Endosymbionts in Paramecium: 12 (Microbiology Monographs)
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In order to understand the molecular mechanisms involved in this mutualistic relationship, experiments to reproduce endosymbiosis are indispensable. The ciliate "Paramecium" is an ideal host for performing such studies. Topics presented in this volume are: the origins of algal and bacterial symbionts in "Paramecium", the diversity of endosymbiotic bacteria, such as "Holospora" bacteria and especially "Chlorella" species, as well as the infection and maintenance processes. The metabolic control, the regulation of circadian rhythms and photobiological aspects of the mutualistic association, as well as the killer effect of "Paramecium" and its causative agents are further points discussed.
Help Centre. My Wishlist Sign In Join. Be the first to write a review. Add to Wishlist. Ships in 15 business days. Link Either by signing into your account or linking your membership details before your order is placed. Caedibacter taeniospiralis apparently increases its host fitness via manipulation of metabolic pathways and cell cycle control.
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New phenotypes and abilities arise from symbioses. Some of those have been studied at the functional and transcriptome level. The association between the eukaryote Paramecium tetraurelia Ciliophora, Alveolata and the cytoplasmic endosymbiont Caedibacter taeniospiralis Gammaproteobacteria might be considered a less complex symbiosis as it includes only two participants. However, complexity is not necessarily restricted to the number and variety of involved partners. The underlying mechanism is not yet completely understood. Caedibacter taeniospiralis residing in the cytoplasm of P.
The R-body is build-up from a large, tightly coiled protein ribbon. Phagocytosis represents the normal feeding behavior of bacterivorous paramecia. Incorporation in a phagosome followed by acidification triggers the R-body to unroll and thereby to destroy bacterial and vacuolar membranes. This action is probably connected with the release of an unidentified toxin, which ultimately leads to the death of the Caedibacter -free Paramecium cell.
Infected paramecia are protected from the killer trait by an unknown mechanism provided by the symbiotic bacteria.
Thus, they gain a selective advantage compared with symbiont-free competitors Schrallhammer and Schweikert Hence, its survival, reproduction, and evolution are coupled to its host. At the first glance, all benefits of expressing the killer trait are gained by Paramecium while Caedibacter pays the costs. It devotes a part of its population to R-body production, an evolutionary dead-end because those bacteria lose the ability to divide. Finally, those cells die when the R-body unrolls, albeit once inside a phagosome Caedibacter would be digested in any case.
However, by sacrificing a proportion of individual members, the host and therewith the rest of the symbiont population can thrive. Interestingly, the alphaproteobacterial symbiont Caedimonas varicaedens basonym Caedibacter varicaedens , Schrallhammer et al. The question regarding the nature of additional effects of these symbionts is controversial. While both studies arrive at the conclusion that C. Thus, cultivation conditions e. We introduce a new perspective on this interaction by applying mRNA-Seq to reveal differential gene expression in Caedibacter -carrying paramecia compared with symbiont-free Paramecium cells.
Fitness assays are used as a methodological independent confirmation of transcriptome results. Paramecium tetraurelia 51K infected by the bacterial symbiont C. The line P.
From now on, the two lines are referred to as 51K and Thereafter, cells were washed individually four times and individual cells were incubated in antibiotic solution as described. Then the cells were washed as before and transferred to 0. The line 51 was established circa seven months prior to the preparation for RNA extraction and fitness experiments by pooling two batches of Caedibacter -free paramecia.
For analysis of the potential killer effect of 51K and 51 an additional line with reported susceptibility to this phenomenon P. Furthermore, to exclude potential undesired side effects of streptomycin on paramecia, 51S was also subjected to the above-described antibiotic treatment as control. Presence or absence of endosymbionts was monitored regularly before and after antibiotic treatment and fitness assays, before killer tests, cell age synchronization, and harvesting for RNA extraction.
Caedibacter- bearing paramecia confer the killer trait to their host and hence kill sensitive cells while infected cells are resistant. Without the symbionts, the Paramecium cells become susceptible. The occurrence of this characteristic behavior was assessed for 51K and 51 using the symbiont-free and reportedly sensitive line P. The lysate was prepared by vortexing the Paramecium suspension with glass beads until no intact paramecia were observed.
Thus, potentially present R-body containing Caedibacter , the lethal agents of the killer trait, were released at least in case of 51K. The number of living 51S was determined at several time points. Six replicates were included for each experiment. The symbiont-free 51 was used as reference. Fluorescence In Situ hybridizations of used cell lines Paramecium tetraurelia 51K infected by Caedibacter taeniospiralis A and symbiont-free P.
Applied probes are the universal EUB labeled with Cy3 and the symbiont-specific Ctaenio fluorescein. Merged images show the presence of numerous cytoplasmic bacteria A respectively bacteria in digestive vacuoles B. To obtain cell age synchronized cells, paramecia were cultivated for circa 25 divisions and then triggered to undergo autogamy, which was confirmed by the observation of fragmented macronuclei after DAPI staining C. In order to avoid differences in gene expression resulting from sexual recombination autogamy , 51K and 51 were subjected to cell age synchronization fig.
The F1 paramecia were then cultivated to obtain the desired cell number before reaching maturity again. Starting from single cells, both 51K and 51 were grown in bacterized 0.
The cells were harvested by centrifugation and resuspended in exhausted 0. Exhausted medium is the cell-free supernatant of bacterized 0. High cell density and starvation triggered the cells to undergo autogamy as microscopically confirmed by visualization of macronuclear fragmentation after DAPI staining fig. Read numbers were: , Reads were demultiplexed with bcl2fastq v1.
Gene expression levels for each individual library were quantified using Sailfish version 0. However, differential expression of genes was determined by DESeq2 version 3. DESeq2 uses negative binomial linear models for dispersion estimates.
Those are concepts to describe gene molecular functions or biological processes. This visualization summarizes biological processes according to their similarity and GO enrichment P -values. The impact of Caedibacter on P. In preparation for the fitness assay, cells were washed and adjusted to exponential growth.
For the actual assay, five replicates per line were included.
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Caedibacter taeniospiralis was removed from infected cells via antibiotic treatment enabling subsequent comparisons between killer and symbiont-free paramecia. Ideally, cured cells should be re-infected and used in parallel for transcriptome or fitness analyses. However, C. By streptomycin treatment of C. In analogously treated 51S cells no physiological effects abnormal cell morphology or division rate, increased mortality caused by the antibiotics were observed. We performed killer tests to verify the presence of the symbiont and hence if 51K can cause the death of sensitive 51S.
Vice versa, in case of 51 we tested for the loss of this ability together with the elimination of C. Therefore, 51K and 51 were mechanically lysed to maximize the number of potentially released bacteria. Exposing 51S cells to these lysates confirmed toxic effects of 51K fig. Those exposed to 51 lysate did not show reduced cell numbers or any prelethal symptoms. On the contrary, 51S multiplied in these replicates fig. Lysates were prepared from P. After exposure, the number of unaffected 51S cells was reported at different time points. RNA sequencing with three biological replicates was carried out to determine the genome-wide gene expression patterns for C.
A hierarchical clustering analysis of these transcriptomes revealed major reproducible differences between both lines as the replicates of infected and symbiont-free cells cluster clearly together fig. Considering symbiont-free 51 as reference, out of 39, annotated genes, slightly more were significantly up- than down-regulated in Caedibacter -infected paramecia fig.