No abstract

Type 51 R bodies are produced by all bacterial endosymbionts (Caedibacter taeniospiralis) of Paramecium tetraurelia that confer the hump-killer trait upon their hosts. Type 51 R-body synthesis by C. taeniospiralis is required for expression of the hump-killer trait. The genetic determinants for type 51 R-body synthesis by C. taeniospiralis 47 have been cloned and expressed in Escherichia coli. In this communication we describe three species of polypeptides required for R-body synthesis and the organization of their genetic determinants. Each polypeptide species is controlled by a separate gene that is expressed as an independent transcriptional unit possessing regulatory signals that are recognized by E. coli. Two polypeptide species of 10 and 18 kilodaltons are required for R-body synthesis but apparently are not structural subunits. The third polypeptide species (13 kilodaltons) is the major structural subunit. R-body assembly involves polymerization reactions that result in high-molecular-mass polypeptide complexes, primarily composed of the 13-kilodalton polypeptide species, that appear to be the result of covalent cross-linking between structural subunits. The results presented here have been suggested to apply to the assembly and structure of all type 51 R bodies, but not necessarily to other R-body types.Bacterial endosymbionts commonly occur in various species of Paramecium. The most notable group of these bacteria comprise the genus Caedibacter, more commonly known as kappa or kappa particles. These bacteria are easily distinguished from other symbionts by their ability to produce refractile inclusion bodies known as R bodies. An R body is a proteinaceous ribbon (approximately 10 to 20 ,um long, 0.5 ,um wide, and 13 nm thick) that is rolled up inside the bacterial cell, appearing as a hollow cylindical structure (1) (Fig. 1). Within any given population of Caedibacter species only a fraction of the individuals possess R bodies. 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. Killer paramecia apparently release some of their endosymbionts into the environment when food vacuoles are emptied via the cytopyge (15). Killing occurs after an R body-containing symbiont is ingested by a sensitive paramecium.In three of the four species of Caedibacter, R-body synthesis appears to be determined by extrachromosomal elements that are thought to be defective bacteriophages (15). In the fourth species, Caedibacter taeniospiralis, Rbody synthesis is determined by plasmids (17). R bodies produced by C. taeniospiralis, type 51 R bodies, are distinguished from those produced by the other species of Caedibacter on the basis of morphology and their response to changes in pH. Type 51 R-body ribbons typically end in acute angles and unroll from the inside, in a telescoping fashion, when the pH is lowered to 6.5 or less. Loss of the ability to assemble type 51 R bodies by C. taeniospiralis is accompanied by...

Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2+ and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.

No abstract

An Ile-136 -* Thr substitution in calmodulin reduces the Ca2+-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene ijected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 104 copies of such fragments consistently restored the phenotypes to near normal; even 103 or fewer copies could still effect weak transformation. The restored phenotypes were stable for >20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.