Molecular Evolution of R Body Production

Caedibacter endocytobionts of paramecia are infected by phages or plasmids, either of which is responsible for the production of R bodies and probably the toxin (Preer et al. 1972, 1974; Pond et al. 1989; Kusch et al. 2000). Bacterio-phages are evidently non-infective, and bacterial lysis does not seem to occur (Pond et al. 1989), with the exception of Caedibacter caryophilus in the host Paramecium caudatum (Schmidt et al. 1987). The R body coding region of the plasmid of C. taeniospiralis is of 1.8 kb and consists of the three genes rebA, rebB, and rebC coding for proteins of 18, 13 and 10 kDa, respectively. A putative fourth gene, rebD, may be involved in R body production and expression of the toxin (Heruth et al. 1994). Extrachromosomal elements coding for R bodies in different Caedibacter species seem to be more closely related than their hosts (Quackenbush et al. 1986a), that are polyphyletic, with e.g., C. caryophilus belonging to the a-Proteobacteria and C. taeniospiralis being a member of the y-Proteobacteria (Beier et al. 2002). R body production occurs in non-endobiotic bacteria, too, including pseudomonads (Pond et al. 1989). It appears that different bacterial genera gained (or were infected by) identical or functionally similar extrachromosomal elements during their evolution.

Among species of the genus Caedibacter, C. taeniospiralis is the only one bearing a plasmid (pKAP) as an R body coding extrachromosomal element (Dilts 1976; Quackenbush 1983; Quackenbush and Burbach 1983). Plasmid pKAP is present in all strains of C. taeniospiralis, but varying in size (41.5 to 50.5 kb). The plasmids of C. taeniospiralis strains differ by the insertion of transposons at different plasmid locations (Quackenbush et al. 1986b). The transposons are present in the genome of the bacterial host, too. This was evident because of spontaneous insertions of those transposons into pKAP plasmids and from hybridizations of the transposons with genomic DNA (Dilts and Quackenbush 1986; Quackenbush et al. 1986b).

The transcriptional activity of the isolated plasmid pKAP298 of C. taeniospiralis strain 298 and its complete sequence (49112 bp) were analyzed (Fig. 4) to gain insight into the constitution of R body coding extrachromosomal elements (Jeblick and Kusch 2005). A significant homology of the total pKAP298 to other plasmids or phages was not detected, indicating a so far unique molecular structure of pKAP298. Yet, the occurrence of sequences with homology to several phage proteins indicate that pKAP298 is derived from a former phage, as was hypothesized by Preer et al. (1974). Stress conditions induce the transcription of R body coding genes. This is why R bodies occur predominantly in the stationary phase of paramecia resulting from a lack of food (Pond et al. 1989). Additional transcription activity of pKAP298 indicates functions besides R body synthesis.

Regions active in transcription contain some of the genes with homology to phage proteins (Jeblick and Kusch 2005). Because of this observation one may assume a synthesis of phages or incomplete phages in Caedibacter taeniospi-ralis, too. Long ago, Preer, Preer and Jurand already discussed the possibility

Fig. 4. Map of the R-body coding plasmid pKAP298 from Caedibacter taeniospiralis as deduced from analysis of its sequence and transcription activity. The only Bam HI restriction site was arbitrarily defined as bp 0. Each of the five double segments gives 10 kbp, except the last one with 9.112 kbp. The upper part of each double segment shows regions with detected transcription activity above the line and transposons below the line. Base pair (Bp) numbers belong to vertical lines below the indicated Bp values. Some restriction sites are also given (below bp numbers). The lower parts of each double segments show the detected open reading frames (ORF), with the direction of transcription indicated by arrows on the left side of the figure. Putative functions are given for some ORFs. (After Jeblick and Kusch 2005, changed)

Fig. 4. Map of the R-body coding plasmid pKAP298 from Caedibacter taeniospiralis as deduced from analysis of its sequence and transcription activity. The only Bam HI restriction site was arbitrarily defined as bp 0. Each of the five double segments gives 10 kbp, except the last one with 9.112 kbp. The upper part of each double segment shows regions with detected transcription activity above the line and transposons below the line. Base pair (Bp) numbers belong to vertical lines below the indicated Bp values. Some restriction sites are also given (below bp numbers). The lower parts of each double segments show the detected open reading frames (ORF), with the direction of transcription indicated by arrows on the left side of the figure. Putative functions are given for some ORFs. (After Jeblick and Kusch 2005, changed)

that R body proteins are related to the proteins of the defective phages of Cae-dibacter (Preer et al. 1974). Prophages of some temperate phages, e.g., of col-iphages P1 (Ikeda and Tomizawa 1968; Walker and Walker 1975) and N15 (Ravin and Shulga 1970) also are autonomous plasmids. Furthermore, Casjens et al. (2004) reported that the Klebsiella oxytoca linear plasmid pKO2 is a prophage. Sequences with homology to proteins that are involved in the phage-induced lysis of a host cell were not detected on pKAP298. Schmidt et al. (1987) presumed from the occurrence of R bodies in the macronucleus of C. caryophilus-infected Paramecium caudatum that bacteriophages of C. caryophilus induced host cell lysis. In C. taeniospiralis induced lysis was never observed. Plasmid pKAP298 may stem from a bacteriophage of the order Caudovirales, to which all bacteriophages that have sequence homology to pKAP298 belong (Jeblick and Kusch 2005). Some of the sequences that were identified to originate from a phage, have homologies to proteins of the order Rhizobiales (Kaneko et al. 2002; Galibert et al. 2001), to which the original host may have belonged. The plasmid pKAP298 may have evolved from a bacteriophage to a broad host range-plasmid, since host strains classified as C. taeniospiralis have only low DNA homologies (Quackenbush 1977, 1978).

Apparently, pKAP298 and its R body genes are related to a bacteriophage that does not have a complete set of phage head and tail genes. In several other bacteria, sequence analysis and electron microscopy revealed a common ancestry for some bacteriocins, bacterial products with specific bactericidal activity, and bacteriophages (e.g., Strauch et al. 2001; Jabrane et al. 2002). These bacteriocins have been regarded as defective bacteriophages, which might have arisen from temperate phages by several successive mutations (Bradley 1967; Daw and Falkiner 1996). The bacterial bacteriocin production obviously evolved from phage structural genes, and it seems that Caedibacter species similarly derived R-body genes from a phage. The gene transfer agent (GTA) of Rhodobacter capsulatus is another example, where bacterial functions may have evolved from phage genes (Marrs 1974; Yen et al. 1979). Therefore, the present genes in pKAP298 should come from an adaptive selection for toxicity that preserved this genetic element during evolution.

Toxicity of R bodies to paramecia ingesting Caedibacter species may be associated with a protein that has homology to the Soj-/ParA-family (ORF 43, Fig. 4) and also has homologies to a membrane associated ATPase (Jeblick and Kusch 2005). This ATPase is involved in eukaryotic ATPase dependent ion carriers (Zhou et al. 2000) and may cause toxic effects on paramecia ingesting this protein. As an ATPase the protein may primarily function in the distribution of pKAP298 on daughter cells after host divisions (Motallebi-Vesharesh et al. 1990). It also may interfere with the distribution of the host genome due to misrelations of ParA and ParB proteins (Easter and Gober 2002), leading to the observed low reproduction rates observed for Caedibac-ter species (Preer et al. 1972). The toxin might disturb the osmoregulatory abilities of Paramecium via effects on the cell membrane (Butzel and Pagliara

1962) and therefore leads to hump-killing or vacuolization as typical killing mechanisms (Preer et al. 1974).

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