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. 2007 Jun;9(6):1604-9.
doi: 10.1111/j.1462-2920.2007.01268.x.

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts

Eva Heinz  1 , Irina Kolarov, Christian Kästner, Elena R Toenshoff, Michael Wagner, Matthias Horn
Affiliations

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts

Eva Heinz et al. Environ Microbiol. 2007 Jun.

Abstract

Acanthamoebae are ubiquitous free-living amoebae and important predators of microbial communities. They frequently contain obligate intracellular bacterial symbionts, which show a worldwide distribution. All Acanthamoeba spp. described so far harboured no or only a single specific endosymbiont phylotype, and in some cases evidence for coevolution between the symbiotic bacteria and the amoeba host has been reported. In this study we have isolated and characterized an Acanthamoeba sp. (strain OEW1) showing a stable symbiotic relationship with two morphologically different endosymbionts. 16S rRNA sequence analysis assigned these symbionts to the candidate genus Procabacter (Betaproteobacteria) and the genus Parachlamydia (Chlamydiae) respectively. Fluorescence in situ hybridization and transmission electron microscopy confirmed the affiliation of the endosymbionts and showed their co-occurrence in the amoeba host cells and their intracellular location within separate compartments enclosed by host-derived membranes. Further analysis of this stable relationship should provide novel insights into the complex interactions of intracellular multiple-partner associations.

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Figures

Fig. 1
Fig. 1
Phylogenetic affiliation of the two endosymbionts of Acanthamoeba sp. OEW1, ‘Cand. Procabacter sp. OEW1’ (A) and Parachlamydia sp. OEW1 (B) based on 16S rRNA sequence analysis. Tree calculations were performed using the maximum likelihood (AxML), neighbour joining and maximum parsimony algorithms implemented in the ARB program package (Ludwig et al., 2004). Maximum likelihood trees are shown, neighbour joining (left triangle) and maximum parsimony (right triangle) bootstrap values (1000 re-samplings) are indicated for supported nodes; black triangles, bootstrap values > 90%; grey triangles, bootstrap values ≥ 75%; white triangles, bootstrap values < 75%. Bar represents 10% estimated evolutionary distance.
Fig. 2
Fig. 2
In situ identification of the two endosymbionts of Acanthamoeba sp. OEW1, Parachlamydia sp. OEW1 and ‘Cand. Procabacter sp. OEW1’. The probes used in this analysis were Proca-438 directly labelled with the hydrophilic sulfoindocyanine fluorescent dye Cy3 specific for ‘Candidatus Procabacter acanthamoebae’ (Horn et al., 2002), Bn9-658 labelled with Cy5 targeting a subgroup of the Parachlamydiaceae (Amann et al., 1997) and EUK-516 labelled with 5(6)-carboxyfluorescein-N-hydroxy-succinimide (FLUOS) targeting most members of the Eukarya (Amann et al., 1990). To ensure specificity hybridization was performed with 20% formamide in the hybridization buffer and corresponding salt concentration in the washing buffer. For further details on oligonucleotide probes, see probeBase at http://www.microbial-ecology.net/probebase (Loy et al., 2007). The overlay of the FISH micrographs, illustrating Cy3 in red, Cy5 in blue, and FLUOS in green, demonstrates the intracellular location of both endosymbionts within the same amoeba host cell. Fluorescence in situ hybridization was performed as described previously (Manz et al., 1992; Horn et al., 2001) and examined by a confocal laser scanning microscope (LSM 510 Meta, Carl Zeiss, Jena, Germany). All experiments were performed at least three times and yielded consistent results. Intervals of at least 1 week separated individual experiments. Bar, 5 μm.
Fig. 3
Fig. 3
Ultrastructure of Acanthamoeba sp. OEW1 and its endosymbionts, Parachlamydia sp. OEW1 and ‘Cand. Procabacter sp. OEW1’. A. Overview of one amoeba trophozoite containing numerous Parachlamydia sp. OEW1 in a large inclusion (white arrowheads) and few rod-shaped ‘Cand. Procabacter sp. OEW1’ (black arrowheads). Bar, 5 μm. B. Close-up on ‘Cand. Procabacter sp. OEW1’ enclosed by a host-derived membrane; a cross and a longitudinal section is depicted. Bar, 0.5 μm. C. Close-up on a Parachlamydia sp. OEW1 inclusion. Reticulate and elementary bodies are readily recognized (black and white arrowheads respectively). Bar, 2 μm. Samples for electron microscopy were fixed in 2.5% glutaraldehyde for 1 h at room temperature, post-fixed in 2% osmium tetroxide for 1 h at room temperature, prestained with 2% aqueous uranyl acetate, dehydrated with an ascending ethanol series and embedded in Low Viscosity Resin (Agar Scientific, UK). After polymerization for 16 h at 65°C, samples were cut on an ultramicrotome (Reichert Ultracut E) with a glass-knife and stained with 1% uranyl acetate for 4 min and 0.3% lead citrate for 2 min. Analysis was performed on a transmission electron microscope (TEM 902, Carl Zeiss, Jena, Germany). Cultures were embedded in five separate experiments, and a total of approximately 50 amoebal cells were examined closely and showed consistent results. Intervals of at least 1 week separated individual experiments.
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