INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1987, p. 459462
0020-7713/87/040459-04$02.OO/O
Copyright 0 1987, International Union of Microbiological Societies
Vol. 37, No. 4
Caedibacter caryophila sp. nov. a Killer Symbiont Inhabiting the
Macronucleus of Paramecium caudatum
HELMUT J. SCHMIDT,l* HANS-DIETER GORTZ,' AND ROBERT L. QUACKENBUSH*
Zoologisches Institint der Universitat Munster, D 4400 Munster, Federal Republic of Germany,' and Department of
Microbiology, University of South Dakota School of Medicine, Vermillion, South Dakota 570692
Caedibacter caryophila sp. nov. lives in the macronucleus of certain strains of Paramecium caudatum. The
type strain is 221, carried in P . caudatum C221. C . caryophila is distinguished from other caedibacteria on the
basis of host specificity, R body morphology, behavior of R bodies, and guanine-plus-cytosinecontent of its
deoxyribonucleic acid.
Bacteria, fungi, algae, and even other protozoa have been
observed as endosymbionts of protozoa (1).Unique morphological or behavioral phenotypes can characterize such
symbiotic relationships (6). Endosymbionts of the genus
Caedibacter cause their paramecium hosts to kill susceptible
paramecia (cells without these symbionts), whereas killer
paramecia themselves are resistant. This is a classic example
of a unique behavioral phenotype associated with endosymbionts. Furthermore, caedibacteria can produce unusual
inclusion bodies (R bodies) which make them morphologically unique.
All members of the genus Caedibacter are obligate
endosymbionts of Paramecium spp. that confer killer traits
upon their ciliate hosts (7). Caedibacter spp. are distinguished from other bacterial endosymbionts of paramecia
that also confer killer traits upon their hosts by their ability
to produce R bodies (7). R bodies are long (up to 20 pm),
proteinaceous ribbons (approximately 0.5 pm wide and 13
pm thick) that are rolled up inside the bacterial cell, forming
a hollow cylindrical structure. All Caedibacter spp. have
been described as cytoplasmic endosymbionts occurring in
Paramecium biaurelia or Paramecium tetraurelia (6, 7). We
report here an obligate bacterial endosymbiont of Paramecium caudatum that confers a killer trait upon its host and
produces R bodies. Furthermore, this bacterium resides in
the macronucleus rather than the cytoplasm (Fig. 1). The
purpose of this paper is to name this organism in accordance
with the rules of the Bacteriological Code. We propose the
name Caedibacter caryophila sp. nov. to indicate that the
new species is a bacterium which kills and lives in a nucleus.
The genus Caedibacter contains bacteria which occur in
P. biaurelia and P . tetraurelia. All Caedibacter spp. are
toxic to certain susceptible strains of paramecia. They are
gram negative and nonmatile, and up to 50% (usually less
than 10%) of the cells in any given population contain single
(rarely double) refractile inclusion bodies (R bodies). Cells
with R bodies are called bright forms (as determined by
phase-contrast microscopy) ( 5 ) , whereas R-body-free cells
are referred to as nonbright forms. Nonbright forms are
considered as the reproductive forms, and some of them
change into bright forms by producing R bodies ( 5 ) . The R
body is a proteinaceous ribbon which is tightly rolled up
within the cell. Cells containing R bodies are usually thicker
(Fig. 2) than cells that do not contain R bodies and have
certain spherical bacteriophagelike structures or covalently
closed circular deoxyribonucleic acid (DNA) plasmids (2).
The type species is Caedibacter taeniospiralis (4). The
detailed description of C. caryophila sp. nov. (see below)
shows that this species complies with all diagnostic characteristics that qualify it for membership in the genus
Caedibacter.
Gram-negative, R-body-producing bacteria similar in appearance to those described by E s t h e (3) were originally
discovered in strains C220 and C221 of P . caudatum. A few
months after their cultivation in the laboratory, the
endosymbionts in strain C220 lost their ability to produce R
bodies. However, this strain still is a host to nonbright
forms. Nonbright forms of both strains are generally uniform
in size, with a diameter of 0.4 pm. Their average length is 1.0
to 1.5 pm, but occasionally much larger rods can be found
which resemble a row of nonbright forms sticking together
end to end. Bright forms are bigger, about 0.7 pm wide and
1.5 to 2.5 pm long (Fig. 3). They are always present in strain
C221 with low numbers in rapidly growing cultures. The
numbers of bright forms per nucleus, however, increased
considerably when such cultures were starved. Bright forms
contain R bodies and bacteriophages (Fig. 3).
C. caryophila is well adapted to its life in a nucleus. The
symbionts were usually seen in clusters which are distributed with the dividing macronuclei. Symbiont-free cells were
never found in cultures of strain C221 or C220. The high
infection rate of 100% may be due to the killing activity; that
is, symbiont-free cells may be killed immediately. However,
no killing activity was detected for strain C220. The infection
rate of 100% in this stock therefore must be due to very
effective (accurate) mechanisms for the balanced distribution of symbionts at the division of the host nucleus. It was
impossible to free paramecia from their symbionts by rapid
growth. Attempts to infect symbiont-free P . caudatum
strains with isolated nonbright forms or homogenates from
C220 were unsuccessful. Paramecia of strain C220 are obviously not harmed by their nonbright symbionts. On the other
hand, strain C221 paramecia suffer and eventually die, but
only in starving cultures. Their death is probably caused by
their nonbright forms. Strain C221 is a killer with prelethal
symptoms of the paralysis type. Cells from strain C220 do
not kill, but they are still resistant against the toxin. It is not
known whether they were killers prior to loosing their bright
forms.
C. caryophila has a guanine-plus-cytosine (G+C) content
of 34.6 mol% (calculated from thermal denaturation profiles
[T,] [S]). When isolated DNA from strain C220 symbionts
and the DNA from C221 nonbright forms was digested with
EcoRI restriction enzymes and electrophoresed on a gel,
their banding patterns were very similar (Fig. 4). In addition,
* Corresponding author
459
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FIG. 1. Electron micrograph of P.caudutum C221. In the macronucleus (Ma), a cluster of bacterial endosymbionts is surrounded by large
areas filled with small chromatin bodies and nucleoli (arrows). The cytoplasm (Cy) is free of bacteria. T, Trichocysts. x 10,000. Bar, 1 pm.
-
both DNAs hybridize strongly with each other on Southern
blots (H. J. Schmidt, H.-D. Gortz, F. R. Pond, and R. L.
Quackenbush, Exp. Cell Res., in press).
These results together with the observations that strain
C220 endosymbionts could produce bright forms when first
cultured in the laboratory and that this strain is still resistant
against killing strongly support the conclusion that both strains
are of the same species, and that an unknown event destroyed
the ability for R body production in C220 endonucleobionts.
Both nonbright and bright forms (and their R bodies) can
be isolated from paramecium homogenates by ion-exchange
chromatography (Ecteola; Serva, Heidelberg, Federal Republic of Germany) or Percoll (Pharmacia Fine Chemicals)
density gradient centrifugation. C. curyophilu DNA, extracted from such preparations, does not hybridize to R body
FIG. 2. C. caryophilci floating in culture medium after the host
cell was crushed with the cover glass. Arrows indicate bright forms.
Unfixed, unstained, phase-contrast, microflash. x 1,600. Bar, 10
I*m.
460
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VOL. 37, 1987
NOTES
461
FIG. 3. Electron micrograph of C. catyophila 221 in the macronucleus of P . caudaturn. B, Bright forms; N , nonbright forms. Arrows
indicate phages associated with R bodies (lamellar structures). ~ 3 9 , 0 0 0 Bar,
.
1 pm.
coding sequences from pKAP47 plasmids (Schmidt et al., in
press).
C. caryophila R bodies are a useful diagnostic characteristic for the species. They are approximately 0.8 pm in width
and diameter and are larger than any other R bodies from
caedibacteria; they are also visible with the light microscope. They are associated with bacteriophages, which are
always seen on the inner terminus of unrolled R bodies (Fig.
5 ) . These inner ends form an acute angle, whereas the outer
one is blunt (Fig. 5A and B). Unrolling takes place in a
telescoping fashion from the inside (Fig. 5C). The R body
proteins exhibit some antigenic cross-reactivity against antisera prepared for type 51 and type 7 R bodies (H. J.
Schmidt, F. R. Pond, and H.-D. Gortz, J. Cell Sci., in press).
A summary of diagnostic characteristics is given in Table 1.
Description of Caedibacter caryophila sp. nov. Caedibacter
caryophila (ca.ry.o’phi.la; Gr. noun caryum, nucleus; Gr.
adj. philus, loving; M. L. adj. caryophila, nucleus loving).
Obligate macronuclear endosymbionts of P . caudatum.
Gram-negative rods with rounded termini, probably best
described as elongated elipsoids. Within any population of
C. caryophila, a small percentage (usually <lo%) of the
individuals contain R bodies. R-body-containing cells are
larger (1.5 to 2.5 pm long by 0.7 pm wide) than cells without
R bodies (1.0 to 1.5 pm by 0.4 pm). C. caryophila R bodies
are about twice as large (approximately 0.8 pm long by 0.5
T A B L E 1. Diagnostic characteristics of C . caryophila sp. nov.
FIG. 4. Banding patterns of DNAs (digested with EcoRI) from
C. caryophila 221 (lane a) and 220 (lane b ) nonbright forms. The
DNAs were separated according to size by electrophoresis o n a
0.8% agarose gel and stained with ethidium bromide. Lane M gives
fragment sizes (in kilobases) of the A DNA marker digested with
HindIII. Arrows indicate the similar banding patterns of some
dominant bands.
Characteristic
Diagnosis
Host species
Site of endosymbiosis
Size of nonbright forms (width
by length)
Size of bright forms (width by
length)
Killing
D N A G + C content
R body width (after isolation)
Mode of unrolling
Outer terminus
Inner terminus
Extrachromosomal elements
P . caudatum
Macronucleus
0.44.5 p m by 1.0-1.5 p m
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0.7 p m by 1.5-2.5 p m
Paralysis type
34.6 mol%
0.8 p m
From inside
Blunt
Acute
Phage
462
INT. J.
NOTES
SYST.
BACTERIOL.
FIG. 5. Electron micrographs of isolated R bodies from C. caryophila. Negative stain, phosphotungstic acid. (A) Acute inner terminus,
showing bacteriophages (arrows), of an unrolled R body (~24,000;bar, 1 pm). (B) Blunt outer terminus of an unrolled R body (~30,000;bar,
1 pm). (C) R body starting to unroll from inside in a telescoping fashion (~24,000;bar, 1 pm).
pm in diameter in the rolled up form inside the bacterial cell)
as those reported to occur in the other species of
Caedibacter. R-body-containing cells are toxic to susceptible strains of paramecia, with paralysis being the only
observable prelethal effect. Appearance of R bodies in C.
caryophila is accompanied by the production of bacteriophage particles. DNA base composition is approximately 35
mol% G+C as determined by the thermal denaturation
method (8). The type strain is 221 carried in P . caudatum
C221 (ATCC 50168).
We thank C. Kulessa and P. Nikolaus for excellent technical
assistance.
This research was supported by grants from the Deutsche
Forschungsgemeinschaft (Schm 544, 2-2,3; SFB 310, C2), awarded
to H.J.S. and H.-D.G., and by grants GM36293 from the National
Institutes of Health and IN8420591 from the National Science
Foundation, awarded to R.L.Q.
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