Marine amoebae with cytoplasmic and perinuclear symbionts deeply branching in the Gammaproteobacteria

Abstract

Amoebae play an important ecological role as predators in microbial communities. They also serve as niche for bacterial replication, harbor endosymbiotic bacteria and have contributed to the evolution of major human pathogens. Despite their high diversity, marine amoebae and their association with bacteria are poorly understood. Here we describe the isolation and characterization of two novel marine amoebae together with their bacterial endosymbionts, tentatively named 'Candidatus Occultobacter vannellae' and 'Candidatus Nucleophilum amoebae'. While one amoeba strain is related to Vannella, a genus common in marine habitats, the other represents a novel lineage in the Amoebozoa. The endosymbionts showed only low similarity to known bacteria (85-88% 16S rRNA sequence similarity) but together with other uncultured marine bacteria form a sister clade to the Coxiellaceae. Using fluorescence in situ hybridization and transmission electron microscopy, identity and intracellular location of both symbionts were confirmed; one was replicating in host-derived vacuoles, whereas the other was located in the perinuclear space of its amoeba host. This study sheds for the first time light on a so far neglected group of protists and their bacterial symbionts. The newly isolated strains represent easily maintainable model systems and pave the way for further studies on marine associations between amoebae and bacterial symbionts.

Figure 1. Vannella sp. A1 and its bacterial endosymbiont ‘Candidatus Occultobacter vannellae’.

(A) Trophozoites as seen in hanging drop preparations (scale bar = 20 μm). (B) Fine structure of Vannella sp. A1 and its bacterial symbiont. (i) Section of an amoeba trophozoite: cell organelles located within granuloplasm; nucleus (n) with laterally located nucleolus, mitochondria (m), vacuoles (v), bacterial endosymbionts (arrowheads) (scale bar = 5 μm). (ii) Cell surface of trophozoite with amorphous glycocalyx (scale bar = 200 nm). (iii) Mitochondria with tubular cristae (scale bar = 500 nm). (iv–vi) Bacterial endosymbionts in detail: (iv, v) host-derived vacuolar membranes (arrowheads) enclosing endosymbionts undergoing cell division, (vi) longitudinal section through an endosymbiont (scale bar = 500 nm). (C) Fluorescence in situ hybridization image showing the intracytoplasmic location of ‘Candidatus Occultobacter vannellae’ (Occultobacter-specific probe A1_1033, pink) in its Vannella sp. A1 host (probe EUK516, grey) with DAPI stained nuclei (blue) and food bacteria (general bacterial probe EUB338-mix, green); scale bar indicates 10 μm.

Figure 2. Phylogenetic relationships of Vannella sp. A1 and amoeba isolate JAMX8 within the Amoebozoa.

Phylogenetic 18S rRNA-based trees of the Amoebozoa (left panel) and Vannellidae (right panel) constructed using the Bayesian inference method. Bayesian posterior probabilities (>0.6) and RaxML bootstrap support values (>60%) are indicated at the nodes; the dashed line indicates a branch shortened by 50% to enhance clarity. Colored squares indicate the typical habitat of the respective amoeba species (left panel). A detailed version of the trees including accession numbers is available as supplementary Figure S1.

Figure 3. Amoeba isolate JAMX8 and its bacterial endosymbiont ‘Candidatus Nucleophilum amoebae’.

(A) Trophozoites as seen in hanging drop preparations (scale bar = 20 μm). (B) Fine structure of JAMX8 and its bacterial endosymbiont inhabiting the perinuclear space. (i) Section of an amoeba trophozoite: vesicular nucleus (n), phagosomes (p), mitochondria (m), bacterial endosymbionts associated with the nuclear envelope (arrowheads) (scale bar = 5 μm). (ii) Amorphous and tenuous cell coat (scale bar = 200 nm). (iii) Mitochondria with tubular cristae (scale bar = 1 μm). (iv) The Golgi complex organized as dictyosome (scale bar = 1 μm). (v,vi) Bacterial endosymbionts located within the perinuclear space, between inner and outer nuclear membrane. (v) Nucleus in detail with numerous endosymbiotic bacteria in transverse section (scale bar = 2 μm). Arrowheads indicate outer nuclear membrane. (vi) Longitudinal section through a rod-shaped bacterial endosymbiont, nucleus (n) (scale bar = 1 μm). (C) Fluorescence in situ hybridization image showing the co-localization of ‘Candidatus Nucleophilum amoebae’ (Nucleophilum-specific probe JAMX8_197, pink) with its host nucleus (DAPI, blue); food bacteria (general bacterial probe EUB338-mix, green) enclosed in the amoeba cytoplasm (probe EUK516, grey); scale bar indicates 10 μm.

Figure 4. Phylogenetic relationship of ‘Candidatus Occultobacter vannellae’ and ‘Candidatus Nucleophilum amoebae’ with the Gammaproteobacteria.

The phylogenetic tree (PhyloBayes, CAT + GTR) is based on the 16S rRNA sequences, Bayesian posterior probabilities are indicated at the nodes (only values < 0.99 are shown). Colored squares indicate the environmental origin of the respective sequence; colored circles indicate host association. A detailed version of this tree including accession numbers is available as supplementary Figure S2.