Wzy connected the five-sugar chains, but it did so with no defined limit to the number of five-sugar units involved, a feature that does not match the natural process. On an actual bacterial cell wall, the length of the polysaccharide falls within a relatively narrow range of the number of chains connected.
So the scientists introduced another protein, called Wzz, to the mixture. This protein is known as a "chain length regulator." With this protein in the mix, the lengths of the resulting polysaccharides were confined to a much more narrow range.
"We were able to replicate the exact polysaccharide biosynthetic pathway in vitro, getting the correct lengths," Woodward said. "This is important because now you can begin to look at a whole host of other properties in the system."
The group already started trying to answer one compelling question: whether the two proteins, Wzy and Wzz, have to interact to fully achieve formation of the polysaccharide.
"We've shown in some preliminary results that they do interact, but we haven't determined whether that interaction has any functional relevance," Woodward said.
With this knowledge in hand, researchers now have access to information about how all three parts of the lipopolysaccharide, the large biomolecule on Gram-negative bacteria cell surfaces, is formed. One thing they already knew is that the entire process takes place on an inner membrane and is then exported to the outer membrane on the cell surface.
Now that scientists can reproduce formation of the lipopolysaccharide, they can more directly characterize the export process a step in the pathway that serves as another potential antibiotic target, Woodward noted.
|Contact: Robert Woodward|
Ohio State University