A nonconserved carboxy-terminal segment of GroEL contributes to reaction temperature.

The role of the C-terminal segment of the GroEL equatorial domain was analyzed. To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another. In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera. Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 degrees C, but also at 10 degrees C, a temperature at which wild-type E. coli GroE is inactive. In a detailed look at the temperature dependence of the GroELs, normal E. coli GroEL and the L524I mutant became highly active above 14 degrees C and 12 degrees C respectively. Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding. These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL. Moreover, E. coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.

Oceanithermus desulfurans sp. nov., a novel thermophilic, sulfur-reducing bacterium isolated from a sulfide chimney in Suiyo Seamount.

A novel thermophilic, microaerophilic, sulfur-reducing bacterium designated strain St55BT was isolated from a sulfide chimney in the hydrothermal field of Suiyo Seamount (Izu-Bonin Arc, Western Pacific). Cells of the isolate were rod-shaped and tended to form a chain-link circular structure (a rotund body) at exponential phase under good growth conditions. The isolate was a chemoheterotroph requiring yeast extract for growth. Although strain St55BT used oxygen as an electron acceptor, it could not form colonies in an oxygen concentration of more than 5 % (v/v). The isolate also used nitrate, nitrite or elemental sulfur in the absence of oxygen. A phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was closely related to Oceanithermus profundus, belonging to the phylum 'Deinococcus-Thermus' (sequence similarity 99.5 %). However, strain St55BT differed from O. profundus in terms of usage of electron donors, cellular fatty acid profile and DNA G + C content. In addition, a DNA-DNA hybridization test indicated low relatedness between the isolate and O. profundus. For the reasons given above, the name Oceanithermus desulfurans sp. nov. is proposed for strain St55BT (= NBRC 100063T = DSM 15757T).

Two bacteria phylotypes are predominant in the Suiyo seamount hydrothermal plume.

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.

Microbial diversity in hydrothermal surface to subsurface environments of Suiyo Seamount, Izu-Bonin Arc, using a catheter-type in situ growth chamber.

After excavation using a portable submarine driller near deep-sea hydrothermal vents in the Suiyo Seamount, Izu-Bonin Arc, microbial diversity was examined in samples collected from inside the boreholes using an in situ growth chamber called a vent catheter. This instrument, which we devised for this study, consists of a heat-tolerant pipe tipped with a titanium mesh entrapment capsule that is packed with sterilized inorganic porous grains, which serve as an adhesion substrate. After this instrument was deployed inside each of the boreholes, as well as a natural vent, for 3-10 days in the vicinity of hot vent fluids (maxima: 156-305 degrees C), DNA was extracted from the adhesion grains, 16S rDNA was amplified, and randomly selected clones were sequenced. In phylogenetic analysis of more than 120 clones, several novel phylotypes were detected within the epsilon-Proteobacteria, photosynthetic bacteria (PSB)-related alpha-Proteobacteria, and Euryarchaeota clusters. Members of epsilon-Proteobacteria were frequently encountered. Half of these were classified between two known groups, Corre's B and D. The other half of the clones were assigned to new groups, SSSV-BE1 and SSSV-BE2 (Suiyo Seamount sub-vent origin, Bacteria domain, epsilon-Proteobacteria, groups 1 and 2). From this hydrothermal vent field, we detected a novel lineage within the PSB cluster, SSNV-BA1 (Suiyo Seamount natural vent origin, Bacteria domain, alpha-Proteobacteria, group 1), which is closely related to Rhodopila globiformis isolated from a hot spring. A number of archaeal clones were also detected from the borehole samples. These clones formed a novel monophyletic clade, SSSV-AE1 (Suiyo Seamount sub-vent origin, Archaea domain, Euryarchaeota, group 1), approximately between methanogenic hyperthermophilic members of Methanococcales and environmental clone members of DHVE Group II. Thus, this hydrothermal vent environment appears to be a noteworthy microbial and genetic resource. It is also noteworthy that some of the findings presented here were made possible by the application of the in situ growth chamber into the hot fluids deep inside the boreholes.