Two bacteria phylotypes are predominant in the Suiyo seamount hydrothermal plume

Abstract

Microbial diversity and populations in a hydrothermal plume that was present inside the caldera of the Suiyo Seamount, a submarine volcano on the Izu-Bonin Arc, were investigated by performing a phylogenetic analysis of the 16S rRNA gene and by using fluorescence in situ hybridization (FISH). Corresponding to transmissivity, an indicator of turbidity, the vertical total cell count as determined by 4',6'-diamidino-2-phenylindole (DAPI) staining varied from 5.6 x 10(4) to 1.1 x 10(5) cells ml(-1), and the apparent plume layer was assessed to be at a depth of 1,050 to 1,200 m inside the caldera and to contain 1.0 x 10(5) to 1.1 x 10(5) cells ml(-1). From microbial samples collected in the plume by an in situ filtration system, the following two major phylogenetic groups, which were closely related to sulfur-oxidizing microbes, were obtained: the SUP05 group belonging to the gamma subclass of the Proteobacteria (13 of 20 clones) and the SUP01 group belonging to the epsilon subclass of the Proteobacteria (5 of 20 clones). Specific oligonucleotide probes for these groups (SUP05-187 and SUP01-63) were designed and were used with various water samples obtained from the Suiyo Seamount. In the apparent plume layer, up to 66% of the total counts of microbial cells were estimated to be Bacteria cells that hybridized to EUB338, and few cells were identified by the archaeal probe ARCH915. Almost all Bacteria cells were hard to identify with the known group-specific probes, such as ALF19, GAM42a, and CF319, while 88 to 90% of the Bacteria cells hybridized with SUP05-187 and >98% of them were considered members of the SUP05 and SUP01 populations. In a low-temperature vent fluid emitted from a bivalve-colonized mound, the SUP05 cells accounted for >99% of the Bacteria cells, suggesting that a portion of the plume cells originated on the surface of the seafloor at a depth of about 1,380 m. From further analysis of cell morphology (i.e., cell size and cell elongation index) we inferred that the SUP05 cells were active in the plume layer at a depth of 1,050 to 1,200 m compared to the activity in a near-bottom layer, while many elongated cells were found between these layers. These findings suggest that the morphology and distribution of SUP05 cells have complex relationships with hydrothermal activities and water circulation. Although growth and production rates remain to be defined, we concluded that this Suiyo Seamount caldera has functioned as a natural continuous incubator for these two phylotypes of Bacteria in an aphotic deep-sea environment.

FIG. 1.

Location of Suiyo Seamount and sampling points. Seawater samples for microscopic analysis were collected at sites indicated by the triangle (28°34′31"N, 140°28′59"E) during the KR01-05 cruise and by the open circle (28°34′27"N, 140°38′61"E) during the NT01-09 cruise, while in situ filtration samples for microbial DNA extraction and phylogenetic analysis were obtained at the site indicated by the square (28°34′35"N, 140°38′64"E) during the KR-01-15 cruise.

FIG. 2.

Phylogenetic positions of microbial 16S rRNA gene clones obtained from the Suiyo Seamount hydrothermal plume at a depth of 1,200 m (i.e., SUP clones). In the SUP01 and SUP05 clone groups, the levels of sequence similarity were >99.7% for 1,467 and 1,446 bp, respectively. Bootstrap confidence values are expressed as percentages of 1,000 replications; the values at the nodes are the values that were greater than 50%. Bacillus subtilis was used as an outgroup. Scale bar = 0.01 nucleotide substitution per sequence position.

FIG. 3.

(A) Vertical profiles of cell numbers and transmissivity. Circles, total cell counts obtained with DAPI; squares, EUB338 probe-hybridized cell counts; triangles, SUP05-187 probe-hybridized cell counts; open symbols, KR-01-15 cruise; solid symbols, NT-01-09 cruise. (B) Vertical profiles of relative abundance of specific microbial cells. Circles, SUP05-187 and SUP01-63 counts in EUB338 counts; multiplication signs, EUB338 counts in DAPI counts; triangles, SUP05-187 counts in EUB338 counts; open symbols, KR-01-15 cruise; solid symbols, NT-01-09 cruise; symbols in circles, samples from low-temperature hydrothermal fluid emitted from a mound heavily colonized with bivalves.

FIG. 4.

Digital images of fluorescently labeled microbial cells. The samples are from different depths (1,163 m, 1,276 m, and 2 m above the seafloor) in the water column inside the Suiyo Seamount caldera and from low-temperature vent fluid from a bivalve colony. Total cells were stained with DAPI (upper panels), and specific cells were detected by FISH analysis (lower panels, which are synthesized digital images) in the same fields for the same samples. The colors indicate microbial cells that hybridized with both the EUB338 and SUP05-187 probes (yellow), with both the EUB338 and SUP01-63 probes (cyan), and with only the EUB338 probe (green). Bars = 10 μm.

FIG. 5.

(A) Vertical profiles of morphological features of SUP05 cells in the water column inside the caldera. Cell size (area) (circles) and the cell elongation index (ratio of the major axis length to the minor axis length of a cell) (triangle) were determined for samples from the KR-01-15 cruise (open symbols) and the NT-01-09 cruise (solid symbols). Each value is the average for more than 200 cells measured by digital imaging analysis. (B) Histograms of the cell elongation indices for SUP05 cells from a depth of 1,163 m, a depth of 1,327m, and the seafloor.