Representative Freshwater Bacterioplankton Isolated from Crater Lake, Oregon
Kathleen A. Page, Stephanie A. Connon, and Stephen J. Giovannoni
High-throughput culturing (HTC) methods that rely on dilution to extinction in very-low-nutrient media were used to obtain bacterial isolates from Crater Lake, Oregon. 16S rRNA sequence determination and phylogenetic reconstruction were used to determine the potential ecological significance of isolated bacteria, both in Crater Lake and globally. Fifty-five Crater Lake isolates yielded 16 different 16S rRNA gene sequences. Thirty of 55 (55%) Crater Lake isolates had 16S rRNA gene sequences with 97% or greater similarity to sequences recovered previously from Crater Lake 16S rRNA gene clone libraries. Furthermore, 36 of 55 (65%) Crater Lake isolates were found to be members of widely distributed freshwater groups. These results confirm that HTC is a significant improvement over traditional isolation techniques that tend to enrich for microorganisms that do not predominate in their environment and rarely correlate with 16S rRNA gene clone library sequences. Although all isolates were obtained under dark, heterotrophic growth conditions, 2 of the 16 different groups showed evidence of photosynthetic capability as assessed by the presence of puf operon sequences, suggesting that photoheterotrophy may be a significant process in this oligotrophic, freshwater habitat.
The use of rRNA-based molecular techniques has revolutionized the field of environmental microbiology in recent decades. Studies of microbial diversity are now dominated by analyses of environmental 16S rRNA by gene cloning, sequencing, and in situ hybridization. As a result of these studies, numerous abundant but uncultured microorganisms have been discovered (11, 12, 32, 41, 44, 53). Phylogenetic reconstruction based on 16S rRNA gene sequences has proven to be a powerful tool for organizing and understanding microbial diversity and has revealed many habitat-specific phylogenetic clusters. In many cases, few or no cultured representatives of some of the most numerically dominant phylogenetic clusters are available (18, 19). In particular, Zwart et al. (51) and Glockner et al. (12) have identified phylogenetic clusters of freshwater bacteria that are frequently found by cloning and sequencing 16S rRNA genes found in lakes around the world. Urbach et al. (44) identified frequently occurring bacterioplankton groups present in Crater Lake. Many of these bacterial groups remain uncultivated. An important goal of environmental microbiology is to understand the ecological roles of the most frequently occurring habitat-specific phylogenetic clusters. To do this, representative organisms from dominant phylogenetic clusters must be identified and cultivated. Studies that link microbial metabolism to environmental variables and biogeochemical cycles may then be initiated.