Association of thioautotrophic bacteria with deep-sea sponges

Abstract

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.

Fig. 1

DGGE profiles of the V3 region of 16S rRNA genes of the sponges from the Sumisu Caldera (a) and the Gulf of Mexico (b), which were amplified by PCR with primers, GC-341F and 534r. a Characella sp. SC-S: O outside surface of the sponge, I inner core, T distal tip region, M mid region, B basal region. b Pachastrella sp. GM-BWS and Poecilosclerida sponge GM-SBS. One major band was found, respectively, in the Characella sp. SC-S and the Poecilosclerida sponge GM-SBS (arrowheads). Four major bands (a–d) were detected from the Pachastrella sp. GM-BWS. Accession numbers of the bands are listed in Table S1

Fig. 2

DGGE profiles of V3 region of bacterial 16S rRNA gene (135–164 bp without primers) amplified by PCR with primers, GC-341F and 534r, from the Characella sp. SC-S, tubeworms, and mussels collected in the Sumisu Caldera. Lanes B, T, and M, respectively, indicate the basal (outer surface), tip (outer surface), and middle (inner core) portions of the Characella sp. SC-S. Mobilities of the major bands from the different parts of the Characella sp. SC-S (band b on lanes B, T, and M) were the same, but differed from those of the four vestimentiferan tubeworm species (arrowheads on lanes 1 to 6) and from those of two mussels (B. septemdierum) individuals (bands a, b, and c on lanes Bsp6 and Bsp8). Three major bands were observed from mussels but sequence analysis showed that the middle band c was a mixture of sequences of a and b. A single major band was obtained from each lane of the sponge and of the vestimentiferan tubeworms [1 Escarpia sp., 2 Lamellibrachia sp. (first individual), 3 Alaysia sp. A1 (first individual), 4 Alaysia sp. A1 (second individual), 5 Alaysia sp. A5, 6 Lamellibrachia sp. (second individual)]. Arrowheads Thioautotrophic symbiotic bacteria-like sequences. Small letters a, b, and c signify excised bands (see Table S1). Accession numbers are shown in Table S1

Fig. 3

Phylogenetic (neighbor-joining) tree of the nearly full length of 16S rRNA gene sequences amplified by PCR and cloned from bacteria in Characella sp. SC-S, Poecilosclerida sponge GM-SBS and B. Septemdierum. Dotted line A clade containing thioautotrophic symbionts of bivalves including Bathymodiolus, Calyptogena, and Maorithyas. Accession numbers of the corresponding sequences are shown after the names of the sequences