Isolation of Hyperthermophilic Archaea Previously Detected by Sequencing rDNA Directly from the Environment

Abstract

Hydrothermal systems are an exciting source of organisms (Blöchl et al., 1995; Stetter, 1992). To date, an unexpectedly large number of unique phenotypes, almost exclusively Archaea, have been isolated from biotypes at a temperature above 85°C, and it seems very likely that the limitations of the conventional enrichment culturing methods have prevented us from assessing the true extent of this number. The first indication of the real variety of Archaea living in hydrothermal ecosystems was given by analyses of 16S rDNA sequences obtained directly without previous cultivation from samples from the Obsidian Pool, a hot spring at Yellowstone National Park. A large number of archaeal sequences could be detected, all of which differed from those of known species (Barns et al., 1994a,b). Knowing the 16S rRNA sequences permits designing specific oligonucleotide probes that can be used in whole cell hybridization experiments (Amann et al., 1990b; Burggraf et al., 1994; DeLong et al., 1989; Stahl and Amann, 1991). This method allows determining the morpho-type of an organism that corresponds to a new sequence, but the examination of its physiological and biochemical properties still requires a growing culture. For more than a century, Koch’s plating technique was the fundamental means to obtain pure cultures of microorganisms (Koch, 1881). However, by this technique, only a small percentage of organisms that form colonies can be obtained in pure culture. Serial dilutions, another isolation technique, works only for organisms that are predominant within environmental samples. A revolutionary new approach is the use of a strongly focused infrared laser beam (“optical tweezers”) to separate single microorganisms (Ashkin and Dziedzic, 1987; Ashkin et al., 1987; Huber et al., 1995).