APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 2014–2021 Vol. 72, No. 3 0099-2240/06/$08.00ϩ0 doi:10.1128/AEM.72.3.2014–2021.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved. “Candidatus Thiobios zoothamnicoli,” an Ectosymbiotic Bacterium Covering the Giant Marine Ciliate Zoothamnium niveum Christian Rinke,1* Stephan Schmitz-Esser,2 Kilian Stoecker,2 Andrea D. Nussbaumer,1 Da´vid A. Molna´r,3 Katrina Vanura,1,4 Michael Wagner,2 Matthias Horn,2 Jo¨rgA.Ott,1 and Monika Bright1 Department of Marine Biology, University of Vienna, 1090 Vienna, Austria1; Department of Microbial Ecology, University of Vienna, 1090 Vienna, Austria2; Department of Microbiology, Eo¨tvo¨s Lora´nd University of Sciences, 1112 Budapest, Hungary3; and Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University Vienna, 1090 Vienna, Austria4 Received 20 September 2005/Accepted 29 November 2005 Zoothamnium niveum is a giant, colonial marine ciliate from sulfide-rich habitats obligatorily covered with chemoautotrophic, sulfide-oxidizing bacteria which appear as coccoid rods and rods with a series of intermediate shapes. Comparative 16S rRNA gene sequence analysis and fluorescence in situ hybridization showed that the ectosymbiont of Z. niveum belongs to only one pleomorphic phylotype. The Z. niveum ectosymbiont is only moder- ately related to previously identified groups of thiotrophic symbionts within the Gammaproteobacteria, and shows highest 16S rRNA sequence similarity with the free-living sulfur-oxidizing bacterial strain ODIII6 from shallow- water hydrothermal vents of the Mediterranean Sea (94.5%) and an endosymbiont from a deep-sea hydrothermal vent gastropod of the Indian Ocean Ridge (93.1%). A replacement of this specific ectosymbiont by a variety of other bacteria was observed only for senescent basal parts of the host colonies. The taxonomic status “Candidatus Thiobios zoothamnicoli” is proposed for the ectosymbiont of Z. niveum based on its ultrastructure, its 16S rRNA gene, the intergenic spacer region, and its partial 23S rRNA gene sequence. Symbioses of eukaryotes with chemolithoautotrophic sulfur- are occupied by a monolayer of rod-shaped bacteria (average oxidizing (thiotrophic) bacteria are widespread and known length, 1.4 ␮m; average width, 0.4 ␮m), whereas bacteria on from diverse marine sulfidic habitats, ranging from shallow the oral side of microzooids are coccoid to rod shaped (average subtidal sands and macrophyte debris to deep sea cold seeps length, 1.9 ␮m; average width, 1.0 ␮m) (5). The series of and hydrothermal vents (14, 24, 25, 45). The giant peritrich transitional morphological stages from rods to coccoid rods ciliate Zoothamnium niveum (Ciliophora, Oligohymenophora) found on the microzooids suggests the presence of a single is host of one of the most remarkable of such symbioses. This ectosymbiotic bacterial species on Z. niveum (5, 7). This reg- sessile, colonial ciliate was originally described more than 150 ular coat, however, appears to be disturbed and replaced by a years ago from the Red Sea (20, 30); later it was also found in variety of other bacterial morphotypes on the basal parts of the Lower Keys of Florida (Gulf of Mexico) and at the Belize large colonies only (44). Barrier Reef (Caribbean Sea) (4). Typically Z. niveum occurs Culture and incubation experiments showed that Z. niveum on mangrove peat walls in groups of more than 100 colonies, is unable to survive without its symbionts (4), and physiological with an estimated group life of approximately 20 days, pre- studies applying 14C-labeled bicarbonate incubations (48) and dominantly at sites where the microbial surface mat has been Cartesian diver experiments (44) indicated that the Z. niveum destroyed (44). ectosymbionts are thiotrophic bacteria. Since none of the The feather-like Z. niveum colonies, which reach sizes up to known thiotrophic symbionts have yet been cultivated without 1.5 cm, consist of a central stalk with branches occurring al- their respective hosts, culture-independent approaches have to ternately on the stalk and three cellular morphotypes on the be employed to reveal the identity of these elusive bacteria (3). branches: (i) microzooids are the feeding stages; (ii) macro- Here we investigated by scanning electron microscopy (SEM), zooids are the dispersal stages, capable of leaving the colony as 16S rRNA gene sequencing, comparative sequence analysis, and large swarmers to build a new colony after settlement; and (iii) specific fluorescence in situ hybridization (FISH) the thiotrophic terminal zooids are responsible for asexual reproduction by ectosymbionts of Z. niveum. We identified a single bacterial phy- longitudinal fission (4, 5) (Fig. 1). lotype representing the Z. niveum ectosymbiont, which is only Z. niveum has been found exclusively in obligatory associa- moderately related to known symbiotic and free-living bacteria tion with ectosymbiotic bacteria that are responsible for the within the Gammaproteobacteria. Only on basal, senescent parts ciliate’s brilliant white color (4, 5). Z. niveum is virtually com- of large colonies was the symbiont replaced by a variety of other pletely covered by these bacteria appearing as rods and coccoid bacteria. For classification of the Z. niveum ectosymbiont we rods arranged in a highly specific and permanently occurring propose the tentative taxonomic status “Candidatus Thiobios pattern (Fig. 2). Macrozooids, terminal zooids, and branches zoothamnicoli.” * Corresponding author. Mailing address: Department of Marine MATERIALS AND METHODS Biology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria. Sample collection and fixation. Samples of Zoothamnium niveum colonies Phone: 431427757120. Fax: 431427754339. E-mail: [email protected]. were cut from peat surfaces in 1 to 3 m depth at different locations in the main 2014 VOL. 72, 2006 ECTOSYMBIOTIC BACTERIUM 2015 FIG. 1. Zoothamnium niveum colony. (A) General view of the entire colony by the central stalk (st) with alternate branches (br) bearing cells. Light microscopy; scale bar, 0.5 mm. (B) Detail of the middle part of the colony, demonstrating the three different Z. niveum cell types: macrozooids (ma), microzooids (mi), and terminal zooids (t) occurring on the branches. Light microscopy, contrast enhanced; scale bar, 50 ␮m. channel (extending 1 km) of the mangrove island Twin Cays (16°48ЈN, 88°05ЈW; osmium tetroxide in natural seawater), and for scanning electron microscopy Belize Barrier Reef, Caribbean Sea) by scuba diving in July 2003. Samples (20 (2% osmium tetroxide in natural seawater). All solutions were sterile filtered. ciliates each) were preserved for DNA analysis (96% ethanol), for FISH (4% Applying osmium tetroxide fixation, natural seawater was evaporated by boiling paraformaldehyde in phosphate-buffered saline [PBS] 0.1 M, pH 7.4, or 1% to half of its volume and mixed 1:1 with 2% (for FISH) or 4% (for SEM) aqueous osmium tetroxide. All specimens preserved for FISH and SEM were rinsed in PBS three times for 5 min each, dehydrated in 30 and 50% ethanol for 5 min each, and stored in 70% ethanol until further treatment up to a year later. Scanning electron microscopy. Specimens fixed for SEM and stored in 70% ethanol were further dehydrated, critical point dried, and sputter coated (4). A Philips XL 20 scanning electron microscope was used to view the colonies. PCR, cloning, and DNA sequencing. Specimens stored in 96% ethanol were transferred to PBS, followed by short pulses of vortexing to mechanically disin- tegrate the large Z. niveum colonies; 5 ␮l of this PBS solution containing a mix of host tissues and symbionts was directly used as the template for PCR without prior DNA extraction. PCRs were performed in a temperature gradient ther- mocycler with a standard PCR cycling program and an annealing temperature of 55°C. A typical PCR mixture contained 2 mM of MgCl2, 0.2 mM of deoxynucleo- side triphosphate, 1.5 U of Taq DNA polymerase (Promega GmbH, Mannheim, Germany), 50 pM of forward and reverse primer, and 5 ␮loftheZ. niveum mix in a total volume of 50 ␮l. The forward primer 616V (5Ј-AGA GTT TGA TYM TGG CTC-3Ј) (33) targeting the 5Ј end of the 16S rRNA gene of all bacteria was used in combi- nation with the unlabeled probe GAM42a (5Ј-GCC TTC CCA CAT CGT TT-3Ј) targeting the 23S rRNA of all Gammaproteobacteria at Escherichia coli position 1027 as the reverse primer (38). PCR products were separated by electrophoresis using a 2% agarose gel. Gels were stained with ethidium bromide and visualized by UV transillumination. Amplification products in the desired size (approximately 3,000 bp) were cut out and purified with GELase (Epicenter, Madison, WI). The TopoXL cloning kit (Invitrogen Life Technologies, Karlsruhe, Germany) was used for all cloning reactions. The nucleotide sequences of cloned DNA fragments were determined by cycle sequencing of purified plasmid DNA with the Thermo Sequenase cycle sequencing kit (Amersham Life Science, Little Chalfont, United FIG. 2. SEM of the Zoothamnium niveum symbiosis. (A) Z. niveum Kingdom) with dye-labeled, vector-specific primers using an automated DNA microzooid with bacterial coat, consisting of coccoid rods (co) on the sequencer (LI-COR 4200; LI-COR Inc., Lincoln, NE) under conditions recom- oral part of the microzooid, rods (ro) on the aboral part, and transi- mended by the manufacturers. tional stages of intermediate shapes (in) in between. (B) Basal part of Phylogenetic analysis and probe design. The nucleotide sequences obtained