INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1987, p. 459462 Vol. 37, No. 4 0020-7713/87/040459-04$02 .OO/O Copyright 0 1987, International Union of Microbiological Societies

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 The type species is Caedibacter taeniospiralis (4). The observed as endosymbionts of protozoa (1).Unique morpho- detailed description of C. caryophila sp. nov. (see below) logical or behavioral phenotypes can characterize such shows that this species complies with all diagnostic charac- symbiotic relationships (6). Endosymbionts of the genus teristics that qualify it for membership in the genus Caedibacter cause their paramecium hosts to kill susceptible Caedibacter. paramecia (cells without these symbionts), whereas killer Gram-negative, R-body-producing bacteria similar in ap- paramecia themselves are resistant. This is a classic example pearance to those described by Esthe (3) were originally of a unique behavioral phenotype associated with endo- discovered in strains C220 and C221 of P. caudatum. A few symbionts. Furthermore, caedibacteria can produce unusual months after their cultivation in the laboratory, the inclusion bodies (R bodies) which make them morphologi- endosymbionts in strain C220 lost their ability to produce R cally unique. bodies. However, this strain still is a host to nonbright All members of the genus Caedibacter are obligate forms. Nonbright forms of both strains are generally uniform endosymbionts of Paramecium spp. that confer killer traits in size, with a diameter of 0.4 pm. Their average length is 1.0 upon their ciliate hosts (7). Caedibacter spp. are distin- to 1.5 pm, but occasionally much larger rods can be found guished from other bacterial endosymbionts of paramecia which resemble a row of nonbright forms sticking together that also confer killer traits upon their hosts by their ability end to end. Bright forms are bigger, about 0.7 pm wide and to produce R bodies (7). R bodies are long (up to 20 pm), 1.5 to 2.5 pm long (Fig. 3). They are always present in strain proteinaceous ribbons (approximately 0.5 pm wide and 13 C221 with low numbers in rapidly growing cultures. The pm thick) that are rolled up inside the bacterial cell, forming numbers of bright forms per nucleus, however, increased a hollow cylindrical structure. All Caedibacter spp. have considerably when such cultures were starved. Bright forms been described as cytoplasmic endosymbionts occurring in contain R bodies and bacteriophages (Fig. 3). Paramecium biaurelia or Paramecium tetraurelia (6, 7). We C. caryophila is well adapted to its life in a nucleus. The report here an obligate bacterial endosymbiont of Parame- symbionts were usually seen in clusters which are distrib- cium caudatum that confers a killer trait upon its host and uted with the dividing macronuclei. Symbiont-free cells were produces R bodies. Furthermore, this bacterium resides in never found in cultures of strain C221 or C220. The high the macronucleus rather than the cytoplasm (Fig. 1). The infection rate of 100% may be due to the killing activity; that purpose of this paper is to name this organism in accordance is, symbiont-free cells may be killed immediately. However, with the rules of the Bacteriological Code. We propose the no killing activity was detected for strain C220. The infection name Caedibacter caryophila sp. nov. to indicate that the rate of 100% in this stock therefore must be due to very new species is a bacterium which kills and lives in a nucleus. effective (accurate) mechanisms for the balanced distribu- The genus Caedibacter contains bacteria which occur in tion of symbionts at the division of the host nucleus. It was P. biaurelia and P. tetraurelia. All Caedibacter spp. are impossible to free paramecia from their symbionts by rapid toxic to certain susceptible strains of paramecia. They are growth. Attempts to infect symbiont-free P. caudatum gram negative and nonmatile, and up to 50% (usually less strains with isolated nonbright forms or homogenates from than 10%) of the cells in any given population contain single C220 were unsuccessful. Paramecia of strain C220 are obvi- (rarely double) refractile inclusion bodies (R bodies). Cells ously not harmed by their nonbright symbionts. On the other with R bodies are called bright forms (as determined by hand, strain C221 paramecia suffer and eventually die, but phase-contrast microscopy) (5), whereas R-body-free cells only in starving cultures. Their death is probably caused by are referred to as nonbright forms. Nonbright forms are their nonbright forms. Strain C221 is a killer with prelethal considered as the reproductive forms, and some of them symptoms of the paralysis type. Cells from strain C220 do change into bright forms by producing R bodies (5). The R not kill, but they are still resistant against the toxin. It is not body is a proteinaceous ribbon which is tightly rolled up known whether they were killers prior to loosing their bright within the cell. Cells containing R bodies are usually thicker forms. (Fig. 2) than cells that do not contain R bodies and have C. caryophila has a guanine-plus-cytosine (G+C) content certain spherical bacteriophagelike structures or covalently of 34.6 mol% (calculated from thermal denaturation profiles closed circular deoxyribonucleic acid (DNA) plasmids (2). [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, * Corresponding author their banding patterns were very similar (Fig. 4). In addition,

459 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 Re- public of Germany) or Percoll (Pharmacia Fine Chemicals) density gradient centrifugation. C. curyophilu DNA, ex- tracted 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 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). ~39,000.Bar, 1 pm.

coding sequences from pKAP47 plasmids (Schmidt et al., in one is blunt (Fig. 5A and B). Unrolling takes place in a press). telescoping fashion from the inside (Fig. 5C). The R body C. caryophila R bodies are a useful diagnostic character- proteins exhibit some antigenic cross-reactivity against an- istic for the species. They are approximately 0.8 pm in width tisera prepared for type 51 and type 7 R bodies (H. J. and diameter and are larger than any other R bodies from Schmidt, F. R. Pond, and H.-D. Gortz, J. Cell Sci., in press). caedibacteria; they are also visible with the light micro- A summary of diagnostic characteristics is given in Table 1. scope. They are associated with bacteriophages, which are Description of Caedibacter caryophila sp. nov. Caedibacter always seen on the inner terminus of unrolled R bodies (Fig. caryophila (ca.ry.o’phi.la; Gr. noun caryum, nucleus; Gr. 5). These inner ends form an acute angle, whereas the outer 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

TABLE 1. Diagnostic characteristics of C. caryophila sp. nov. Characteristic Diagnosis Host species P. caudatum Site of endosymbiosis Macronucleus Size of nonbright forms (width 0.44.5 pm by 1.0-1.5 pm by length) Size of bright forms (width by 0.7 pm by 1.5-2.5 pm length) Killing Paralysis type FIG. 4. Banding patterns of DNAs (digested with EcoRI) from DNA G + C content 34.6 mol% C. caryophila 221 (lane a) and 220 (lane b) nonbright forms. The R body width (after isolation) 0.8 pm DNAs were separated according to size by electrophoresis on a Mode of unrolling From inside 0.8% agarose gel and stained with ethidium bromide. Lane M gives Outer terminus Blunt fragment sizes (in kilobases) of the A DNA marker digested with Inner terminus Acute HindIII. Arrows indicate the similar banding patterns of some Extrachromosomal elements Phage dominant bands. 462 NOTES INT. J. 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) Pergamon Press, New York. as those reported to occur in the other species of 2. Dilts, J. A. 1976. Covalently closed circular DNA in kappa Caedibacter. R-body-containing cells are toxic to suscepti- endosymbionts of Paramecium. Genet. Res. 27:161-170. ble strains of paramecia, with paralysis being the only 3. Esteve, J.-C. 1978. Une population de type (killer) chez Parame- observable prelethal effect. Appearance of R bodies in C. cium caudutum (Ehrenberg). Protistologica 14:201-207. 4. Preer, J. R., Jr., and L. B. Preer. 1982. Revival of names of caryophila is accompanied by the production of bacterio- protozoan endosymbionts and proposal of Holospora caryophila phage particles. DNA base composition is approximately 35 nom. nov. Int. J. Syst. Bacteriol. 32:14&141. mol% G+C as determined by the thermal denaturation 5. Preer, J. R., Jr., and L. B. Preer. 1984. Endosymbionts, p. method (8). The type strain is 221 carried in P. caudatum 795-942. In N. R. Krieg (ed.), Bergey’s manual of systematic C221 (ATCC 50168). bacteriology, vol. 1. Williams & Wilkins, Baltimore. 6. Preer, J. R., Jr., L. B. Preer, and A. Jurand. 1974. Kappa and We thank C. Kulessa and P. Nikolaus for excellent technical other endosymbionts in Paramecium aurelia. Bacteriol. Rev. assistance. 38: 113-163. This research was supported by grants from the Deutsche 7. Quackenbush, R. L., B. J. Cox, and J. A. Kanabrocki. 1986. Forschungsgemeinschaft (Schm 544, 2-2,3; SFB 310, C2), awarded Extrachromosomal elements of extrachromosomal elements of to H.J.S. and H.-D.G., and by grants GM36293 from the National Paramecium and their extrachromosomal elements, p. 265-278. Institutes of Health and IN8420591 from the National Science In R. B. Wickner, A. Hinnebusch, A. M. Lambowitz, I. C. Foundation, awarded to R.L.Q. Gunsalus, and A. Hollaender (ed.), Extrachromosomal elements in lower eukaryotes. Plenum Publishing Corp., New York. LITERATURE CITED 8. Schmidt, H. J. 1982. Isolation of omikron-endosymbionts from 1. Ball, G. H. 1969. Organisms living on and in protozoa, p. mass cultures of Euplotes aediculatus and characterization of 565-718. In T.-T. Chen (ed.), Research in protozoology. their DNA. Exp. Cell Res. 140:417425.