoids are not surrounded bymembranes and that of P. myojinensis issurrounded by a single layered membrane with
scattered gaps. Furthermore, this membrane differsfrom the eukaryotic nuclear membrane, which isdouble-layered and complete. This nucleoid
membrane could be a form of primitive nuclear
membrane. If this is the case, it follows that
nuclear membranes could have evolved from thecytomembranes of prokaryotes that had developed
inner membrane systems.
There is still a question about whether anucleus was formed when before mitochondrial
ancestors started the process of endosymbiosis(Roger, 1999; Gray et al., 1999; Poole and Penny,
2006; Martijin and Ettema, 2013). The nuclear
region of P. myojinensis is not a completely
formed eukaryotic nucleus but internalized endosymbionts are already in the host cell. Thissuggests that a fully formed and differentiatednucleus was not necessary before eubacteria could
start to be integrated within the prokaryote hostcell. Thus, the formation of the eukaryotic nucleus
might not have been linked in any way to thetransformation of bacteria into mitochondria.

44

Deep-sea microorganisms and evolution

Fig. 16. A model of eukaryotic origins and a possible position of P. myojinensis. C, chloroplast; E, endosymbiont;
M, mitochondrion; N, nucleus or nucleoid.

A MODEL OF EUKARYOTE ORIGINS
Fig. 16 shows a model of eukaryote origins
and a plausible evolutionary position of P.
myojinensis partly adapted from Whittaker (1969)
and Woese et al. (1990). Primitive Archea with nocell wall may have engulfed α-proteobacteria byphagocytosis and become the ancestor of protists,
fungi, animals, plants, and the parakaryote. The
ancestor of plants may have engulfed cyanobacteria at a later time to become modern plants. Cell
walls likely arose independently in the plant, fungi,
and parakaryote lineages after the endosymbiosis
process was complete.

At this point, the phylogenic position of theparakaryote is a matter of pure conjecture. Further
genetic studies and biochemical analysis are necessary to elucidate the nature and phylogenetic
position of the parakaryote as these are impossible
to perform on specimens prepared for electron
microscopy. Whatever the true position of theparakaryote in the tree of life, the existence of anapparently transitional life form between prokaryote and eukaryote provides a useful model of what
the ancestor to all eukaryotic life could have been
like and thus how the transition from prokaryote toeukaryote could have proceeded. It is likely thatthe cellular physiology and functioning of theJpn. J. Protozool. Vol. 47, No. 1, 2. (2014)

parakaryote are as unique as is its morphology andby providing a third perspective could potentiallygive a much deeper understanding of cellularmechanisms in general.
THE PROBLEMS AND POTENTIAL OF
DEEP-SEA MICROORGANISM STUDIES
There are a few studies of newly discovered
deep-sea microorganisms that were characterizedmorphologically, genetically and/or biochemicallyafter being cultured in the lab (e.g. Takishita et al.,2007; Wu et al., 2014). However, culturing practices are always biased towards certain types of
microorganisms with particular tolerances. Considering the fact that standard methods fail tosuccessfully culture most microbes (Pace, 1997),
most organisms are overlooked by these methods.
Our 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 tothrive in particular culturing conditions.
Standard methods of identifying microorganisms through culture and genetic and biochemical characterization are some of the greatest
successes of modern biology. However, the natural
bias inherent to cell culture techniques severely
limits the exploration of unique microorganisms
from extreme environments or with unusual
requirements. Rather than ignoring these microorganisms and their potentially paradigm-altering
structures and adaptations, we believe that careful
preparation and morphological characterization
can be a useful and important first step in
discovering and describing new and uniquemicroorganisms. To do so effectively, wedeveloped the CF-FS method to observe thenatural morphology of microorganisms at highresolution by using freeze-substitution electron
microscopy (Yamaguchi et al., 2011b). To identify
different microorganisms morphologically, weused a serial ultrathin sectioning technique to45

conduct structome analysis (Yamaguchi et al.,2009, 2011a). These methods enabled us toobserve and record a variety of microorganisms
from the deep sea, many of which exhibit unusualmorphologies. These unusual morphologies arelikely mirrored by unusual biochemistry andevolutionary histories and may hold clues toimportant questions in evolutionary biology, suchas the transition from prokaryote to eukaryote, theorigins of mitochondria and nuclei, and the origins
of centrioles, spindle pole bodies, flagella, andother organelles. The deep sea is an extremely
stable environment in which there might be very
little selective pressure for change and low levelsof competition, leading to still surviving “living
fossils” that may retain features long absent from
more typical lineages in more “normal” environments (Yamaguchi et al., 2012; Yamaguchi andWorman, 2012; Yamaguchi, 2013).
PERSPECTIVES ON FUTURE RESEARCH
As discussed above, it is difficult to culture
deep-sea microorganisms under laboratory
conditions. Yet, it is important to obtain genetic
data for each microorganism observed with anelectron microscope to clarify its phylogenetic
position. This presents a tremendous problem
because, generally, electron microscopy specimens
are fixed with chemicals, embedded in a resin,thinly sliced, stained with heavy metals, andradiated by an electron beam for observation.
These harsh treatments must be avoided if the
DNA is to remain intact for analysis.
Matching up morphology with phylogenetics
would be possible if the same individual cell wereable to be used for both types of analysis. This
could be achieved by using serial sections of thetarget microorganism. That is, the specimen is
freeze-substituted with ethanol only, embedded,
thinly sliced, photographed by electron microscope, and the target is mapped. The next section
is cut thick for light microscopy, the target micro-

46

Deep-sea microorganisms and evolution

organism is isolated by laser microdissection, andthe DNA/RNA is sequenced. In this technique,since the DNA/RNA of the target microorganism
is never treated with chemicals like osmium
tetroxide and never irradiated by electron beams, it
could be used for sequencing. We are now starting
experiments, using the yeast Saccharomyces as amodel, to develop techniques that make it possible
to sequence ribosomal RNA of a microorganismthat is also observed under an electron microscope.
If this technique is perfected, research on deep-sea
microorganisms would advance significantly. This
technique would be also useful for other researchwhere the microorganisms cannot be cultured. As
it stands now, the partnership between morphology
and genetics of microbes lags far behind that ofmulticellular organisms, but hopefully, this willrapidly change in the near future.
ACKNOWLEDGEMENTS
This work is the result of joint research with
Yoshimichi Kozuka, Yuko Mori, Hitoshi Okada,
Katsuyuki Uematsu, Akihiro Tame, Hiromitu
Furukawa, Tadashi Maruyama, and Koji
Yokoyama. We sincerely thank them. We also
thank the crew of the research vessel Natsushima,
the operation team of Hyper-Dolphin, and the staffof the Japan Marine Science and Technology
Center for their support of the NT10-08 cruise.
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