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Although the SAR11 clade of the Alphaproteobacteria represents the most abundant and ubiquitous bacterioplankton in the ocean, very few laboratories have successfully cultured SAR11 cells. All of the SAR11 strains isolated thus far have been retrieved from the Oregon coast and the Sargasso Sea. In this study, a modified dilution-to-extinction culturing with prolonged incubation at low temperature was applied in an effort to cultivate major bacterioplankton lineages in the East Sea, Western Pacific Ocean. Five to 10 cells were inoculated into each well of 48-well plates, followed by the incubation of the plates at 10 degrees C for 4, 8, 20, and 24 weeks. Among a total of 35 isolated strains, 18 strains assigned to the SAR11 clade were isolated after 8, 20, and 24 weeks of incubation, whereas no SAR11 cells were detected in the samples after 4 weeks of incubation. The SAR11 isolates, noticeably, comprised 64-82% of the total isolates from the plates incubated for 20 and 24 weeks. Extinction cultures belonging to the Roseobacter, OM43, and SAR92 clades were also cultivated. The results of this study suggest that long-term incubation at low temperatures might prove an alternative for the efficient cultivation of new variants of the members of the SAR11 clade.

The introduction of high-throughput dilution-to-extinction culturing (HTC) of marine bacterioplankton using sterilized natural sea water as media yielded isolates of many abundant but previously uncultured marine bacterial clades. In early experiments, bacteria from the SAR11 cluster (class Alphaproteobacteria), which are presumed to be the most abundant prokaryotes on earth, were cultured. Although many additional attempts were made, no further strains of the SAR11 clade were obtained. Here, we describe improvements to the HTC technique, which led to the isolation of 17 new SAR11 strains from the Oregon coast and the Sargasso Sea, accounting for 28% and 31% of all isolates in these experiments. Phylogenetic analysis of the internal transcribed spacer (ITS) region showed that the isolates from the Oregon coast represent three different subclusters of SAR11, while isolates from the Sargasso Sea were more uniform and represented a single ITS cluster. A PCR assay proved the presence of proteorhodopsin (PR) in nearly all SAR11 isolates. Analysis of PR amino-acid sequences indicated that isolates from the Oregon coast were tuned to either green or blue light, while PRs from strains obtained from the Sargasso Sea were exclusively tuned to maximum absorbance in the blue. Interestingly, phylogenies based on PR and ITS did not correlate, suggesting lateral gene transfer. In addition to the new SAR11 strains, many novel strains belonging to clusters of previously uncultured or undescribed species of different bacterial phyla, including the first strain of the highly abundant alphaproteobacterial SAR116 clade, were isolated using the modified methods.

The alpha-proteobacterial lineage that contains SAR11 and related ribosomal RNA gene clones was among the first groups of organisms to be identified when cultivation-independent approaches based on rRNA gene cloning and sequencing were applied to survey microbial diversity in natural ecosystems. This group accounts for 26% of all ribosomal RNA genes that have been identified in sea water and has been found in nearly every pelagic marine bacterioplankton community studied by these methods. The SAR11 clade represents a pervasive problem in microbiology: despite its ubiquity, it has defied cultivation efforts. Genetic evidence suggests that diverse uncultivated microbial taxa dominate most natural ecosystems, which has prompted widespread efforts to elucidate the geochemical activities of these organisms without the benefit of cultures for study. Here we report the isolation of representatives of the SAR11 clade. Eighteen cultures were initially obtained by means of high-throughput procedures for isolating cell cultures through the dilution of natural microbial communities into very low nutrient media. Eleven of these cultures have been successfully passaged and cryopreserved for future study. The volume of these cells, about 0.01 micro m(3), places them among the smallest free-living cells in culture.