Caedibacter taeniospiralis, an obligate bacterial endosymbiont ofParamecium tetraurelia, confers a killing trait upon its host paramecium. Type 51 R bodies (refractile inclusion bodies) are synthesized by these endosymbionts and are required for expression of the killing trait. The nucleotide sequence of the genetic determinants for type 51 R body synthesis and assembly was determined for C. taeniospiralis 47 and 116. Three independently transcribed genes (rebA, rebB, and rebC) were characterized. To date these are the only genes from C. taeniospiralis to be sequenced and characterized. DNA regulatory regions are recognized by Escherichia coli, and codon usage appears similar to that in E. coli. A fourth open reading frame with appropriate regulatory sequences was found within the reb locus, but no evidence was obtained to suggest that this putative gene is expressed in E. coli. The R body-encoding sequences from both strains are identical. Two-dimensional gel electrophoresis of deletion derivatives shows that two polymerization events are involved in R body assembly. One polymerization event requires only RebB and RebC; the other requires all three proteins. Expression of RebC is necessary for the posttranslational modification of RebA and RebB into species with three and two different molecular weights, respectively. In the presence of RebC, each species of RebB with a different molecular weight has six different isoelectric points.Refractile inclusion bodies, known as R bodies, are produced by only a few species of bacteria. These inclusion bodies are highly insoluble protein ribbons, typically seen coiled into cylindrical structures within the cell. Five classes or types of R bodies have been described and are distinguished on the basis of physical dimensions, morphology, and behavior in response to certain physical and chemical treatments (16,21,22).Several classes of R bodies are produced by Caedibacter species, more commonly known as kappa particles. Caedibacter species are obligate bacterial endosymbionts of Paramecium species and are characterized by their ability to produce both R bodies and a toxin. R body-containing forms of Caedibacter species are toxic to sensitive strains of paramecia. Thus, paramecia that carry any Caedibacter strain are referred to as killers. Killing occurs when a sensitive paramecium ingests an R body-containing kappa particle that has been released into the environment by a killer paramecium (6).Five Caedibacter species, producing three of the five known types of R bodies (types 7, 51, and Cc [18,19]), have been identified. R body synthesis in four of the species is determined by extrachromosomal elements thought to be defective phages (17). In the fifth species, Caedibacter taeniospiralis, R body synthesis is directed by plasmid DNA (23). The R bodies of C. taeniospiralis are type 51. They are 0.4 p.m wide, have a maximum length of 20 ,um, possess acute angles at each end, and unroll in a telescopic fashion when exposed to a pH of 6.5 or lower.The R body-encoding region from...

Caedobacter taeniospiralis (kappa), a bacterial endosymbiont isolated from Paramecium tetraurelia stock 51, contains, in addition to the bacterial chromosome, covalently closed circular DNA molecules as shown by isolation on dye-buoyant-density gradients. The closed circular molecule has a contour length of 13-75 + 0-04 /im with a buoyant density of 1-698 g/cm 3 . The buoyant density of the bacterial chromosome is 1-700-1-701 g/cm 3 . Kappa of the 51 group isolated from stock 298 and stock 6g2, P. tetraurelia, also contain the closed circular DNA. Two forms of kappa coexist in paramecia: brights and nonbrights. Examination by density-gradient centrifugation of the DNA of brights and nonbrights shows the extrachromosomal DNA to be associated mainly with brights. It is suggested that the extrachromosomal DNA might be the determinant for the refractile bodies and the helical phage-like structures found in brights.

Investigative-and cooperative-based learning strategies have been used effectively in a variety of classrooms to enhance student learning and engagement. In the General Microbiology laboratory for juniors and seniors at James Madison University, these strategies were combined to make a semester-long, investigative, cooperative learning experience involving culture and identification of microbial isolates that the students obtained from various environments. To assess whether this strategy was successful, students were asked to complete a survey at the beginning and at the end of the semester regarding their comfort level with a variety of topics. For most of the topics queried, the students reported that their comfort had increased significantly during the semester. Furthermore, this group of students thought that the quality of this investigative lab experience was much better than that of any of their previous lab experiences.

This report describes a mutant strain of Caedibacter taeniospiralis 169 that does not produce refractile (R) bodies or kill sensitive paramecia, but still renders its host resistant to killing by wild-type strains of Caedibacter taeniospiralis. The mutation is due to insertion of a 7.5-kilobase, transposon-like element into the R body-coding region of the plasmid pKAP169. The results provide strong evidence that R body synthesis is required for expression of the killer trait.

We report that the 1.5-and 7.5-kilobase-pair (kbp) transposonlike sequences present in the R-body-coding plasmids of Caedibacter taeniospiralis share homology. The R-body-coding plasmids of two new strains of C. taeniospiralis, derived from strains 169 and A30, carry the 7.5-and 1.5-kbp elements, respectively, inserted at new positions. Sequences homologous to the 7.5-kbp sequence from C. taeniospiralis 47 were detected in the chromosomes of three other strains of C. taeniospiralis.All members of the bacterial species Caedibacter taeniospiralis are obligate cytoplasmic endosymbionts of the freshwater ciliate Paramecium tetraurelia. These bacteria, commonly known as hump killer kappa particles, confer the hump killer trait on their protozoan hosts. In addition, C. taeniospiralis is notable in its ability to synthesize a large inclusion body known as an R body. R bodies are long (approximately 10 ,um), proteinaceous ribbons about 0.5 ,um wide and 13 nm thick, which are rolled up inside the bacterial cell, forming hollow cylinders (1). All strains of C. taeniospiralis carry plasmids (2,7,8). The functions of these plasmids are, for the most part, unknown. However, one function (R-body production) has been demonstrated (9), and another (the killer trait) has been suggested (2).Restriction endonuclease analysis has shown that the R-body-coding plasmids of six strains of C. taeniospiralis have very similar physical maps (8). In every case, the differences observed among these plasmids can be accounted for by single insertion events involving DNA sequences that are about 1.5 or 7.5 kilobase pairs (kbp) in length. In this communication, we report that these inserted sequences share homology with each other and that they appear to be transposable genetic elements.The strains of C. taeniospiralis used in this study are listed in Table 1. Plasmid DNA was purified from these strains with ethidium bromide-cesium chloride gradients as previously described (10). Both upper and lower bands were collected from gradients. Conditions for restriction endonuclease digestions and agarose gel electrophoresis were as previously described (10). Agarose gels were stained for approximately 5 min in ethidium bromide (10 jig/ml) and photographed. To obtain Southern blots (14) of agarose gels, we denatured restriction endonuclease-generated DNA fragments in situ and then transferred them to Gene Screen membranes (New England Nuclear Corp., Boston, Mass.) per the manufacturer's instructions. The recombinant plasmid pBQ54 (9) was used as a probe in Southern blot hybridizations. Probe DNA was radiolabeled by nick translation with 32P-dCTP (ICN Pharmaceuticals, Inc., Irvine, Calif.) as previously described (6 radiolabeled pBQ54 were carried out at 60°C by the method described by Maniatis et al. (5). Physical maps of pKAP30-1 and pKAP169-1 were determined by restriction endonuclease analysis as previously described (10), in which DNA fragment profiles generated by single and double restriction endonuclease digestions were compared. Restriction en...