Akinori IGUCHI

Development of a new separation method for uncultured microorganisms using a phage display technique.

Akiyoshi OHASHI, Hideki HARADA

In order to isolate the yet-to-be cultured microorganisms in natural ecosystems, we developed a new separation method combined with 16S rRNA-based fluorescence in situ hybridization (FISH) and phage display technique.
The proposal technique is described as following 4 steps; 1) Detection of the yet-to-be cultured microbial cells in environmental samples using 16S rRNA-targeted FISH. 2) Collection of the detected microbial cells using fluorescence-activated cell sorter, 3) Selection of the specific peptides binding to the target cells in combinatorial phage display peptide library. 4) Separation and isolation of the targeted microbial cells in environments using the selected peptides.
In this study, we explored specific peptides tightly binding to a test organism Gemmatimonas aurantiaca (a fastidious microorganisms) in our phage display peptide libraries, and applied the peptide for separation of G. aurantiaca microbial cells from microbial community.
Through our screening, we found 53 types of the peptides (7-mer or 12-mer) likely binding to G. aurantiaca cells. Finally, we selected the peptide (WPHAPWGFSAFS) showing the highest specificity to G. aurantiaca based on the tyramide signal amplification (TSA) analysis. The selected peptide (WPHAPWGFSAFS) was chemically synthesized. Separation of G. aurantiaca cells was performed using the synthesized peptide coupled with magnetic beads. Consequently, The peptide-mediated magnetic separation allowed the separation of G. aurantiaca cells efficiently.
These results suggest that the proposal technique can be the effective method for cultivation of uncultured microorganisms in natural ecosystems.