Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116.

Caedibacter taeniospiralis, an obligate bacterial endosymbiont of Paramecium 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.

A mutation in the R body-coding sequence destroys expression of the killer trait in P. tetraurelia.

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.

Transposonlike elements in Caedibacter taeniospiralis.

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.

Physical map of a plasmid from Caedibacter taeniospiralis 51.

Caedibacter taeniospiralis 51 carries at least two plasmids, pKAP51 and pKAP52. The smaller plasmid, pKAP51, contains 43 kilobase pairs. The larger plasmid, pKAP52, contains more than 110 kilobase pairs. Relative positions of recognition sequences for seven different restriction endonucleases were determined, and a physical map of pKAP51, consisting of a total of 28 restriction sites, was constructed.

Chromosomal and extrachromosomal deoxyribonucleic acid from four bacterial endosymbionts derived from stock 51 of Paramecium tetraurelia.

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 10(7), 9.7 X 10(7), and 11.8 X 10(7) 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 10(7) and 4.4 X 10(7) 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 51m1 pi: 0.3 X 10(7), 2.3 X 10(7), and 4.5 X 10(7) daltons. The buoyant densities of both the chromosoaml DNA and the CC DNA were 1.694 to 1.695 g/cm3. It is not known whether 51m1 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 10(7) 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.