Transformation of Diatoms

      Diatoms represent a ubiquitous group of photosynthetic eukaryotes that are most noted for their highly patterned silica shells or frustules. We have isolated and analyzed several genes from the diatom Phaeodactylum tricornutum that encode the fucoxanthin-chlorophyll binding light harvesting polypeptides (FCPs) (Apt and Grossman, 1996; Zaslavskaia et al., 2000).  The promoters and terminators of these genes were fused to genes such as sh ble, which encodes a protein that confers Zeocin resistance to the alga, and reporter genes such as cat, to generate diatom transformation vectors. Introduction of the vector into P. tricornutum makes the organism resistant to the antibiotic Zeocin.  The transformation vectors are being used to analyze biological processes in diatoms and more specifically, to determine mechanisms by which polypeptides are transported into plastids (Apt et al., 2002).  In addition, these vectors are becoming instrumental in engineering diatoms for increased production of commercially valuable metabolites and faster growth under specific environmental conditions.  For example, transformation technology has recently been employed to engineer a trophic conversion of P. tricornutum from an obligate photoautotroph to an organism that can grow heterotrophically in the dark (Zaslavskaia et al., 2001). This was accomplished by introducing a gene encoding a glucose transporter (GLUT1 from red blood cells) into the diatom cells. As shown in Figure 1, the transporter becomes localized to the plasma membrane, where it can function to efficiently transport glucose into the cells.

Figure 1. The top panels show light microscopy of wild type cells (top, left), fluorescence of the chlorophyll within the plastids (top, middle) and the low level green fluorescence in the cells (top, right).  When GFP is synthesized in the cells, most of the protein localizes in and around the nucleus (bottom, left).  However, when the GFP is fused to Glut1, most of the fluorescence is associated with the plasma membrane (bottom, right).

References

Apt, K.E. and A. R. Grossman (1996) Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol Gen Genet 252:572-579.

Apt, K. E., L. Zaslavkaia, C. Lippmeier, J. C. Lang, O. Kilian, R. Wetherbee, A. R. Grossman and P. G. Kroth (2002) In vivo characterization of the diatom mulitpartite plastid targeting signals. J Cell Sci 115: 4061-4069.

Zaslavskaia, L., J. C. Lippmeier, C. Shih, A. R. Grossman and K. E. Apt (2001) Trophic conversion of an obligate photoautotrophic alga through metabolic engineering. Science 292: 2073-2075.

Zaslavskaia, L. A., J. C. Lippmeier, P. G. Kroth, A. R. Grossman and K. A. Apt (2000) Transformation of the diatom Phaeodactylum tricornutum with a variety of selectable marker and reporter genes.  J Phycol 36:379-386.