Genome sequence of the sulfur-oxidizing Bathymodiolus thermophilus gill endosymbiont

Abstract

Bathymodiolus thermophilus, a mytilid mussel inhabiting the deep-sea hydrothermal vents of the East Pacific Rise, lives in symbiosis with chemosynthetic Gammaproteobacteria within its gills. The intracellular symbiont population synthesizes nutrients for the bivalve host using the reduced sulfur compounds emanating from the vents as energy source. As the symbiont is uncultured, comprehensive and detailed insights into its metabolism and its interactions with the host can only be obtained from culture-independent approaches such as genomics and proteomics. In this study, we report the first draft genome sequence of the sulfur-oxidizing symbiont of B. thermophilus, here tentatively named Candidatus Thioglobus thermophilus. The draft genome (3.1 Mb) harbors 3045 protein-coding genes. It revealed pathways for the use of sulfide and thiosulfate as energy sources and encodes the Calvin-Benson-Bassham cycle for CO₂ fixation. Enzymes required for the synthesis of the tricarboxylic acid cycle intermediates oxaloacetate and succinate were absent, suggesting that these intermediates may be substituted by metabolites from external sources. We also detected a repertoire of genes associated with cell surface adhesion, bacteriotoxicity and phage immunity, which may perform symbiosis-specific roles in the B. thermophilus symbiosis.

Keywords: Autotrophy; Hydrothermal vents; Marine invertebrate symbiosis; Thiotrophy; Uncultured endosymbiont.

Fig. 1

Transmission electron micrographs of Candidatus Thioglobus thermophilus. B. thermophilus gill tissue was homogenized in a glass tissue grinder and subjected to crude density gradient centrifugation using Histodenz™ gradient medium. Subsamples were taken from two visible bands and fixed for electron microscopy (a and b). Both subsamples contained numerous free symbiont cells (S) as well as some intact host vacuoles (V) containing several symbiont cells, besides various other cellular components and host tissue debris. L: Lipid drop or mucus. Scale bar: 5 μm. Electron microscopy method details: samples were fixed in a) 1% glutaraldehyde, 2% paraformaldehyde in IBS (imidazole-buffered saline; 0.49 M NaCl, 30 mM MgSO₄*7H₂O, 11 mM CaCl₂*2H₂O, 3 mM KCl, 50 mM imidazole) and b) in 2.5% glutaraldehyde, 1.25% paraformaldehyde in IBS. After embedding in low-gelling agarose and postfixation in 1% osmium tetroxide in cacodylate buffer (0.1 M cacodylate; pH 7.0), samples were dehydrated in a graded ethanol series (30 to 100%) and embedded in a mixture of Epon and Spurr (1:2). Sections were cut on an ultramicrotome (Reichert Ultracut, Leica UK Ltd., Milton Keynes, UK), stained with 4% aqueous uranyl acetate for 5 min followed by lead citrate for 1 min and analyzed with a transmission electron microscope LEO 906 (Zeiss, Oberkochen, Germany)

Fig. 2

Phylogenetic tree of Candidatus Thioglobus thermophilus and related free-living and host-associated sulfur oxidizers. Ca. T. thermophilus, the thiotrophic symbiont of Bathymodiolus thermophilus, is displayed in bold. The tree was inferred from closely related 16S rRNA gene sequences obtained from the SILVA database using the SILVA Incremental Aligner (SINA) [51] and was estimated with the 16S rRNA sequence of 46 bacteria. The final alignment covered 1138 nucleotides. Sequence alignment and phylogenetic analysis were performed using the MEGA7 software tool [52]. The phylogenetic tree was constructed using the Maximum Likelihood method based on the Tamura-Nei model implemented in MEGA7 [53]. Branch bootstrap support values were calculated using 1000 replicates and are displayed as circles (black: ≥ 90%, white: ≥ 60%). For the sake of clarity some organisms were merged into groups (wedges): ^auncultured clones (KC682721, KC682765, JQ678401, AB193934); ^bwhale fall symbionts (HE814589, HE814588, HE814591 HE814585); ^cuncultured clones (FM246509, FM246513); ^duncultured clones (JQ678344, JQ678392); ^eMytilidae symbionts (AM503921, AM503923); ^fVesicomyidae symbionts (EU403432, EU403431, CP000488* 1081274–1,082,807, AP009247* 948400–949,934); ^gLucinidae symbionts (X84979, M99448, M90415); ^htube worm symbionts (NZ_AFOC01000137* 503–2033, DQ660821, NZ_AFZB01000059* 4132–5662). The lucinid clam symbionts, the vestimentiferan tube worm symbionts, and the free-living Thiomicrospira crunogena XCL-2 were included as outgroup. Branches that are not highlighted by colors represent free-living relatives. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. *these NCBI accession numbers refer to whole genome submissions and not to individually submitted 16S rRNA gene sequences (start and stop positions of the 16S rRNA gene are given after the asterisk). JdFR: Juan de Fuca Ridge, EPR: East Pacific Rise, MAR: Mid-Atlantic Ridge, OMZ: oxygen minimum zone, MFZ: Mendocino Fracture Zone, SBB: Santa Barbara Basin, WH: Woods Hole