Marine Microbial Discovery and Analysis
Written by Darcie Miller and Clem Fortman
In 2001, Prof. David Sherman and a team of researchers traveled to Papua New Guinea to search the Bismarck Sea for sponges known to produce bioactive metabolites. The sponge pictured, Jaspis splendens, produces jasplakinolide (jaspamide), a cyclic depsipeptide shown to bind actin. Actin is known to disrupt the cell cycle and activity related to its binding signifies potential anti-cancer activity. Field studies performed by scientists using J. splendens included cell separations for compound localization and DNA isolation.
The task of moving mere milliliters of solution from vial to vial is never an easy one, however, the team faced the added difficulty of performing these intricate experiments on a rocking boat. "This localization of jasplakinolide (or other metabolite) to a specific cell type is a useful piece of information in determining the biosynthetic origin of a compound," said Clem Fortman [pictured].
First, samples were collected from the Bismarck Sea and labeled as shown here. Prof. Sherman's team was fortunate to find several J. splendens samples on this expedition.
The team put this sponge through a cell dissociation process in order to purify different cell types by size. The first step is to cut up the sponge into small pieces to help release the cells from the sponge's internal matrix. It is necessary to mechanically liberate the individual cells from the whole organism.
Next, the chopped sponge is wrapped in permeable cheesecloth and squeezed into a solution of artificial seawater.
Artificial seawater is used instead of real seawater because sponge cells require divalent cations to aggregate. "The artificial seawater lacks calcium or magnesium. This helps the sponge cells to remain dissociated," reports Clem Fortman. When squeezed, remnants of the sponge can be seen passing through the cheesecloth and into the solution.
Next, the solution is put through a process to achieve cell suspension. The mixture is centrifuged to pellet it and is then re-suspended in a smaller volume of artificial seawater to obtain a higher concentration of cells. The resuspended cells are then placed on top of a step gradient of polyvinylpyrrolidine. The tubes are centrifuged resulting in the initial separation of cell types.
The top layers contain the unicellular bacteria and the smallest sponge cells. Filamentous bacteria (when present) and larger cells collect in the lower layers. This process allows for the separation of bacteria from sponge cells. Repeating this process with a finer density gradient can result in the purification of distinct cell types.
This process of enrichment is done in order to localize jasplakinolide to a specific cell type. The resulting cell fractions can be further enriched for either sponge cells or bacteria. This segregation is essential for metagenomic library building and screening conducted at our lab at the University of Michigan.