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FEMS MicrobiologyEcology31 (1985) 187-192
Published by Elsevier
187
FEC 00023
Endosymbiofic meth ogenic bacteria of the sapropelic
amoeba Mastigella
(Flagellated amoebae; Pelomyxa palustris," anaerobic protozoa)
Johan J.A. van Bruggen, Claudius K. Stumm, Kor B. Zwart and
Godfried D. Vogels
Departmentof Microbiology,Facultyof Science, Universityof Nijmegen, Toernooivela~NL-6525 El) Nijmegen, The Netherlands
Received11 April 1985
Revisionreceivedand accepted26 April 1985
1. SUMMARY
Large numbers of Gram-positive methanogenic
bacteria, which morphologically resembled Methanobacterium formicicum, were found in the flagellated amoeba Mastigella from fresh water sapropel. A non-methanogenic Gram-positive bacterium
with a characteristic axial cleft was found to live in
close association with the amoebal nucleus. The
similarity between the symbiotic system of Mastigella and that of the giant amoeba Pelomyxa is
discussed, as well as the contribution of sapropelic
amoebae to the methanogenesis of the sapropel
ecosystem.
2. INTRODUCTION
Intracellular bacteria in sapropelic protozoa
have been known to exist for many years [1-4].
Recently, it was shown in 8 sapropelic protozoa
investigated, that the majority of these .endosymbionts were methanogenic bacteria [5]. The identification of the methanogens was documented by
fluorescence microscopy [6], demonstration of
coenzyme F420 and 7-methylpterin, and methane
production by an isolated cell of the giant amoeba
Pelomyxa palustris. Moreover, Methanobacterium
formicicum was isolated from the ciliate Metopus
striatus [7]. P. palustris was found to contain 2
types of methanogens [5] and a non-methanogenic
Gram-positive rod with a characteristic axial cleft
[8,9], whereas several organdies normally present
in eukaryotic cells were absent.
In this paper, we describe the endosymbionts of
another sapropelic amoeba, Mastigella. Striking
similarities were observed with the endosymbionts
of the (taxonomically unrelated) amoeba P.
palustris.
3. MATERIALS AND METHODS
3.1. Organisms
The amoeba was studied in sapropel samples
taken from several eutrophic ponds near Nijmegen
and--as a constant source of the organism--from
a mud-filled 200-1 aquarium in the laboratory. The
organism was identified according to Goldschmidt
[1] and Lemmermann [10].
3.2. Microscopical procedures
For a rapid identification of methanogenic
bacteria, Leitz epifluorescence microscopy, according to Doddema and Vogets [6], was used. Differential interference contrast (DIC) microscopy
0168-6496/85/$03.30 © 1985 Federation of European MicrobiologicalSocieties
188
was performed with the Leitz T-device on an
Ortholux microscope.
Electron microscope preparations were made as
reported previously [7] and were studied in a
Philips EM300.
3.3. Quantification of amoebae
Using a pipette, 27.7 ml water containing a
small quantity of sapropel was sampled from the
muddy surface of the aquarium. In a second
experiment, 6 sapropel samples were taken from
different places in the aquarium and pooled (total
volume approx. 50 ml) and 4 samples (2.2 ml)
were studied. The material was transferred to a
petri dish and examined under a dissection microscope. Using a small pipette, virtually all the
Mastigella and P. palustris cells present were collected and counted. The dry weight of the sapropel
was determined after collection by membrane
filtration and subsequent drying for 24 h at 75°C
in vacuo.
3.4. Quantification of endosymbionts
200 Mastigella t,ells were subjected to 3 pipette
transfers of 10 mM NazHPOa/KH2PO4 buffer,
pH 7.0. The mean volume of the endoplasm of the
cells was estimated by measuring the diameter of
the cells, which had rounded after pipetting. Subsequently, the endosymbionts were released from
the Mastigella cells by homogenisation in a defined buffer volume, and counted in a Bi~rker-T'urk
counting chamber (W. Schreck, Hofheim/Ts,
F.R.G.) by means of phase-contrast microscopy.
4. RESULTS
4.1. Description of the amoeba
Under the light microscope, the uninucleate
amoeba, with a diameter of 133 + 14 #m, showed
a granular endoplasm with many ingested plant
particles. The ectoplasm was hyaline with numerous pointed or blunt, erupting pseudopodia. The
cell possessed a short flagellum (Fig. 1) which
showed sweeping and curling movements; no contribution to the mobility of the amoeba by this
organelle was observed. The prominent nucleus,
with a diameter of about 35 /~m, showed periph-
f
Fig. 1. Living,migratingMastigellacellwithpointedand blunt
pseudopodia, clear ecto- and granular endoplasm,and flagellum. ( --, ) Differentialinterferencecontrast (DIC), electronic
flash. Bar represents20/~m.
eral nucleoli (dispersed type, Fig. 2); no connection Was observed between the nucleus and the
flagellum origin. Division stages of the nucleus
were not observed. However, ceils with 2 nuclei
were sometimes found, presumably representing a
stage shortly prior to cytokinesis (Fig. 3). On the
basis of these characteristics, the organism was
identified as a member of the genus Mastigella, a
group of flagellated amoebae. (Order, Pantostomatinae; family, Rhizomastigaceae. Lemmermann [10]). Its morphological characteristics were
compared with those of some other Mastigella
species (Table 1). Except for the diameter of the
nucleus, the present organism most resembled M.
vitrea. The presence of endosymbiotic bacteria in
this organism had already been reported in 1907
by Goldschmidt [1], who also noticed the similarity of these endosymbionts with those found in P.
palustris [8,9].
4.2. Endosymbionts
Throughout the endoplasm of the Mastigella
cells, numerous slender rods (3.4 x 0.3 /~m) were
189
?
i
1
!i!~ ii~!iii~/!~ !i~....
Fig. 2. Optical transverse section of the nucleus of a riving
Mastigella cell. The round bodies inside the nucleus are
nucleolar granules. The surface of the nucleus is covered with
thick rods, As a result of the rapid streaming of the cytoplasm
the thickness of the bacterial layer is not uniform. DIC, electronic flash. Bar represents 10/~m.
always observed. Flattening of the Mastigella cells
by a slight pressure on the cover glass facilitated
the visibility of these endosymbionts, which showed
the characteristic bluish fluorescence of methanogenic bacteria (Fig. 4) and stained Gram-positively.
Fig. 3. Binucleate Mastigella cell. The lower nucleus is slightly
out of focus in this Giemsa-stained preparation. Both nuclei are
surrounded by masses of thick rods. Bar represents 20/~m.
Another endosymbiont always present was a
thick rod (4.5 × 0.8 #m) surrounding the nucleus
in a uni- or multicellular layer (Figs. 2 and 3).
Aggregates of these bacteria were sometimes found
separated from the nucleus in the endoplasm. This
organism also stained Gram-positively but did not
show fluorescence.
The mean number of slender and thick rods per
Table 1
Morphological properties of some Mastigella species
Organism
Cell
diameter
(~tm)
Nucleus
diameter
(~m)
Endosymbionts
Flagellum
length
Mastigella sp.
Mastigella vitrea
Mastigella nitens
Mastigellapolymastix
120-150
125-150
80,90
32-80
35
10-15
_a
18-20
present
present
_a
absent
40-70
120-250
45
> 80 b
* This paper
Not mentioned.
b 1-4 Flagella present.
a
References
(~tm)
[*]
[1,10]
[10]
[11]
190
Table 2
N u m b e r s of amoebae and amoebae-derived methanogenic
bacteria in the aquarium sapropel
Mastigella Pelomyxa
A m o e b a e / m l sapropel
A m o e b a e / m g dried sapropel
Methanogens/cell
M e t h a n o g e n s / m g dried sapropel
M e t h a n o g e n s / m l sapropel
52 _+29 a
106
22 x 103
2.3 x 106
1.1 x 106
9 +_7 a
16
50 x 103 b
0.8 >( 106
0.4 x 106
Mean + S D of 5 samples.
b The mean diameter of Pelomyxa cells present was considered
as 200 lam. The concentration of methanogenic bacteria is
1.2 x 101° per ml cytoplasm [5].
a
Fig. 4. Epifluorescence micrograph of a single unfixed and
slightly flattened Mastigella cell. The methanogenic bacteria
are visible as thin rods. Thick rods are non-fluorescent and not
visible in this micrograph. Bar represents 10/xm.
Fig. 5. Ultra-thin section of Mastigella demonstrating the 2 endosymbiotic bacteria. The thick rod-shaped bacteria contain a typical
axial cleft ( ---, ) and are separated from the nucleus only by a thin layer of vesicles. The thin rods are highly osmiophilic and, as shown
in the inset, contain mesosome-like structures ( ~ ). Bar represents 1 #m. N = nucleus.
191
cell were estimated at 22000 and 6500, respectively. Since the amoeba endoplasm had a mean
volume of 0.7 x 10 -6 ml, the numbers of bacteria
correspond to a concentration per ml endoplasm
of 3 × 101° and 0.9 × 101° for slender and thick
rods, respectively.
4.3. Ultrastructure of Mastigella
Electron microscopy revealed that Mastigella
cells lack mitochondria, Golgi apparatus and microbodies. The ultrastructure of the nucleus is
similar to that of P. palustris [8,9], but inclusions
like sand and paraglycogen grains, which are typical for the giant amoeba P. palustris, were not
found in Mastigella. 2 Types of endosymbionts
were observed in the cytoplasm. Slender,
osmiophilic, Gram-positive rods, with conically
pointed ends, containing mesosome-like structures,
were encountered throughout the cytoplasm. These
slender endosymbionts, considered to be methanogenic bacteria, strongly resembled Methano-
bacterium formicicum.
The second endosymbiont, a thick Gram-positive rod, had a rectangular shape and a deep,
incision-like cleft along the whole length of the cell
(Fig. 5). Many thick rods were observed in close
association with the nucleus, separated from it
only by a thin layer of vesicles. This bacterium is
probably identical to the thick rod found in P.
palustris [8,9].
Both endosymbionts were separated from the
amoeba cytoplasm by a membrane and were actively growing, as was apparent from the frequent
observation of dividing bacteria.
4.4. Numbers of amoebae in the sapropel
Both Mastigella and P. palustris cells were present in great numbers in the upper layer of the
aquarium sapropel (Table 2). Mastigella cells were
fairly constant in size, whereas the P. palustris cell
diameter ranged from 100-1000 #m.
The mean concentration of amoeba-derived
methanogens in the upper layer of the sapropel
was estimated to be 1.5 × 106/ml. This is a
minimum concentration, since it is likely that some
amoebae escaped collection from the mud samples.
5. DISCUSSION
5.1. Characterization of Mastigella
The amoeba described here and found
ubiquitously in the upper layers of the sapropel
was identified as a Mastigella species and resembled M. vitrea. The cells contained large numbers
of methanogenic and non-methanogenic endosymbiotic bacteria. The methanogens resembled morphologically the Gram-positive methanogens occurring in P. palustris (manuscript in preparation),
another sapropel amoeba. The non-methanogenic
thick rods are most probably identical to the thick
rods of P. palustris [9].
Apart from the endosymbionts, the ultrastructures of both anaerobic amoebae share many features. Mitochondria, Golgi apparatus and microbodies are absent. In contrast to the multinucleated Pelomyxa, Mastigella has a single
nucleus and contains no paraglycogen. The presence of methanogenic endosymbionts is not unique
to anaerobic amoebae, but seems to be a common
feature among sapropel protozoa [5].
5.2. Function of the endosymbionts
No attempts have yet been made to isolate and
characterise the methanogenic endosymbionts of
Mastigella. Therefore, no information is available
on the substrate specificity of this organism. However, hydrogen or formate are common substrates
for methanogenic rods, and probably also for the
methanogenic rods of Mastigella. As a result of
methane production by these endosymbionts, the
intracellular hydrogen levels may remain very low
and, as a consequence, the eukaryote can easily
reoxidize its reduced electron acceptors and profit
from an energetically favourable situation. The
question arises as to where the reduction equivalents are produced, as hydrogen or formate, in
the Mastigella cell. Except for P. palustris and the
now described Mastigella species, all anaerobic
sapropel protozoa so far investigated ([7] and unpublished results) contain hydrogenosome-like microbodies which are usually closely associated with
the methanogenic bacteria in the cells. It is not
likely that hydrogen has a cytoplasmic origin in
both anaerobic amoebae, since hydrogenase appears to be organelle-bound in all eukaryotic cells
192
studied. For that reason, it is tempting to suggest
that the non-methanogenic, thick rod serves as a
hydrogen source for the methanogens. Such a
metabolic interaction could be a remarkable example of inter-endosymbiotic hydrogen transfer,
avoiding the disadvantage of mass transfer resistance. Unfortunately, all attempts so far to grow
the thick rod in pure or mixed cultures have failed,
and its substrate spectrum remains unknown. The
close association of these bacteria with the nucleus
strongly suggests a mutual metabolic relationship.
In spite of the presence of a flagellum, Mastigella and Pelomyxa must be considered as descendants of early stages in eukaryotic evolution, since
organelles (except for the nucleus) are absent and
endosymbiotic bacteria seem to be essential for
growth of these primitive eukaryotes. This view
differs strongly from that of Bovee and Jahn [12],
who suggested that Mastigella might have evolved
from mitochondria-containing amoebae.
5.3. Contribution of amoeba-derived methanogens to
total methane production
The numbers of methanogenic bacteria reported in the sapropel vary with the methods
applied and with the kind of sediment analysed.
Approx. 1.6 × 105 methanogens per g wet sediment were found by applying a most-probablenumber method [13], whereas 5 × 104 methanogens per ml wet sediment were found by determining the number of colony-forming units [14] and
105-109 methanogens per g dry sediment were
estimated by means of a direct fluorescent-antibody technique [15].
The presence of at least 1.5 × 106 amoeba-derived endosymbiotic methanogenic bacteria per ml
of the upper layer of the sapropel clearly indicates
that such methanogens contribute considerably to
the methane production in this part of the sapropel.
ACKNOWLEDGEMENTS
We thank Dr. C. Chapman-Andresen, Dr. F.C.
Page and Mr. F.J. Siemensma for their valuable
suggestions with regard to the identification of the
organism.
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