FEMS Microbiology Letters 110 (1993) 21-25
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
21
FEMSLE 05446
Endosymbiotic purple non-sulphur bacteria
in an anaerobic ciliated protozoon
Tom Fenchel
and Catherine
Bernard
Marine Biological Laboratory, University of Copenhagen, HelsingOr, Denmark
(Received 17 February 1993; revision received 11 March 1993; accepted 12 March 1993)
Abstract: The marine ciliate Strombidium purpureum Kahl harbours endosymbiotic purple non-sulphur bacteria. The bacteria
contain bacteriochlorophyll a and the carotenoid spirilloxanthin, and they have photosynthetic membranes and cell walls. The
ciliates require light for survival and growth under anaerobic conditions; in the dark the cells prefer microaerobic conditions. The
ciliates show a photosensory behaviour, and they accumulate in light at wave lengths corresponding to the absorption spectrum of
the symbionts. The findings are discussed in terms of theories on the endosymbiotic origin of mitochondria.
Key words': Anaerobic ciliates; Endosymbionts; Purple non-sulphur bacteria; Mitochondrial origins
Introduction
In 1932 K a h l [1] briefly d e s c r i b e d t h e ciliate
p u r p u r e u m a n d s u g g e s t e d that its
p u r p l e c o l o u r was d u e to symbiotic ' r h o d o b a c t e r i a ' ; the species s e e m s not to have b e e n
m e n t i o n e d in t h e l i t e r a t u r e since t h e original
d e s c r i p t i o n . In the s u m m e r of 1992 we n o t i c e d a
mass o c c u r r e n c e of the ciliate in s a m p l e s o f m a rine s a n d with p u r p l e s u l p h u r b a c t e r i a which h a d
b e e n i n c u b a t e d a n a e r o b i c a l l y in glass j a r s a n d left
in an east facing window. S u b s e q u e n t l y we w e r e
able to grow the cells in i l l u m i n a t e d a n a e r o b i c
cultures. T h e e n d o s y m b i o n t s p r o v e d to be p u r p l e
n o n - s u l p h u r b a c t e r i a . H e r e we d e s c r i b e the basic
p r o p e r t i e s of w h a t s e e m s to be the first e x a m p l e
of a symbiotic r e l a t i o n s h i p b e t w e e n non-oxygenic
p h o t o s y n t h e t i c b a c t e r i a a n d eukaryotes.
Strombidium
Correspondence to: T. Fenchel, Marine Biological Laboratory,
University of Copenhagen, DK-3000 Helsing0r, Denmark.
Materials and Methods
S a m p l e s o f s a n d with p a t c h e s o f p u r p l e o r
white s u l p h u r b a c t e r i a w e r e c o l l e c t e d in Niv~t Bay
(approx. 25 k m n o r t h of C o p e n h a g e n , D e n m a r k ) .
In the l a b o r a t o r y the jars w e r e filled with O2-free
s e a w a t e r a n d left in the light. A f t e r a few days,
large n u m b e r s of S t r o m b i d i u m p u r p u r e u m cells
a c c u m u l a t e d along the i l l u m i n a t e d side of the
jars. T h e cells w e r e grown in 15- or 100-ml s e r u m
vials with 10 or 50 ml O2-free s e a w a t e r a n d one
o r t h r e e b o i l e d w h e a t grains a n d with O2-free N 2
gas in the h e a d s p a c e . T h e vials w e r e e x p o s e d to
c o n s t a n t i l l u m i n a t i o n from a 60 W a t t i n c a n d e s -
22
cent light bulb at a distance of 30-50 cm. Initially
the cultures attained cell densities of > 1000 cells
ml-1, but after several generations the cell yield
declined (after 2 - 3 months) and the ciliate had to
be re-isolated.
The in vivo absorption spectrum of the ciliate
was obtained by collecting ceils on 4 m m 2 of a
glass fibre filter which was scanned in a LKBUltrospec Plus Spectrometer using a moist glass
fibre filter as a blank [2]. An absorption spectrum
of a methanol extract of the ciliates was also
obtained. For TEM, cells were fixed in a solution
containing 1% OSO4, 2.5% glutaraldehyde and 10
mM sucrose in 0.2 M phosphate buffer (7.5),
washed, blockstained in uranyl acetate in 70%
ethanol, dehydrated, embedded in epon and sectioned on a LKB microtome. Some sections were
also stained in lead citrate. The sections were
observed in a Zeiss EM900 transmission electron
microscope. Estimates on the number and volume of the endosymbionts are based on serial
sections and on standard stereological techniques
nm) after 10 min, The response to 0 2 was studied
by placing capillaries with cell suspensions in
stoppered test tubes with an O2-free N 2 atmosphere. By injecting known amounts of air into
the test tube, the retreat from or accumulation at
the meniscus could be followed under a dissection microscope.
Results and Discussion
The ciliate is a typical m e m b e r of the genus
Strombidium (Oligotrichida), a genus which com-
prises planktonic as well as benthic species [8],
but anaerobic species have not been described
previously. Cells of S. purpureum appeared red to
reddish brown in transmitted light and pink in
reflected light. The individual symbionts are
mainly situated along the periphery of the cell;
they are ovoid and contain a varying number of
refractile granules (Fig. 1). The latter do not
[3].
The methods used for studying the behavioural
response to oxygen tension and light were described earlier [4-7]. Briefly, anaerobic cell suspensions were placed in 50 m m long capillary
microslides (Camlab) with the internal dimensions 4 × 0.4 mm. For studying the accumulation
in light, only a slit (corresponding to 6% of the
length of the capillary) was illuminated at time
zero and the rest was kept in the dark. The
number of cells in the illuminated patch was then
counted at 1- or 2-min intervals from video
recordings through a dissection microscope. The
light source was a 12 V halogen lamp with an
intensity of about 1 klux at the preparation. This
experiment was also carried out after this light
had passed an infrared filter (Wratten No. 87
gelatine filter, no transmission < 740 nm and
> 70% transmission above 820 nm) and with a
green filter (IF 550 interference filter, Olympus).
In order to study the preferred wave lengths in
the visible spectrum, capillaries with cell suspensions were placed on the top of a calibrated
wedge interference filter (350-750 nm, Schott)
illuminated from below and the number of cells
counted at intervals of 2 m m (corresponding to 15
Fig. 1. A living Strombidium purpureum cell showing the
symbionts with refractile storage vacuoles. Scale bar: 10/xm.
23
consist of sulphur and they are probably storage
vacuoles containing poly-/3-hydroxybutyrate since
they stain intensely with Sudan black B (data not
shown). The average diameter of the symbionts
was 2.7/~m in cells collected in the field, but only
1.5 /~m in cultured ciliates, the symbionts of
which contained fewer storage vacuoles. There
were between 200 and 700 symbionts per ciliate,
always corresponding to 10-15% of the host cell
volume. The ciliates also contain 200-300 mitochondria which, as in other anaerobic ciliates,
contain few tubular cristae and an electron-dense
matrix [9] (Fig. 2a, b. Attempts to demonstrate
the presence of hydrogenase (indicating that the
mitochondria are capable of fermenting pyruvate
into hydrogen and acetate as found in some other
anaerobic ciliates [10]) with cytochemical methods [9] yielded ambiguous results. The mitochondria are found close to the symbionts. The ciliate
is a phagotroph and it always contains food vacuoles with partly digested bacteria.
The symbionts are not surrounded by vacuoles.
They are covered by a membrane with a thickness
of 11-12 nm. In addition to a unit membrane, the
membrane must include the outer membrane
a n d / o r a reduced peptidoglycan layer; occasionally a space between an inner and an outer layer
is seen (Fig. 2d). Invaginations of the cell membrane are evident in 5-10 places on the cell
surface; these invaginations form irregular stacks
of internal vesicles. From the vesicles, membranous tubules (diameter 20 nm) radiate through
the cell (Fig. 2a, c). In some cases, the tubules
transform into 25-35 nm vesicles which may fill
almost the entire cell (Fig. 2b, d). Variation in the
amount of photosynthetic membrane is probably
related to light intensity, but this was not studied.
A considerable variation with respect to the
Fig. 2. (a) A symbiont with a relatively small a m o u n t of photosynthetic m e m b r a n e occurring mainly as tubules (arrow), arrow
marked: m, mitochondrion; v, storage vacuoles not lined with membranes. (b) Two symbionts filled with vesicular photosyntheiic
vesicles and a few tubules; the upper cell is in the process of budding; m, mitochondrion. (c) Invagination of stacked vesicles from
the cell surface; from these large vesicles tubules extend into the cell (arrow). (d) A symbiont adjacent to a mitochondrion; at the
arrow the two-layered cell wall is visible. Scale bars: a, b, 0.5 # m , c, d, 0.1 /zm.
24
amount of photosynthetic m e m b r a n e and with
respect to the simultaneous presence of tubules
and vesicles has previously been described for
species of Rhodospirillurn and Rhodopseudomonas [11-13]. The variation in the structure
of internal membranes and the considerable variation in cell size do leave the possibility that
there were two different kinds of symbionts.
However, intermediate forms did occur. A few
sections (Fig. 2b) suggest that the symbionts multiply by budding.
In vivo light absorption spectra (Fig. 3) indicated the presence of bacteriochlorophyll a with
absorption maxima at 589, 801, 850 nm and a
'shoulder' around 890 nm [14]. A carotenoid peak
at 486 nm indicates the presence of spirilloxanthin, which has been suggested to be diagnostic of
some species belonging to the genera Rhodospirillum, Rhodopseudomonas and Rhodomicrobium
[15]. Methanol-extracted ciliates showed a bacteriochlorophyll a absorption p e a k at 769 nm.
U n d e r anaerobic conditions the ciliates rapidly
accumulate in white and in infrared ( > 750 nm)
light, but they show a weaker response to green
(around 550 nm) light (Fig. 4). Cells in a glass
capillary placed on the top of the wedge interference filter and illuminated from below accumulated between 450 and 500 nm and between 580
and 600 nm. This corresponds to carotenoid absorption and to the orange absorption peak of
bacteriochlorophyll, respectively. This and the re100
o white
olR
-~ 80
r~
o
~ green
c~
o.
o
o
~
~o
- o
o
o/
60
"--
0
_~
•
/
0
i
0
i
2
o ~
n
I
4
I
i
6
~°
I
I
8
I
i
10
I
12
I
rain
Fig. 3. In vivo absorption spectrum of Strombidium purpureum
cells.
o3
I02
......
01
0
400
i
580
n
600
700
800
900
nm
Fig. 4. Accumulation of S. p u r p u r e u m ceils in white light
(intensity about I klux) and with the same light source after
passing an infrared and a green filter, respectively. The anaerobic cell suspension contained about 100 cells and the graph
shows the percentage of cells accumulating in the illuminated
patch which constitutes 6% of the total area of the preparation.
sponse to infrared light suggest that the photobehaviour of the ciliate is controlled by the
metabolic activity of the symbionts. The photosensory response is weaker under microaerobic
(pO2: 5% atm. sat.) conditions. The ciliates die
within about 30 min when exposed to atmospheric pO 2. In the light, the ciliates avoid even
traces of 0 2, but in the dark the ciliates accumulate in water with a pO 2 of 1 - 4 % atm. sat.
We have so far been able to grow the ciliate
only under strictly anaerobic conditions and under constant illumination. Kept in the dark, all
cells in anaerobic cultures are dead within 36-48
h, but if small amounts of air ( 5 - 1 0 % ) are injected into the headspace, the cells survive 2 - 3
times longer. In the field we have found the
ciliate exclusively together with purple sulphur
bacteria in the upper 5 m m of anaerobic sand.
They were most numerous during the summer,
but could be isolated from sediment samples at
least as late as November.
We suggest that the protozoon, like many other
ciliates belonging to a diversity of taxonomic
25
g r o u p s [10] has evolved an a n a e r o b i c lifestyle
from a e r o b i c a n c e s t o r s a n d t h a t t h e symbiosis
with t h e p h o t o s y n t h e t i c b a c t e r i u m evolved in an
a n a e r o b i c host. T h e f u n c t i o n a l significance of t h e
symbionts m a y r e s e m b l e that o f e n d o s y m b i o t i c
m e t h a n o g e n i c b a c t e r i a o f a n a e r o b i c p r o t o z o a in
that t h e b a c t e r i a r e m o v e m e t a b o l i c a l l y p r o d u c e d
h y d r o g e n a n d thus e n h a n c e the f e r m e n t a t i v e energy m e t a b o l i s m of the host cell [7]. T h e i n t e r a c tion with the p h o t o t r o p h i c b a c t e r i a , however, is
m o r e complex. A n a e r o b i c a l l y in the light t h e p u r ple non-sulphur bacteria perform photosynthesis
using h y d r o g e n a n d various low m o l e c u l a r organic c o m p o u n d s (e.g. a c e t a t e , l a c t a t e ) as h y d r o gen d o n o r s [11,16] a n d so they a r e likely to utilize
o t h e r m e t a b o l i c f e r m e n t a t i o n p r o d u c t s o f the host
in a d d i t i o n to H 2. In the d a r k a n d u n d e r a e r o b i c
conditions, the p h o t o t r o p h i c n o n - s u l p h u r b a c t e r i a
carry o u t oxidative p h o s p h o r y l a t i o n using hydrogen a n d volatile fatty acids as substrates. T h u s
t h e symbionts m a y s e c o n d a r i l y have t r a n s f o r m e d
an a n a e r o b i c p r o t o z o o n into an o r g a n i s m which is
m i c r o a e r o p h i l i c in t h e dark.
T h e findings d e m o n s t r a t e t h a t e n d o s y m b i o t i c
p h o t o t r o p h i c b a c t e r i a d o occur. T h e y also supp o r t s o m e s p e c u l a t i o n s a b o u t how t h e origin of
m i t o c h o n d r i a actually t o o k place. T h e s e a r e believed to b e d e s c e n d e d from e n d o s y m b i o t i c eub a c t e r i a b e l o n g i n g to t h e a - g r o u p of the p u r p l e
b a c t e r i a , a n d a m o n g them, t h e p h o t o t r o p h i c
n o n - s u l p h u r b a c t e r i a have b e e n c o n s i d e r e d the
m o s t likely a n c e s t o r s [17-19]. It has b e e n sugg e s t e d t h a t the o u t e r m e m b r a n e of the m i t o c h o n d r i o n d o e s not r e p r e s e n t a v a c u o l e of the host
cell, b u t r a t h e r t h e o u t e r m e m b r a n e of a G r a m negative b a c t e r i u m [20] a n d t h e symbionts o f
Strombidium purpureum a r e not e n c l o s e d in a
vacuole. It has also b e e n s u g g e s t e d t h a t t h e mitoc h o n d r i o n o r i g i n a t e d as an e n d o s y m b i o t i c p h o t o t r o p h i c b a c t e r i u m in an a n a e r o b i c host a n d t h a t
its significance in t e r m s o f r e s p i r a t i o n a r o s e l a t e r
[17]. This s p e c u l a t i o n is attractive in t h a t it explains how t h e symbiotic r e l a t i o n s h i p could initially b e a d a p t i v e for an a n a e r o b i c host as well as
for a (facultatively) a e r o b i c e n d o s y m b i o n t . T h e
d e s c r i b e d symbiotic r e l a t i o n r e p r e s e n t s an analogy for such events.
Acknowledgements
W e a r e grateful to Dr. B.J. F i n l a y for his
c o m m e n t s . T h e study was s u p p o r t e d by grants
from T h e D a n i s h N a t u r a l Science R e s e a r c h
C o u n c i l (11-8391 a n d 11-9608), from the E u r o pean Community (MAST programme, contract
no. 0044) to T.F. a n d from E E R O to C.B.
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