Molecular Diversity of New Thermococcales Isolates from a Single Area of Hydrothermal Deep-Sea Vents as Revealed by Randomly Amplified Polymorphic DNA Fingerprinting and 16S rRNA Gene Sequence Analysis

Abstract

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.All hyperthermophiles that thrive at temperatures above 95°C belong to the domain Archaea (19, 45). These extremophiles have been extensively studied during the last two decades for both academic and biotechnological reasons (16,34,40). Many type species that belong to new genera, orders, and families in the kingdoms Crenarchaea and Euryarchaea have been described. Some of these organisms, members of the order Thermococcales (genera Thermococcus, Pyrococcus, and Palaeococcus) (15,17,47), have been widely used as model organisms for biochemical or physiological studies (1). Members of the Thermococcales are anaerobic heterotrophs that grow at temperatures between 70 and 105°C, depending on the species, and their optimal growth temperatures range from 80 to 100°C. The genomes of three Pyrococcus species (Pyrococcus horikoshii, Pyrococcus abyssi, and Pyrococcus furiosus) have been completely sequenced (9,20,30), which allowed the first comparative studies of the genomes of several closely related species in the archaeal domain (22,48). Two plasmids from Pyrococcus species have been characterized (13, 42). Plasmid pGT5 from P. abyssi strain GE5T was used as a reporter extrachromosomal element to study DNA topology and DNA replication (7,26).Many type species of Thermococcales were isolated from either shallow-water hydrothermal regions or deep-sea hydrothermal vents, and in one case an organism was isolated from a terrestrial hot spring; 19 species, including 15 Thermococcus species and 4 Pyrococcus spec...

Metabolic stratification driven by surface and subsurface interactions in a terrestrial mud volcano

“…(Sorokin et al, 2002) were also detected in the sediments (Figure 4). Despite variations in sediment community assemblage, sequences affiliated with thermophilic Thermococcus mexicalis (Lepage et al, 2004), Methanosaeta thermophila (Kamagata et al, 1992), Pelotomaculum thermopropionicum (Imachi et al, 2002) and Thermodesulfovibrio yellowstoni (Henry et al, 1994) were either exclusively present or more abundant in the bubbling fluid.…”

Thermococcus nautili sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal deep-sea vent

“…Strain 30-1 T was purified by isolation (three times) in ZB medium solidified with 8 % Gelrite gellan gum (Sigma-Aldrich) incubated in anaerobic jars under a CO 2 (50 kPa)/H 2 (20 kPa) atmosphere for 48 h at 80 u C. Single colonies were picked up, suspended in 0.5 ml of reduced ZB medium and transferred into fresh liquid medium for 24 h at 90 uC. The same conditions (described by Lepage et al, 2004) were used for the routine cultivation of strain 30-1 T .…”

“…The 16S rRNA gene sequence was amplified by PCR as described by Lepage et al (2004). This sequence (1440 bp) was first compared with those in available databases by using the BLAST program (Altschul et al, 1997).…”

Biodiversity and Emerging Biogeography of the Neutrophilic Iron-Oxidizing Zetaproteobacteria

“…Though perhaps highly simplified, this statement offers a good null hypothesis for microbial biogeography: that only the modern effects of environmental parameters and not historical events such as dispersal or past habitat characteristics play a significant role in the current distribution of microorganisms. Although some studies have supported this hypothesis (23,41), many studies have been able to detect a nonrandom distribution of the microbe under investigation (e.g., see reference 5), with some also showing geographically significant distribution patterns with little correspondence to observed environmental parameters (52,70). Ultimately, the ability to detect the presence of extant biogeography has been shown to be dependent upon the spatial scale studied and the resolution of the selected molecular method (5,31,52).…”