Vol. 129, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 888-894 Copyright © 1977 American Society for Microbiology
Chromosomal and Extrachromosomal Deoxyribonucleic Acid from Four Bacterial Endosymbionts Derived from Stock 51 of Paramecium tetraurelial JUDITH A. DILTS2 Department of Zoology, Indiana University, Bloomington, Indiana 47401 Received for publication 6 August 1976
Four variant lines of stock 51 kappa (Paramecium tetraurelia) were screened for the presence of covalently closed circular (CCC) deoxyribonucleic acid (DNA). Stock 51m43 kappa, a nonkiller resistant to 51 killing, contained four classes of CCC DNA: 2.9 x 107, 5.9 x 107, 9.7x 107, and 11.8 x 107 daltons. The buoyant densities of 51m43 kappa chromosomal and CCC DNA were 1.700 and 1.698 g/cm3, respectively. Stock 51m43 pi, a sensitive nonkiller, contained two CCC species: 0.3 x 107 and 4.4 x 107 daltons. The buoyant densities of both the chromosomal and CCC DNA were 1.694 to 1.695 g/cm3. Three sizes of CCC DNA were found in 51ml pi: 0.3 x 107, 2.3 x 107, and 4.5 x 107 daltons. The buoyant densities of both the chromosomal DNA and the CCC DNA were 1.694 to 1.695 g/ cm3. It is not known whether 51ml kappa, a sensitive spinner killer, contains CCC DNA. The buoyant density of its chromosomal DNA was 1.703 g/cm3. Of the four variant lines, only 51m43 kappa appears to be a mutant of 51 kappa. The chromosomal and CCC DNAs of 51m43 kappa have the same buoyant densities as those of 51 kappa; in addition, 51m43 kappa contain a CCC molecule the same size as that found in 51 kappa (2.8 x 107 daltons). The three other lines are probably bacterial species that are distinct from 51 kappa and which, at one time, were co-inhabitants with 51 kappa in stock 51 paramecia.
Bacterial endosymbionts are found in many naturally occurring stocks of the Paramecium aurelia complex. Those that remain under laboratory conditions have been classified taxonomically into genera and species (7). Caedobacter taeniospiralis, the species commonly known as kappa, is characterized by having two morphological forms, brights and nonbrights. A bright is a kappa particle with an intracellular refractile body (R body), a coiled ribbon of protein. On the inner end of the coil are found either icosahedral or helical defective phage. For example, stock 7 kappa contain icosahedral phage (Sp7), and stock 51 kappa contain helical phage (He5l). Nonbrights are the reproductive forms of kappa and can, under certain conditions, give rise to brights. Kappa are responsible for the killing trait first reported by Sonneborn (14). Paramecia containing kappa kill paramecia without kappa (sensitives) and are themselves resistant to the killing. Resistance is specific; e.g., stock 51 kappa impart resistance only to stock 51 kappa killing. Brights have been found to be the agent I Contribution 1044 from the Department of Zoology, Indiana University, Bloomington. 2 Present address: Department of Biology, William Jewell College, Liberty, MO 64068.
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of killing. In the 51 group, the killing is classified as hump killing, because the brights cause the formation of prelethal aboral humps in sensitives. The maintenance of kappa is dependent on the dominant gene, K, in the nucleus of Paramecium. Stocks of the genotype KK or Kk will maintain kappa; stocks kk will not. Genic maintenance is generally specific for each species of endosymbiont. Variant lines of stock 51 kappa from P. tetraurelia, assumed by earlier workers to be mutants, show such changes as loss of killing, differing prelethal effects on sensitives, and loss or change of resistance. Genetic analysis has shown that the changes reside not in the paramecium genome but in the kappa (2-4, 16). It is not clear whether the Paramecium variants are the result of mutation, or whether the original killer stock 51 contained multiple types of endosymbionts and the variation observed actually reflects loss of one or more resident bacterial endosymbionts. Recently, it was discovered that stock 51 kappa contained covalently closed circular (CCC) deoxyribonucleic acid (DNA) having a contour length of about 13.75 Am (1). The DNA was found to be associated with bright formation and was thought to code for the R body and
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He5l. The discovery of the plasmid DNA led Preer et al. (7) to suggest that the mutations of kappa might lie not in the kappa genome, as supposed previously, but in the plasmid DNA, even to loss of the plasmid. To shed some light on the origin of the kappa mutants, the variant lines, 51ml (killer causing prelethal spinning on longitudinal axis), 51m43 (resistant nonkiller), and 51ml pi and 51m43 pi (both nonresistant and nonkillers), were screened for the presence of plasmid DNA by dye buoyant density centrifugation. In addition, electron microscope studies and buoyant density determinations were performed. This paper reports the results of such studies, which suggest that 51m43, the resistant nonkiller, is a mutant of 51 kappa, but that the other kappa strains are more likely of independent origin. (This work was taken in part from the dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree, Indiana University, Bloomington, 1976.) MATERIALS AND METHODS
Strains. The strains of P. tetraurelia used in the experiments are listed below, with a brief description of their origin and characteristics: (i) HB. High brights (HB), from the collection of J. R. Preer, Jr., are stock 51 containing wild-type kappa. (Kappas maintain a high proportion of brights, about 35% [7].) (ii) 51m43. 51m43, from the collection of J. R. Preer, Jr., is a strain of stock 51 carrying a mutant kappa and was isolated from a subculture of 51m42 (16). The strain was described originally as a nonkiller, resistant strain evidencing no R bodies, but has since been reported to produce rare R bodies and weak killing (7). (iii) 51m43 pi. 51m43 pi, from the J. R. Preer, Jr., collection, is a strain of stock 51 carrying pi and was isolated from 51m43 by Preer (personal communication). It is a nonkiller, sensitive strain and does not produce R bodies. (iv) 51ml. 51ml, from the J. R. Preer, Jr., collection, is a strain of stock 51 carrying mutant kappa and was isolated from a subculture of stock 51 containing wild-type kappa (2). It is a spinner killer (2) and contains seven type R bodies and spherical phage (9). (v) 51ml pi. 51ml pi, from the T. M. Sonneborn collection, is a strain of stock 51 carrying pi and was isolated from 51ml (8). It is a nonkiller, sensitive strain and does not produce R bodies (8). Culture methods. Strains HB and 51m43 were grown initially in bacteria-free medium containing Chlamydomonas reinhardi and transferred to Cerophyl containing Klebsiella pneumoniae as described previously (1). The paramecia were cultivated at 19 to 20°C. Strain 51ml was cultured continuously in bacterized Cerophyl at 300C and fed for one fission every
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other day. The cultures were not kept free from extraneous bacteria, since attempts to clean the cultures resulted in loss of the mutant kappa. To decrease the number of contaminating bacteria before isolation of the endosymbionts, the cultures were cleaned by a modification of the Singler-Bastiaans method (personal communication); after harvesting, the paramecia were resuspended in 500 ml of bacterized Cerophyl and allowed to sit at room temperature for 1 h. The process was repeated three times. Kappa were then isolated. Strains 51ml pi and 51m43 pi were cultured in bacteria-free medium containing C. reinhardi. The paramecia were made bacteria-free as described previously (10) and cultured at room temperature (about 20°C) in test tubes containing 8 to 10 ml of medium inoculated with C. reinhardi. The paramecia, in 0.5-ml portions, were transferred to new inoculated medium every 2 to 3 days. When large quantities of paramecia were needed, 3-liter flasks containing 1,500 ml of inoculated media were sterilely inoculated with 8-ml samples of paramecia from the test tubes. When the paramecia had cleared the medium they were harvested. All strains of paramecia were harvested and homogenized as described by Sonneborn (15). Isolation of kappa. Isolation of HB, 51m43, and 51ml kappa from anion-exchange epichlorhydrin triethanolamine-cellulose (ECTEOLA) columns was as described by Sonneborn (15). Pi was also isolated from epichlorhydrin triethanolamine-cellulose columns, but, since it was retained on the column, it required eluting with a pH 8.0 buffer of 5 ,uM sodium phosphate and 0.5 M sodium chloride (pH 8 buffer) (L. B. Preer, personal communication). All solutions used were first sterilized by passage through sterile membrane filters (0.45-,um pore size; Millipore Corp., Bedford, Mass.). Samples of isolated kappas and pi were plated on nutrient agar to check for bacterial contamination. Total contamination was always less than 4 x 105 bacteria, a contamination factor of less than 0.1% (105 bacteria: 108 to 1012 kappa). Preparation of DNA. DNA was isolated from the endosymbionts by dye buoyant density centrifugation with ethidium bromide as described previously (1). Techniques used to lyse the strains of kappa in preparation for centrifugation are as follows. Isolated HB kappa or 51m43 kappa in 0.01 M sodium-potassium phosphate buffer at pH 7 (pH 7 buffer) were centrifuged at 48,200 x g for 5 min and suspended in 1.9 ml of 0.15 M NaCl-0.1 M ethylenediaminetetraacetic acid (pH 8.0; saline-EDTA). An equal volume of 1% Sarkosyl (NL-97, K & K Laboratories, Inc., Jamaica, N.Y.) in saline-EDTA was added to the suspension, and the mixture was allowed to sit at room temperature for 10 min, allowing lysis to take place. The pi endosymbionts, 51ml pi and 51m43 pi, isolated in pH 8 buffer were centrifuged at 48,200 x g for 5 min and suspended in 1.5 ml of saline-EDTA. To the suspension was added 0.4 ml of lysozyme (purified six times; Miles Research Laboratories, Elkhart, Ind.) (5 mg/ml in saline-EDTA), and the mixture was allowed to sit in ice for 5 min. Sarkosyl
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(2% in saline-EDTA) was then added to the preparation. The mixture remained at room temperature for 10 min for final lysis. The DNA for 51ml kappa was isolated in the same manner as that of pi, except that 4% Sarkosyl was used, followed by incubation in a 60°C water bath for 10 min. The technique allowed lysis of about 80% of the cells. Electron microscopy and density gradient centrifugation. Electron microscopy and density gradient centrifugation were performed as described previously (1).
less than 2 x 10-6 g of DNA was observed in one isolation of 51ml kappa DNA; however, a subsequent isolation did not yield such a band. Thus, further study is necessary to confirm the presence of the extrachromosomal band. After isolation, the CCC and the linear DNAs were analyzed by buoyant density centrifugation. The control DNA was Micrococcus lysodeikticus (1.731 g/cm3). In addition, electron microscopy was employed to determine the
lengths of the closed circular molecules. The results of the analyses described below are sumRESULTS marized in Table 1. (i) 51 kappa (HB)-Wild-type resistant Analysis of DNA samples of variant endosymbionts was undertaken to shed light on the killer. The buoyant density of the chromosomal origin of the variant lines. The endosymbionts DNA (1.700 to 1.701 g/cm3 ) and of the CCC DNA (1.698 g/cm3) and the contour length of were screened for the presence of extrachromosomal DNA by centrifugation of their DNA on the CCC DNA molecule (13.75 0.04 ,Am) for 51 kappa were reported elsewhere (1). The data for an ethidium bromide-cesium chloride gradient, 51 kappa are included in Table 1 to provide a a process that separates CCC DNA from nicked circular and linear DNA (12). After centrifuga- standard for comparison of the variant strains tion, the CCC DNA is the denser of the two with the wild type. (ii) 51m43 kappa -Resistant nonkiller. The DNA bands found. The majority of the DNA, the linear DNA, is located in the less dense buoyant density determined for 51m43 kappa band. This band is presumed to consist primar- chromosomal DNA was 1.700 g/cm3; that for ily of chromosomal DNA, because it represents CCC DNA was 1.698 g/cm3. Electron microscope analysis of the CCC DNA revealed the a major fraction of the DNA and because of its appearance, in an electron microscope, as long presence of at least four different sizes of molelinear strands of DNA of variable length (1). cules. Their molecular masses calculated from Wild-type (HB) kappa, as reported previously the contour lengths, assuming 1 ,um equals 2.07 (1), contained CCC DNA. In addition, as shown x 106 daltons (6), are: 2.9 x 107, 5.9 x 107, 9.7 x in the present study, 51m43 kappa, 51m43 pi, 107, and 11.8 x 107 daltons. 51m43 kappa has been assumed to be a muand 51ml pi each contained a single band of CCC DNA. To date, there is no conclusive evi- tant of stock 51 kappa (15), with the mutation dence indicating whether 51ml kappa do or do most likely lying in the phage DNA (7). The not harbor CCC DNA. A CCC band containing present results are in agreement with this ±
TABLE 1. Results of buoyant density and electron microscope analyses of the DNA of the kappa mutants Buoyant density (g/cm3) Mean contour length of Mutant plasmid DNA (Am) ± stanMol wtb (x 107) Chromosomal
Plasmid
dard errora
1.700-1.701
1.698
13.75 ± 0.04 (18)
1.700
1.698
14.20 ± 0.2 28.45 ± 0.19 46.95 ± 0.75 56.93 ± 0.96
51m43 pi
1.694-1.695
1.694-1.695
1.51 ± 0.007 (20) 21.02 ± 0.7 (10)
0.3 4.4
51ml pi
1.694-1.695
1.694-1.695
1.53 + 0.007 (30) 11.24 ± 0.14 (16) 21.69 ± 0.34 (25)
0.3 2.3 4.5
51 kappac
51m43 kappa
51ml kappa 1.703 Figures in parentheses indicate number of molecules measured. I Based on 2.07 x 106 daltons/,um (6). c See reference 1. a
(5) (9) (7) (3)
2.8
2.9 5.9 9.7 11.8
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view. The buoyant densities of stock 51 kappa (HB) CCC DNA and 51m43 kappa CCC DNA, measured separately are 1.698 g/cm3. Figure 1, the tracing of the mixture of the two DNA species, shows one band at 1.698 g/cm3. (iii) 51m43 pi-Sensitive nonkiller. Both the chromosomal and the CCC DNA of 51m43 pi had a buoyant density of 1.694 to 1.695 g/cm3. Two sizes of molecules were seen by electron microscopy in the CCC preparations: a very small circle with a molecular weight of 0.3 x 107, and a larger molecule with a molecular weight of 4.4 x 107. Endosymbiont 51m43 pi appeared in a paramecium stock previously carrying 51m43 kappa and was, therefore, thought to have been a mutant of 51m43 kappa, again possibly a change in, or loss of, the phage DNA (7). The buoyant density determinations on both the chromosomal and the CCC DNAs do not support this view. Buoyant density determinations made on mixtures of 51m43 kappa and pi linear and circular DNA confirmed the distinct species found in individual determinations. Figure 2, a tracing of a mixture of kappa and pi chromosomal DNA, shows two well-separated peaks: one (pi) at 1.695 g/cm3 and the other (kappa) at 1.701 g/cm3. The results of the buoyant density determinations of a mixture of kappa and pi CCC DNA are shown in Fig. 3. The major band, at 1.698 g/cm3, is skewed towards a lower density, indicating the presence of two species of DNA. (iv) 51ml kappa-Spinner killer. The buoyant density of chromosomal DNA, isolated from 51ml kappa by ethidium bromide-cesium chloride centrifugation, was 1.703 g/cm3. As with 51m43 kappa, 51ml kappa has been assumed to be a mutant of stock 51 kappa (2), with the mutation occurring in the phage DNA
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l
1.731 l
1.695
.701
/ |
FIG. 2. Densitometer tracing of a mixture of 51m43 kappa and 51m43 pi chromosomal DNAs. The kappa DNA banded at 1.701 g/cm3; the pi DNA banded at 1.695 gcm3. The reference DNA from M.
ly8OdeiktiCUs banded at 1.731 g/cm3 *
/3
,
6 1.6Y8 | /
\
FIG. 3. Densitometer tracing of a mixture of 51m43 kappa and 51m43 pi CCC DNAs. The major band at 1.698 glcm3 is skewed towards a lower density, the presence two distinct species of DNA.indicating reference DNA offrom M. lysodeikticus bandedThe at 1.731 g/cm3.
(7). Since extrachromosomal DNA has not yet been isolated from 51ml kappa, it was not posI sible to compare the extrachromosomal DNAs. Chromosomal DNA was available, however, .73/ 31 and the chromosomal DNA from 51ml kappa I|l was compared with that of stock 51 kappa (HB). I . 1.A /698 51ml kappa chromosomal DNA has a buoyant density of 1.703 g/cm3, whereas that of stock 51 has a buoyant density of 1.700 to 1.701 g/cm3. l l ll Figure 4 is a tracing resulting from a mixture of the two samples. Although two distinct peaks | \ are not present, the peak at 1.703 g/cm3 is Il l skewed towards a lower density, indicating the presence of two DNA species. It appears likely that 51ml kappa did not result simply from a FIG. 1. Densitometer tracing of a mixture of 51 change in the phage DNA, but is a species kappa (HB) and 51m43 kappa CCC DNAs. The distinct from 51 kappa. (v) 51ml pi-Sensitive nonkiller. The buoyDNAs banded at 1.698 glcm3. The reference DNA ant density of both 51ml pi chromosomal and from M. lysodeikticus banded at 1.731 glcm3.
l1I l698*
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presence of brights and hump killing (7), the data presented here and the hybridization studies indicate strongly that 51m43 kappa and 51 1.731 kappa are related. The mutational event leading to the forma1.703 tion of 51m43 kappa may have been caused by X rays. The original paramecium isolate containing 51m43 kappa was isolated from a line (51m42, a weak hump killer) derived from Xray-treated paramecia (16). Whether the mutation lies in the kappa genome or in the plasmid genome is unknown. In 51 kappa, the CCC DNA is associated mainly with brights, indicating that induction, DNA synthesis, and syntheFIG. 4. Densitometer tracing of a mixture of 51 sis of phage and R body protein have occurred kappa and 51ml kappa chromosomal DNAs. The in a normal fashion. In 51m43 kappa, the CCC major band at 1.703 glcm3 is skewed towards a lower DNA is associated with nonbrights, since there density, indicating the presence of two distinct species are few, if any, brights. The size and buoyant of DNA. The reference DNA from M. lysodeikticus density of the CCC DNA molecules in 51m43 banded at 1.731 g/cm3. kappa indicate that the molecules may be concatenates of the 2.9 x 107-dalton molecule. The CCC DNA was calculated at 1.694 to 1.695 g/ presence of the concatenated CCC DNA and the cm3. When the CCC DNA was examined by lack of brights imply that the mutational event electron microscopy, three sizes of molecules affects stages after the initial induction and were identified: 0.3 x 107, 2.3 x 107, and 4.5 x DNA replication process (probably the stages of 107 daltons. DNA cleavage and synthesis of the R body and 51ml pi was isolated from a paramecium phage protein products). The presence of a very stock originally containing 51ml kappa (8). few brights in 51m43 kappa may indicate that Consequently, 51ml pi was thought to be a the mutation is leaky; conversely, the brights mutant of 51ml kappa. 51ml pi and 51ml may represent back mutations. kappa chromosomal DNAs are compared in While indicating that 51m43 kappa is probaFig. 5. Two distinct peaks are present. The pi bly a mutant, the data also provide additional DNA has a buoyant density of 1.695 g/cm3; the evidence supporting the hypothesis that the 51 kappa DNA has a density of 1.703 g/cm3. Again, plasmid is the genetic determinant for the R comparison of extrachromosomal DNAs was bodies and helical structures. The phenotype not possible at this time; however, the chromo- lent to the paramecium by 51m43 kappa is that somal buoyant densities of the two strains are of a nonkiller; very few brights are ever obso different that 51ml pi appears to be a totally served in a preparation of 51m43 kappa. The different endosymbiont.
I
DISCUSSION 1.731 Three of the four endosymbionts examined (51ml pi, 51m43 kappa, and 51m43 pi) contained CCC DNA. Electron microscope studies revealed three species of CCC molecules in 1.703 51ml pi, two species in 51m43 pi, and four species in 51m43 kappa. At present, no infor.6 95 mation is available indicating whether 51ml kappa contains CCC DNA. Of the four "mutants" of stock 51 kappa examined, probably only 51m43 kappa is a true mutant. The chromosomal buoyant densities agree with those of 51 kappa, as do the plasmid densities. In addition, a plasmid molecule of the same size as that found in 51 kappa was found FIG. 5. Densitometer tracing of a mixture of 51 ml in 51m43 kappa. In recent hybridization studies kappa and 51 ml pi chromosomal DNAs. 51 ml kappa on 51m43 kappa DNA, 90% homology with 51 DNA banded at 1.703 g/cm3 and that of 51ml pi kappa DNA was shown (11). In conjunction banded at 1.695 g/cm3. The reference DNA from M. with the similar bacterial morphology and the lysodeikticus banded at 1.731 g/cm3.
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lack of R bodies and, consequently, of brights is in conjunction with the physical change of the plasmid DNA and further demonstrates the role of the plasmid. It seems unlikely that either pi strain represents a mutant of kappa. As this paper has shown, the buoyant densities of their chromosomal DNAs differ significantly from that of 51 kappa. The phenotype of pi also differs greatly from that of 51 kappa; e.g., pi is longer and thinner, it has no brights, it does not kill, it does not confer resistance, and it shows some serological differences (3, 5). In addition, the plasmid species do not correspond at all in either buoyant density or size to the 51 plasmid. Pi appears to be a different bacterial endosymbiont species, a hypothesis supported by recent data (11) showing that the DNA of the two pi strains hybridizes less than 20% with the DNA of 51 kappa. The data presented on buoyant density and, more importantly, recent studies on hybridization (11) showing 100% homology between the two pi strains indicate that 51ml pi and 51m43 pi are probably descendants of the same clone of bacteria. The origin of the paramecia containing the two pi endosymbionts does not rule out this identification. The strains of paramecia from which the pi strains were isolated were sublines of stock 51 containing 51 kappa, originally a single isolate from nature. If the two pi strains are derived from the same clone, it is probable, on the basis of their sizes and buoyant densities, that the plasmids in 51m43 pi are homologous to the 1.5- and 21.7,tm species in 51ml pi. Whether the third plasmid species in 51ml pi, the 11.2-,tm molecule, is truly absent from 51m43 pi or is present in a very small proportion has not been determined. If it is absent and the two pi strains arose from the same clone, the molecule has been lost or gained some time during the 30 years it has been maintained in the laboratory. The 21.7-,um molecule may represent two 11.2-,um molecules arranged tandemly, but the exact relationship between the two molecular species needs to be determined by hybridization studies. The function of all three plasmid species is cryptic at present. Endosymbiont 51ml kappa also is probably not a mutant of 51 kappa. The arguments are analogous to those for pi. As has been showp, the buoyant density of 51ml kappa DNA is different from that of 51 kappa. In addition, Quackenbush (11) hybridized 51ml kappa DNA to 51 kappa DNA and showed only about 23% homology. Finally, the phenotype of 51ml kappa is very different from that of 51 kappa. 51ml kappa cause spinner killing, will kill paramecia containing 51 kappa, and do not impart
893
resistance to 51 kappa killing (2). They maintain a different fission rate and are more slender than 51 (2). In addition, 51ml brights contain morphologically different R bodies and spherical, rather than helical, phage (9). Serological differences also exist (13). If 51ml kappa and pi are not mutants of 51 kappa, how did they arise? It must be concluded that the paramecia of stock 51, when originally isolated from nature, probably contained more than one endosymbiotic bacterial species. Although the other species were present in the paramecia, the phenotype as originally expressed was that of 51 kappa, i.e., a hump killer. Pi lent no visible phenotype to the cell. 51ml kappa, although a killer particle, was probably in the minority, as has been found in stocks 298 and 116, hump killers containing a few kappa having the same type of R bodies as those in 51ml kappa (9). Since conditions in the laboratory varied, the environment favored the other endosymbionts over 51 kappa, and the "mutant" kappa was discovered. It appears, then, that pi and 51ml kappa are most likely independent endosymbiotic bacterial species which, at one time, were co-inhabitants with 51 kappa in P. tetraurelia stock 51. Plasmids have been found in many free-living species of bacteria in roles such as sex factors, R factors, colicinogenic factors, and phage. In addition, many cryptic plasmids have been discovered. The bacterial endosymbionts of paramecia appear to be no different from freeliving species of bacteria in their possession of plasmids. Other endosymbionts are known in paramecia, some of which kill sensitive paramecia and others of which do not. The majority of the other killer endosymbionts do not contain phage structures or subunits; it would be of interest to investigate the role of plasmids, if any, in such killers. ACKNOWLEDGMENTS I wish to express my gratitude to John R. Preer, Jr., for his encouragement, guidance, and friendship throughout the course of this work. Special thanks is also due to Robert Quackenbush for his helpful discussions of the paper, to D. Lang for his instruction in the electron microscopy of DNA, and to Larry Prather for his assistance with the ultracentrifuge. This work was supported by National Science Foundation grant GB-27609 and Public Health Service grant GM20038 from the National Institute of General Medical Sciences (to John R. Preer, Jr.) and by Public Health Service training grant 82 to J. Dilts.
LITERATURE CITED 1. Dilts, J. A. 1976. Covalently closed, circular DNA in kappa endosymbionts of Paramecium. Genet. Res. 27:161-170. 2. Dippell, R. V. 1950. Mutation of the killer cytoplasmic factor in Paramecium aurelia. Heredity 4:165-187. 3. Hanson, E. D. 1954. Studies on kappa-like particles in
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sensitives of Paramecium aurelia, variety 4. Genetics 39:229-239. 4. Hanson, E. D. 1956. Spontaneous mutations affecting the killer character in Paramecium aurelia, variety 4. Genetics 41:21-30. 5. Hanson, E. D. 1958. Test for genetic recombination in kappa particles of Paramecium aurelia, variety 4. Science 128:254. 6. Lang, D. 1970. Molecular weights of coliphages and coliphage DNA. III. Contour length and molecular weight of DNA from bacteriophages T4, T5 and T7 and from Bovine Papilloma Virus. J. Mol. Biol.
54:557-565. 7. Preer, J. R., Jr., L. B. Preer, and A. Jurand. 1974. Kappa and other endosymbionts in Paramecium aurelia. Bacteriol. Rev. 38:113-163. 8. Preer, J. R., Jr., R. W. Siegel, and P. S. Stark. 1953. The relationship between kappa and paramecium in Paramecium aurelia. Proc. Natl. Acad. Sci. U.S.A. 39:1228-1233. 9. Preer, L. B., A. Jurand, J. R. Preer, Jr., and B. M. Rudman. 1972. The classes of kappa in Paramecium aurelia. J. Cell Sci. 11:581-600.
J. BACTERIOL. 10. Preer, L. B., B. M. Rudman, J. R. Preer, Jr., aind A.
11.
12.
13. 14. 15.
16.
Jurand. 1974. Induction of R bodies by ultraviolet light in killer paramecia. J. Gen. Microbiol. 80:209215. Quackenbush, R. L. 1977. Phylogenetic relationships of bacterial endosymbionts of Paramecium aurelia: polynucleotide sequence relationships of 51 kappa and its mutants. J. Bacteriol. 129:895-900. Radloff, R., E. Bauer, and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1501-1521. Siegel, R. W., and J. R. Preer, Jr. 1957. Antigenic relationship among Feulgen positive cytoplasmic particles in Paramecium. Am. Nat. 91:253-257. Sonneborn, T. M. 1938. Mating types, toxic interactions and heredity in Paramecium aurelia. Science 88:503. Sonneborn, T. M. 1970. Methods in Paramecium research. Methods Cell Physiol. 4:241-339. Widmayer, D. J. 1965. A non-killer resistant kappa and its bearing on the interpretation of kappa in Paramecium aurelia. Genetics 51:613-623.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 888-894 Copyright © 1977 American Society for Microbiology
Chromosomal and Extrachromosomal Deoxyribonucleic Acid from Four Bacterial Endosymbionts Derived from Stock 51 of Paramecium tetraurelial JUDITH A. DILTS2 Department of Zoology, Indiana University, Bloomington, Indiana 47401 Received for publication 6 August 1976
Four variant lines of stock 51 kappa (Paramecium tetraurelia) were screened for the presence of covalently closed circular (CCC) deoxyribonucleic acid (DNA). Stock 51m43 kappa, a nonkiller resistant to 51 killing, contained four classes of CCC DNA: 2.9 x 107, 5.9 x 107, 9.7x 107, and 11.8 x 107 daltons. The buoyant densities of 51m43 kappa chromosomal and CCC DNA were 1.700 and 1.698 g/cm3, respectively. Stock 51m43 pi, a sensitive nonkiller, contained two CCC species: 0.3 x 107 and 4.4 x 107 daltons. The buoyant densities of both the chromosomal and CCC DNA were 1.694 to 1.695 g/cm3. Three sizes of CCC DNA were found in 51ml pi: 0.3 x 107, 2.3 x 107, and 4.5 x 107 daltons. The buoyant densities of both the chromosomal DNA and the CCC DNA were 1.694 to 1.695 g/ cm3. It is not known whether 51ml kappa, a sensitive spinner killer, contains CCC DNA. The buoyant density of its chromosomal DNA was 1.703 g/cm3. Of the four variant lines, only 51m43 kappa appears to be a mutant of 51 kappa. The chromosomal and CCC DNAs of 51m43 kappa have the same buoyant densities as those of 51 kappa; in addition, 51m43 kappa contain a CCC molecule the same size as that found in 51 kappa (2.8 x 107 daltons). The three other lines are probably bacterial species that are distinct from 51 kappa and which, at one time, were co-inhabitants with 51 kappa in stock 51 paramecia.
Bacterial endosymbionts are found in many naturally occurring stocks of the Paramecium aurelia complex. Those that remain under laboratory conditions have been classified taxonomically into genera and species (7). Caedobacter taeniospiralis, the species commonly known as kappa, is characterized by having two morphological forms, brights and nonbrights. A bright is a kappa particle with an intracellular refractile body (R body), a coiled ribbon of protein. On the inner end of the coil are found either icosahedral or helical defective phage. For example, stock 7 kappa contain icosahedral phage (Sp7), and stock 51 kappa contain helical phage (He5l). Nonbrights are the reproductive forms of kappa and can, under certain conditions, give rise to brights. Kappa are responsible for the killing trait first reported by Sonneborn (14). Paramecia containing kappa kill paramecia without kappa (sensitives) and are themselves resistant to the killing. Resistance is specific; e.g., stock 51 kappa impart resistance only to stock 51 kappa killing. Brights have been found to be the agent I Contribution 1044 from the Department of Zoology, Indiana University, Bloomington. 2 Present address: Department of Biology, William Jewell College, Liberty, MO 64068.
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of killing. In the 51 group, the killing is classified as hump killing, because the brights cause the formation of prelethal aboral humps in sensitives. The maintenance of kappa is dependent on the dominant gene, K, in the nucleus of Paramecium. Stocks of the genotype KK or Kk will maintain kappa; stocks kk will not. Genic maintenance is generally specific for each species of endosymbiont. Variant lines of stock 51 kappa from P. tetraurelia, assumed by earlier workers to be mutants, show such changes as loss of killing, differing prelethal effects on sensitives, and loss or change of resistance. Genetic analysis has shown that the changes reside not in the paramecium genome but in the kappa (2-4, 16). It is not clear whether the Paramecium variants are the result of mutation, or whether the original killer stock 51 contained multiple types of endosymbionts and the variation observed actually reflects loss of one or more resident bacterial endosymbionts. Recently, it was discovered that stock 51 kappa contained covalently closed circular (CCC) deoxyribonucleic acid (DNA) having a contour length of about 13.75 Am (1). The DNA was found to be associated with bright formation and was thought to code for the R body and
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He5l. The discovery of the plasmid DNA led Preer et al. (7) to suggest that the mutations of kappa might lie not in the kappa genome, as supposed previously, but in the plasmid DNA, even to loss of the plasmid. To shed some light on the origin of the kappa mutants, the variant lines, 51ml (killer causing prelethal spinning on longitudinal axis), 51m43 (resistant nonkiller), and 51ml pi and 51m43 pi (both nonresistant and nonkillers), were screened for the presence of plasmid DNA by dye buoyant density centrifugation. In addition, electron microscope studies and buoyant density determinations were performed. This paper reports the results of such studies, which suggest that 51m43, the resistant nonkiller, is a mutant of 51 kappa, but that the other kappa strains are more likely of independent origin. (This work was taken in part from the dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree, Indiana University, Bloomington, 1976.) MATERIALS AND METHODS
Strains. The strains of P. tetraurelia used in the experiments are listed below, with a brief description of their origin and characteristics: (i) HB. High brights (HB), from the collection of J. R. Preer, Jr., are stock 51 containing wild-type kappa. (Kappas maintain a high proportion of brights, about 35% [7].) (ii) 51m43. 51m43, from the collection of J. R. Preer, Jr., is a strain of stock 51 carrying a mutant kappa and was isolated from a subculture of 51m42 (16). The strain was described originally as a nonkiller, resistant strain evidencing no R bodies, but has since been reported to produce rare R bodies and weak killing (7). (iii) 51m43 pi. 51m43 pi, from the J. R. Preer, Jr., collection, is a strain of stock 51 carrying pi and was isolated from 51m43 by Preer (personal communication). It is a nonkiller, sensitive strain and does not produce R bodies. (iv) 51ml. 51ml, from the J. R. Preer, Jr., collection, is a strain of stock 51 carrying mutant kappa and was isolated from a subculture of stock 51 containing wild-type kappa (2). It is a spinner killer (2) and contains seven type R bodies and spherical phage (9). (v) 51ml pi. 51ml pi, from the T. M. Sonneborn collection, is a strain of stock 51 carrying pi and was isolated from 51ml (8). It is a nonkiller, sensitive strain and does not produce R bodies (8). Culture methods. Strains HB and 51m43 were grown initially in bacteria-free medium containing Chlamydomonas reinhardi and transferred to Cerophyl containing Klebsiella pneumoniae as described previously (1). The paramecia were cultivated at 19 to 20°C. Strain 51ml was cultured continuously in bacterized Cerophyl at 300C and fed for one fission every
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other day. The cultures were not kept free from extraneous bacteria, since attempts to clean the cultures resulted in loss of the mutant kappa. To decrease the number of contaminating bacteria before isolation of the endosymbionts, the cultures were cleaned by a modification of the Singler-Bastiaans method (personal communication); after harvesting, the paramecia were resuspended in 500 ml of bacterized Cerophyl and allowed to sit at room temperature for 1 h. The process was repeated three times. Kappa were then isolated. Strains 51ml pi and 51m43 pi were cultured in bacteria-free medium containing C. reinhardi. The paramecia were made bacteria-free as described previously (10) and cultured at room temperature (about 20°C) in test tubes containing 8 to 10 ml of medium inoculated with C. reinhardi. The paramecia, in 0.5-ml portions, were transferred to new inoculated medium every 2 to 3 days. When large quantities of paramecia were needed, 3-liter flasks containing 1,500 ml of inoculated media were sterilely inoculated with 8-ml samples of paramecia from the test tubes. When the paramecia had cleared the medium they were harvested. All strains of paramecia were harvested and homogenized as described by Sonneborn (15). Isolation of kappa. Isolation of HB, 51m43, and 51ml kappa from anion-exchange epichlorhydrin triethanolamine-cellulose (ECTEOLA) columns was as described by Sonneborn (15). Pi was also isolated from epichlorhydrin triethanolamine-cellulose columns, but, since it was retained on the column, it required eluting with a pH 8.0 buffer of 5 ,uM sodium phosphate and 0.5 M sodium chloride (pH 8 buffer) (L. B. Preer, personal communication). All solutions used were first sterilized by passage through sterile membrane filters (0.45-,um pore size; Millipore Corp., Bedford, Mass.). Samples of isolated kappas and pi were plated on nutrient agar to check for bacterial contamination. Total contamination was always less than 4 x 105 bacteria, a contamination factor of less than 0.1% (105 bacteria: 108 to 1012 kappa). Preparation of DNA. DNA was isolated from the endosymbionts by dye buoyant density centrifugation with ethidium bromide as described previously (1). Techniques used to lyse the strains of kappa in preparation for centrifugation are as follows. Isolated HB kappa or 51m43 kappa in 0.01 M sodium-potassium phosphate buffer at pH 7 (pH 7 buffer) were centrifuged at 48,200 x g for 5 min and suspended in 1.9 ml of 0.15 M NaCl-0.1 M ethylenediaminetetraacetic acid (pH 8.0; saline-EDTA). An equal volume of 1% Sarkosyl (NL-97, K & K Laboratories, Inc., Jamaica, N.Y.) in saline-EDTA was added to the suspension, and the mixture was allowed to sit at room temperature for 10 min, allowing lysis to take place. The pi endosymbionts, 51ml pi and 51m43 pi, isolated in pH 8 buffer were centrifuged at 48,200 x g for 5 min and suspended in 1.5 ml of saline-EDTA. To the suspension was added 0.4 ml of lysozyme (purified six times; Miles Research Laboratories, Elkhart, Ind.) (5 mg/ml in saline-EDTA), and the mixture was allowed to sit in ice for 5 min. Sarkosyl
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(2% in saline-EDTA) was then added to the preparation. The mixture remained at room temperature for 10 min for final lysis. The DNA for 51ml kappa was isolated in the same manner as that of pi, except that 4% Sarkosyl was used, followed by incubation in a 60°C water bath for 10 min. The technique allowed lysis of about 80% of the cells. Electron microscopy and density gradient centrifugation. Electron microscopy and density gradient centrifugation were performed as described previously (1).
less than 2 x 10-6 g of DNA was observed in one isolation of 51ml kappa DNA; however, a subsequent isolation did not yield such a band. Thus, further study is necessary to confirm the presence of the extrachromosomal band. After isolation, the CCC and the linear DNAs were analyzed by buoyant density centrifugation. The control DNA was Micrococcus lysodeikticus (1.731 g/cm3). In addition, electron microscopy was employed to determine the
lengths of the closed circular molecules. The results of the analyses described below are sumRESULTS marized in Table 1. (i) 51 kappa (HB)-Wild-type resistant Analysis of DNA samples of variant endosymbionts was undertaken to shed light on the killer. The buoyant density of the chromosomal origin of the variant lines. The endosymbionts DNA (1.700 to 1.701 g/cm3 ) and of the CCC DNA (1.698 g/cm3) and the contour length of were screened for the presence of extrachromosomal DNA by centrifugation of their DNA on the CCC DNA molecule (13.75 0.04 ,Am) for 51 kappa were reported elsewhere (1). The data for an ethidium bromide-cesium chloride gradient, 51 kappa are included in Table 1 to provide a a process that separates CCC DNA from nicked circular and linear DNA (12). After centrifuga- standard for comparison of the variant strains tion, the CCC DNA is the denser of the two with the wild type. (ii) 51m43 kappa -Resistant nonkiller. The DNA bands found. The majority of the DNA, the linear DNA, is located in the less dense buoyant density determined for 51m43 kappa band. This band is presumed to consist primar- chromosomal DNA was 1.700 g/cm3; that for ily of chromosomal DNA, because it represents CCC DNA was 1.698 g/cm3. Electron microscope analysis of the CCC DNA revealed the a major fraction of the DNA and because of its appearance, in an electron microscope, as long presence of at least four different sizes of molelinear strands of DNA of variable length (1). cules. Their molecular masses calculated from Wild-type (HB) kappa, as reported previously the contour lengths, assuming 1 ,um equals 2.07 (1), contained CCC DNA. In addition, as shown x 106 daltons (6), are: 2.9 x 107, 5.9 x 107, 9.7 x in the present study, 51m43 kappa, 51m43 pi, 107, and 11.8 x 107 daltons. 51m43 kappa has been assumed to be a muand 51ml pi each contained a single band of CCC DNA. To date, there is no conclusive evi- tant of stock 51 kappa (15), with the mutation dence indicating whether 51ml kappa do or do most likely lying in the phage DNA (7). The not harbor CCC DNA. A CCC band containing present results are in agreement with this ±
TABLE 1. Results of buoyant density and electron microscope analyses of the DNA of the kappa mutants Buoyant density (g/cm3) Mean contour length of Mutant plasmid DNA (Am) ± stanMol wtb (x 107) Chromosomal
Plasmid
dard errora
1.700-1.701
1.698
13.75 ± 0.04 (18)
1.700
1.698
14.20 ± 0.2 28.45 ± 0.19 46.95 ± 0.75 56.93 ± 0.96
51m43 pi
1.694-1.695
1.694-1.695
1.51 ± 0.007 (20) 21.02 ± 0.7 (10)
0.3 4.4
51ml pi
1.694-1.695
1.694-1.695
1.53 + 0.007 (30) 11.24 ± 0.14 (16) 21.69 ± 0.34 (25)
0.3 2.3 4.5
51 kappac
51m43 kappa
51ml kappa 1.703 Figures in parentheses indicate number of molecules measured. I Based on 2.07 x 106 daltons/,um (6). c See reference 1. a
(5) (9) (7) (3)
2.8
2.9 5.9 9.7 11.8
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view. The buoyant densities of stock 51 kappa (HB) CCC DNA and 51m43 kappa CCC DNA, measured separately are 1.698 g/cm3. Figure 1, the tracing of the mixture of the two DNA species, shows one band at 1.698 g/cm3. (iii) 51m43 pi-Sensitive nonkiller. Both the chromosomal and the CCC DNA of 51m43 pi had a buoyant density of 1.694 to 1.695 g/cm3. Two sizes of molecules were seen by electron microscopy in the CCC preparations: a very small circle with a molecular weight of 0.3 x 107, and a larger molecule with a molecular weight of 4.4 x 107. Endosymbiont 51m43 pi appeared in a paramecium stock previously carrying 51m43 kappa and was, therefore, thought to have been a mutant of 51m43 kappa, again possibly a change in, or loss of, the phage DNA (7). The buoyant density determinations on both the chromosomal and the CCC DNAs do not support this view. Buoyant density determinations made on mixtures of 51m43 kappa and pi linear and circular DNA confirmed the distinct species found in individual determinations. Figure 2, a tracing of a mixture of kappa and pi chromosomal DNA, shows two well-separated peaks: one (pi) at 1.695 g/cm3 and the other (kappa) at 1.701 g/cm3. The results of the buoyant density determinations of a mixture of kappa and pi CCC DNA are shown in Fig. 3. The major band, at 1.698 g/cm3, is skewed towards a lower density, indicating the presence of two species of DNA. (iv) 51ml kappa-Spinner killer. The buoyant density of chromosomal DNA, isolated from 51ml kappa by ethidium bromide-cesium chloride centrifugation, was 1.703 g/cm3. As with 51m43 kappa, 51ml kappa has been assumed to be a mutant of stock 51 kappa (2), with the mutation occurring in the phage DNA
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l
1.731 l
1.695
.701
/ |
FIG. 2. Densitometer tracing of a mixture of 51m43 kappa and 51m43 pi chromosomal DNAs. The kappa DNA banded at 1.701 g/cm3; the pi DNA banded at 1.695 gcm3. The reference DNA from M.
ly8OdeiktiCUs banded at 1.731 g/cm3 *
/3
,
6 1.6Y8 | /
\
FIG. 3. Densitometer tracing of a mixture of 51m43 kappa and 51m43 pi CCC DNAs. The major band at 1.698 glcm3 is skewed towards a lower density, the presence two distinct species of DNA.indicating reference DNA offrom M. lysodeikticus bandedThe at 1.731 g/cm3.
(7). Since extrachromosomal DNA has not yet been isolated from 51ml kappa, it was not posI sible to compare the extrachromosomal DNAs. Chromosomal DNA was available, however, .73/ 31 and the chromosomal DNA from 51ml kappa I|l was compared with that of stock 51 kappa (HB). I . 1.A /698 51ml kappa chromosomal DNA has a buoyant density of 1.703 g/cm3, whereas that of stock 51 has a buoyant density of 1.700 to 1.701 g/cm3. l l ll Figure 4 is a tracing resulting from a mixture of the two samples. Although two distinct peaks | \ are not present, the peak at 1.703 g/cm3 is Il l skewed towards a lower density, indicating the presence of two DNA species. It appears likely that 51ml kappa did not result simply from a FIG. 1. Densitometer tracing of a mixture of 51 change in the phage DNA, but is a species kappa (HB) and 51m43 kappa CCC DNAs. The distinct from 51 kappa. (v) 51ml pi-Sensitive nonkiller. The buoyDNAs banded at 1.698 glcm3. The reference DNA ant density of both 51ml pi chromosomal and from M. lysodeikticus banded at 1.731 glcm3.
l1I l698*
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presence of brights and hump killing (7), the data presented here and the hybridization studies indicate strongly that 51m43 kappa and 51 1.731 kappa are related. The mutational event leading to the forma1.703 tion of 51m43 kappa may have been caused by X rays. The original paramecium isolate containing 51m43 kappa was isolated from a line (51m42, a weak hump killer) derived from Xray-treated paramecia (16). Whether the mutation lies in the kappa genome or in the plasmid genome is unknown. In 51 kappa, the CCC DNA is associated mainly with brights, indicating that induction, DNA synthesis, and syntheFIG. 4. Densitometer tracing of a mixture of 51 sis of phage and R body protein have occurred kappa and 51ml kappa chromosomal DNAs. The in a normal fashion. In 51m43 kappa, the CCC major band at 1.703 glcm3 is skewed towards a lower DNA is associated with nonbrights, since there density, indicating the presence of two distinct species are few, if any, brights. The size and buoyant of DNA. The reference DNA from M. lysodeikticus density of the CCC DNA molecules in 51m43 banded at 1.731 g/cm3. kappa indicate that the molecules may be concatenates of the 2.9 x 107-dalton molecule. The CCC DNA was calculated at 1.694 to 1.695 g/ presence of the concatenated CCC DNA and the cm3. When the CCC DNA was examined by lack of brights imply that the mutational event electron microscopy, three sizes of molecules affects stages after the initial induction and were identified: 0.3 x 107, 2.3 x 107, and 4.5 x DNA replication process (probably the stages of 107 daltons. DNA cleavage and synthesis of the R body and 51ml pi was isolated from a paramecium phage protein products). The presence of a very stock originally containing 51ml kappa (8). few brights in 51m43 kappa may indicate that Consequently, 51ml pi was thought to be a the mutation is leaky; conversely, the brights mutant of 51ml kappa. 51ml pi and 51ml may represent back mutations. kappa chromosomal DNAs are compared in While indicating that 51m43 kappa is probaFig. 5. Two distinct peaks are present. The pi bly a mutant, the data also provide additional DNA has a buoyant density of 1.695 g/cm3; the evidence supporting the hypothesis that the 51 kappa DNA has a density of 1.703 g/cm3. Again, plasmid is the genetic determinant for the R comparison of extrachromosomal DNAs was bodies and helical structures. The phenotype not possible at this time; however, the chromo- lent to the paramecium by 51m43 kappa is that somal buoyant densities of the two strains are of a nonkiller; very few brights are ever obso different that 51ml pi appears to be a totally served in a preparation of 51m43 kappa. The different endosymbiont.
I
DISCUSSION 1.731 Three of the four endosymbionts examined (51ml pi, 51m43 kappa, and 51m43 pi) contained CCC DNA. Electron microscope studies revealed three species of CCC molecules in 1.703 51ml pi, two species in 51m43 pi, and four species in 51m43 kappa. At present, no infor.6 95 mation is available indicating whether 51ml kappa contains CCC DNA. Of the four "mutants" of stock 51 kappa examined, probably only 51m43 kappa is a true mutant. The chromosomal buoyant densities agree with those of 51 kappa, as do the plasmid densities. In addition, a plasmid molecule of the same size as that found in 51 kappa was found FIG. 5. Densitometer tracing of a mixture of 51 ml in 51m43 kappa. In recent hybridization studies kappa and 51 ml pi chromosomal DNAs. 51 ml kappa on 51m43 kappa DNA, 90% homology with 51 DNA banded at 1.703 g/cm3 and that of 51ml pi kappa DNA was shown (11). In conjunction banded at 1.695 g/cm3. The reference DNA from M. with the similar bacterial morphology and the lysodeikticus banded at 1.731 g/cm3.
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lack of R bodies and, consequently, of brights is in conjunction with the physical change of the plasmid DNA and further demonstrates the role of the plasmid. It seems unlikely that either pi strain represents a mutant of kappa. As this paper has shown, the buoyant densities of their chromosomal DNAs differ significantly from that of 51 kappa. The phenotype of pi also differs greatly from that of 51 kappa; e.g., pi is longer and thinner, it has no brights, it does not kill, it does not confer resistance, and it shows some serological differences (3, 5). In addition, the plasmid species do not correspond at all in either buoyant density or size to the 51 plasmid. Pi appears to be a different bacterial endosymbiont species, a hypothesis supported by recent data (11) showing that the DNA of the two pi strains hybridizes less than 20% with the DNA of 51 kappa. The data presented on buoyant density and, more importantly, recent studies on hybridization (11) showing 100% homology between the two pi strains indicate that 51ml pi and 51m43 pi are probably descendants of the same clone of bacteria. The origin of the paramecia containing the two pi endosymbionts does not rule out this identification. The strains of paramecia from which the pi strains were isolated were sublines of stock 51 containing 51 kappa, originally a single isolate from nature. If the two pi strains are derived from the same clone, it is probable, on the basis of their sizes and buoyant densities, that the plasmids in 51m43 pi are homologous to the 1.5- and 21.7,tm species in 51ml pi. Whether the third plasmid species in 51ml pi, the 11.2-,tm molecule, is truly absent from 51m43 pi or is present in a very small proportion has not been determined. If it is absent and the two pi strains arose from the same clone, the molecule has been lost or gained some time during the 30 years it has been maintained in the laboratory. The 21.7-,um molecule may represent two 11.2-,um molecules arranged tandemly, but the exact relationship between the two molecular species needs to be determined by hybridization studies. The function of all three plasmid species is cryptic at present. Endosymbiont 51ml kappa also is probably not a mutant of 51 kappa. The arguments are analogous to those for pi. As has been showp, the buoyant density of 51ml kappa DNA is different from that of 51 kappa. In addition, Quackenbush (11) hybridized 51ml kappa DNA to 51 kappa DNA and showed only about 23% homology. Finally, the phenotype of 51ml kappa is very different from that of 51 kappa. 51ml kappa cause spinner killing, will kill paramecia containing 51 kappa, and do not impart
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resistance to 51 kappa killing (2). They maintain a different fission rate and are more slender than 51 (2). In addition, 51ml brights contain morphologically different R bodies and spherical, rather than helical, phage (9). Serological differences also exist (13). If 51ml kappa and pi are not mutants of 51 kappa, how did they arise? It must be concluded that the paramecia of stock 51, when originally isolated from nature, probably contained more than one endosymbiotic bacterial species. Although the other species were present in the paramecia, the phenotype as originally expressed was that of 51 kappa, i.e., a hump killer. Pi lent no visible phenotype to the cell. 51ml kappa, although a killer particle, was probably in the minority, as has been found in stocks 298 and 116, hump killers containing a few kappa having the same type of R bodies as those in 51ml kappa (9). Since conditions in the laboratory varied, the environment favored the other endosymbionts over 51 kappa, and the "mutant" kappa was discovered. It appears, then, that pi and 51ml kappa are most likely independent endosymbiotic bacterial species which, at one time, were co-inhabitants with 51 kappa in P. tetraurelia stock 51. Plasmids have been found in many free-living species of bacteria in roles such as sex factors, R factors, colicinogenic factors, and phage. In addition, many cryptic plasmids have been discovered. The bacterial endosymbionts of paramecia appear to be no different from freeliving species of bacteria in their possession of plasmids. Other endosymbionts are known in paramecia, some of which kill sensitive paramecia and others of which do not. The majority of the other killer endosymbionts do not contain phage structures or subunits; it would be of interest to investigate the role of plasmids, if any, in such killers. ACKNOWLEDGMENTS I wish to express my gratitude to John R. Preer, Jr., for his encouragement, guidance, and friendship throughout the course of this work. Special thanks is also due to Robert Quackenbush for his helpful discussions of the paper, to D. Lang for his instruction in the electron microscopy of DNA, and to Larry Prather for his assistance with the ultracentrifuge. This work was supported by National Science Foundation grant GB-27609 and Public Health Service grant GM20038 from the National Institute of General Medical Sciences (to John R. Preer, Jr.) and by Public Health Service training grant 82 to J. Dilts.
LITERATURE CITED 1. Dilts, J. A. 1976. Covalently closed, circular DNA in kappa endosymbionts of Paramecium. Genet. Res. 27:161-170. 2. Dippell, R. V. 1950. Mutation of the killer cytoplasmic factor in Paramecium aurelia. Heredity 4:165-187. 3. Hanson, E. D. 1954. Studies on kappa-like particles in
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sensitives of Paramecium aurelia, variety 4. Genetics 39:229-239. 4. Hanson, E. D. 1956. Spontaneous mutations affecting the killer character in Paramecium aurelia, variety 4. Genetics 41:21-30. 5. Hanson, E. D. 1958. Test for genetic recombination in kappa particles of Paramecium aurelia, variety 4. Science 128:254. 6. Lang, D. 1970. Molecular weights of coliphages and coliphage DNA. III. Contour length and molecular weight of DNA from bacteriophages T4, T5 and T7 and from Bovine Papilloma Virus. J. Mol. Biol.
54:557-565. 7. Preer, J. R., Jr., L. B. Preer, and A. Jurand. 1974. Kappa and other endosymbionts in Paramecium aurelia. Bacteriol. Rev. 38:113-163. 8. Preer, J. R., Jr., R. W. Siegel, and P. S. Stark. 1953. The relationship between kappa and paramecium in Paramecium aurelia. Proc. Natl. Acad. Sci. U.S.A. 39:1228-1233. 9. Preer, L. B., A. Jurand, J. R. Preer, Jr., and B. M. Rudman. 1972. The classes of kappa in Paramecium aurelia. J. Cell Sci. 11:581-600.
J. BACTERIOL. 10. Preer, L. B., B. M. Rudman, J. R. Preer, Jr., aind A.
11.
12.
13. 14. 15.
16.
Jurand. 1974. Induction of R bodies by ultraviolet light in killer paramecia. J. Gen. Microbiol. 80:209215. Quackenbush, R. L. 1977. Phylogenetic relationships of bacterial endosymbionts of Paramecium aurelia: polynucleotide sequence relationships of 51 kappa and its mutants. J. Bacteriol. 129:895-900. Radloff, R., E. Bauer, and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1501-1521. Siegel, R. W., and J. R. Preer, Jr. 1957. Antigenic relationship among Feulgen positive cytoplasmic particles in Paramecium. Am. Nat. 91:253-257. Sonneborn, T. M. 1938. Mating types, toxic interactions and heredity in Paramecium aurelia. Science 88:503. Sonneborn, T. M. 1970. Methods in Paramecium research. Methods Cell Physiol. 4:241-339. Widmayer, D. J. 1965. A non-killer resistant kappa and its bearing on the interpretation of kappa in Paramecium aurelia. Genetics 51:613-623.
