Evolutionary Ecology of the Caedibacter Symbiosis and R Body Production

Intracellular bacteria are favorable for the host and may be regarded as mutu-alists when uninfected competitors are killed due to these bacterial symbionts. This view, however, may be too simple, as ecological and molecular studies revealed fascinating details on the evolutionary ecology of this long known occurrence of "killer"-bacteria in paramecia. The bacteria are closely related to pathogens and exploit the host cells just as invasive agents in metazoans do. Caedibacter species are energy parasites that depend on the import of metabolites from the host cytoplasm (Linka et al. 2003), and therefore limit the reproduction of their host; under conditions that are unfavorable for para-mecia Caedibacter may even overgrow the host cell, finally causing its death (Schmidt et al. 1987; Kusch et al. 2002). A carrier-mediated transport of nucleotides occurs for an acquisition of energy from the host cell. Recently, the evolutionary origin of the nucleotide transporters (NTT) of Caedibacter cary-ophilus that are specific ATP / ADP antiporters, has been elucidated. The phy-logeny of bacterial NTT appears highly complex (Fig. 3) with NTT of C. caryophilus exhibiting significant sequence similarity with NTTs in intracellular pathogens of humans (Rickettsia). It was unexpected that NTTs of C. caryophilus and Holospora obtusa are more distantly related to one another, although these two endonucleobionts of Paramecium are close relatives in 16S rRNA trees. It is therefore suggested that earlier horizontal gene transfer is responsible for the distribution of NTT paralogs in bacterial endocytobi-onts of Paramecium (Linka et al. 2003).

For each parasite-host system there may be an optimal strategy of host exploitation maximizing the parasite's propagation (Poulin 1998). Caedibacter endocytobionts limit growth of their host due to energy theft. Too fast growth with subsequent virulence (host damage) of Caedibacter should kill

Intracellular Bacteria Phylogeny

Fig. 3. Phylogeny of nucleotide transport proteins (NTT) in intracellular bacteria and Chlamydomonas chloroplasts. Bootstrap support (calculated from 1,000 replicates) is given as numbers at branches (%) for tree construction with maximum likelihood (TREE-PUZZLE; upper numbers) or distance (PHYLIP; lower numbers) approaches. Support of different tree topology at one branch is indicated by a '-'. The scale bar (0.10) represents mean number of substitutions per site. (After Linka et al. 2003, with changes)

Fig. 3. Phylogeny of nucleotide transport proteins (NTT) in intracellular bacteria and Chlamydomonas chloroplasts. Bootstrap support (calculated from 1,000 replicates) is given as numbers at branches (%) for tree construction with maximum likelihood (TREE-PUZZLE; upper numbers) or distance (PHYLIP; lower numbers) approaches. Support of different tree topology at one branch is indicated by a '-'. The scale bar (0.10) represents mean number of substitutions per site. (After Linka et al. 2003, with changes)

host paramecia. The observation that infections of paramecia by Caedibacter are rare (Preer et al. 1974; Landis 1981, 1987; Kusch et al. 2002) may indicate that killing of host cells can result in the extinction of the responsible bacterial genotype. On the other hand, Caedibacter endocytobionts, besides limiting host reproduction, are giving competitive advantage to host paramecia under certain circumstances (Landis 1981, 1987; Kusch et al. 2002). Competition for food occurs within Paramecium species, when food is scarce, and interspecific competition, too, should occur in case of overlapping resources (Gause 1934; Maruyama et al. 2001).

It is the evolution of R body production by Caedibacter that has resulted in a mechanism for competition by allelopathic interference for paramecia bearing these bacterial endocytobionts. Individuals of both, the same species as well as closely related paramecia may be killed by toxins of Caedibacter when the paramecia are not bearing these bacteria. In Caedibacter, the toxins are associated with R body production. Parasites, in general, depend on the abundance of host organisms and on mechanisms for their successful infection. The rate at which an infected host is transmitting the parasite to susceptible hosts depends on host density. The increase in host abundance should result in an increase of parasite abundance in the next generation, and propagation of parasites is reciprocal to the mortality rate of parasite-free hosts (Anderson and May 1982, 1991; Poulin 1998). Toxic Caedibacter bacteria, instead, kill potential hosts. Infections and a subsequent growth of the bacteria are rare. Therefore, propagation of Caedibacter cells predominantly occurs via vertical transmission by vegetative reproduction of their hosts. Lethal effects on competitors increase the relative population growth rate of infected paramecia and their bacterial parasites. Indeed, infected strains could outcompete genetically identical but uninfected strains in mixed cultures of either of the species, P. tetraurelia or P. novaurelia (Kusch et al. 2002). Reproduction rates of Caedibacter and virulence effects on hosts should be well adapted to interference competition effects by R body production.

Only Caedibacter with R bodies are toxic, and R bodies are expressed only in part of the bacteria. R bodies are refractile in phase contrast microscopy. R body-bearing bacteria are therefore called "bright bodies" or simply "brights". Caedibacter without R bodies are called "non-brights". Since non-brights are not toxic but nevertheless may contain plasmids or phages genomes, it may happen that paramecia not bearing Caedibacter become infected by non-brights. Yet, this may be a rare case, since any brights that are co-ingested by non-infected paramecia will kill the cells. Sometimes, infection of formerly uninfected host paramecia may occur during conjugation, the type of sexual reproduction in ciliates. Paramecia undergoing conjugation do not feed and therefore do not take up the toxin released by an infected mating partner. Sensitive paramecia are therefore protected and can mate with infected "killer" paramecia. During conjugation of ciliates, a cytoplasmic bridge forms, that enables exchange of migrating pronuclei between the two partner cells.

Eventually, some cytoplasm and even cell organells or intracellular bacteria may be exchanged, too (Balsley 1967; Landis 1981). Infection during this cytoplasmic exchange at conjugation would increase the proportion of killers in a population (Landis 1987). For Caedibacter paraconjugatus this possibility generally does not exist, as it is a so-called mate killer symbiont. The toxic principle only acts on sensitive paramecia during mating with C. paraconjuga-tus-bearing ciliates.

Fokin et al. (2004) observed that non-infective bacteria infected the macro-nucleus of Paramecium caudatum, when infectious Holospora obtusa were present, too. If co-infections of Paramecium by Caedibacter were frequent in the presence of Holospora species, then the release of Caedibacter by its host paramecia could significantly increase the transmission rate and prevalence. Otherwise the reproduction rate of Caedibacter should be as low as to ensure stably infected host populations (so, growth of Caedibacter should equal its dilution by growth and reproduction of its hosts). Thereby, virulence effects should be minimized. If infection of new host paramecia would be a predominant way for reproduction and spreading of Caedibacter endocytobionts, selection should favor Caedibacter genotypes without toxin production, as toxic Caedibacter kill their potential hosts. Competitive advantages of host parame-cia due to bacterial toxin production, in combination with vegetative reproduction of the host, renders infection of new hosts by the bacterial parasites unnecessary for their propagation, and should be responsible for the persistence of "killer paramecia" in nature.

Concerning the ecological significance of Caedibacter infections, it is especially interesting that the genetic condition of potential hosts is crucial for the maintenance of Caedibacter in Paramecium. In genetic crossings, Sonneborn (1943) observed that only paramecia with certain genotypes may harbor Caedibacter. Namely, for one special gene it turned out that an allele K is crucial for the persistence of the bacteria in host cells, while Caedibacter could not live in cells with k in the homozygous state. It is not determined, whether K is actively supporting growth of the endocytobionts or k is suppressing growth. In this context, the question arises whether the parasites affect the rate of conjugation or autogamie in host paramecia, and whether genetical recombination of hosts affects the prevalence of Caedibacter in a host population.

In various cases of host-specific intracellular microorganisms, evidences for gene transfer from endocytobionts to host cell have been obtained, e.g., in certain Euplotes and in Amoebaproteus (Heckmann 1983; Jeon and Jeon 1997). It is a first indication that many intracellular bacteria possess small genomes. However, Euplotes and A. proteus became dependent upon their symbionts, but this is not the case for Paramecium bearing Caedibacter. The demands of the bacteria, on the other hand, may not be very specific. The most important need may be ATP, which is not a specific compound. So, at the time being, there may be no real indication for a gene transfer between Caedibacter and Paramecium.

Depending on the environmental conditions given, the infection of Paramecium by Caedibacter may be regarded as parasitic or even mutualistic. Infected hosts should have a selective advantage over uninfected and, thereby, sensitive paramecia. In interspecific competition of Caedibacter-bearing cells with other bacterial feeders like tiny rotifers, nematods, or unrelated ciliates the toxins may not help. In these cases the bacterial load with Caedibacter being energy parasites may be a severe burden and of disadvantage. This conflict of intraspecific versus interspecific competition may be the reason why some natural populations of Paramecium are infected by Caedibacter while others are not. In the case of very low abundance of Caedibacter-bearing paramecia, cells that have lost their bacteria and thereby have become sensitive to the toxin may not be harmed, because only low amounts of toxin are present. Uninfected paramecia grow faster and namely under nutrient-poor conditions may outcompete infected ones.

We do not yet understand the mechanisms, and how resistance against the toxins is achieved. Caedibacter-bearing paramecia are resistant against the toxins, and resistance is even mediated by Caedibacter that have lost the ability for R body expression (Schmidt et al. 1987). Schmidt et al. observed that host cells are less affected by Caedibacter that are no longer able to express R bodies, these paramecia bearing only non-brights grow faster than paramecia with brights. One might therefore expect that Caedibacter-lacking phages or plasmids are frequent in nature, but this doesn't seem to be the case. While R body-free Caedibacter were found in small free-living amoeba, in nature they have not been detected in Paramecium. However, there are good evidences that we only know a few of the bacterial endocytobionts in ciliates, and perhaps R body-free Caedibacter have simply been overlooked. It is therefore of interest to look systematically for bacterial infections in paramecia and other ciliates, the more so as we may expect potential pathogens among the bacterial endocytobionts of protozoa (Görtz and Michel 2003).

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