Tripartite Symbiosis of an Anaerobic Scuticociliate with Two Hydrogenosome-Associated Endosymbionts, a Holospora-Related Alphaproteobacterium and a Methanogenic Archaeon

Abstract

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, "Candidatus Hydrogenosomobacter endosymbioticus."IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

Keywords: anaerobic ciliate; hydrogenosome; methanogen; symbiosis; “Candidatus Hydrogenosomobacter endosymbioticus,” sewage treatment reactor.

FIG 1

An anaerobic scuticociliate strain, GW7. (A and B) Phase-contrast micrograph (A) and fluorescence micrograph (B) of a cell of strain GW7. Autofluorescence of coenzyme F₄₂₀ (light blue) was detected. (C) Growth curve of strain GW7. The timing of supplementation of the food bacterium (0.05 g) is indicated by an arrow. Different-colored lines show each replicate (n = 7). Bars, 10 μm (A and B).

FIG 2

Molecular phylogeny of an anaerobic scuticociliate strain, GW7. The tree displays a maximum likelihood (ML) phylogeny of ciliates belonging to the class Oligohymenophorea. An alignment of 1,524 nucleotide sites of the eukaryotic 18S rRNA gene was used. Accession numbers in the DDBJ/EMBL/GenBank DNA database are shown in square brackets. Bootstrap support values higher than 70% are depicted on or below the internal branches. Boldface type indicates the members of the family Cyclidiidae, which is a systematically problematic taxon. Other systematically problematic taxa are underlined.

FIG 3

Transmission electron micrographs of an anaerobic scuticociliate strain, GW7. (A) Whole cell of strain GW7. (B) Enlarged view of the boxed region in panel A. Methanogenic archaeal and bacterial endosymbionts are indicated by black and white arrows, respectively. A dividing cell of the bacterial endosymbiont is indicated by a white arrow with an asterisk. (C) Dividing cells of the methanogenic archaeal endosymbiont. (D) Cross section of a methanogenic endosymbiont cell. The cell shows two discernible membranes. (E) Longitudinal section of a bacterial endosymbiont cell. Abbreviations: N, nucleus; H, hydrogenosome; FV, food vacuole. Bars, 5 μm (A), 1 μm (B), 500 nm (C), and 100 nm (D and E).

FIG 4

Detection of endosymbionts in an anaerobic scuticociliate strain, GW7. Endosymbionts of strain GW7 were detected by FISH with universal and specific probes. Shown are differential interference contrast (left) and confocal laser scanning (right) micrographs. FISH was performed with EUB338 (Alexa 488) (green) and ARC915 (Alexa 568) (red) probes (A) and with GW7Met1 (Alexa 488) (green) and GW7Bac2 (Alexa 568) (red) probes (B). The host’s nucleus was stained with DAPI (blue). Formamide concentrations were 35% (A) and 20% (B). Bars, 10 μm.

FIG 5

Molecular phylogeny of the methanogenic archaeal endosymbiont of an anaerobic scuticociliate strain, GW7. The tree displays an ML phylogeny of seven clones of the endosymbiotic methanogenic archaeon derived from an anaerobic ciliate strain, GW7, together with related species/clones of the family Methanoregulaceae. An alignment of 1,298 nucleotide sites of the archaeal 16S rRNA gene was used. The origins or sources of isolation of species/clones are presented in parentheses. Accession numbers in the DDBJ/EMBL/GenBank DNA database are shown in square brackets. Bootstrap support values higher than 70% are depicted on or below the internal branches. *TCE, trichloroethene.

FIG 6

Molecular phylogeny of the bacterial endosymbiont of an anaerobic scuticociliate strain, GW7. The tree displays an ML phylogeny of seven clones of the endosymbiotic bacterium “Candidatus Hydrogenosomobacter endosymbioticus” derived from an anaerobic ciliate strain, GW7, together with related species/clones of the order Holosporales. An alignment of 1,333 nucleotide sites of the bacterial 16S rRNA gene was used. The origins or sources of isolation of species/clones are presented in parentheses. Accession numbers in the DDBJ/EMBL/GenBank DNA database are shown in square brackets. Bootstrap support values higher than 70% are depicted on or below the internal branches.