How Bugs Bag Plastic

Commercial interest in polyhydroxyalkanoates--a diverse family of environmentally friendly, biodegradable polyesters harvested from bacteria--is booming as firms look for an alternative to petroleum-based plastics. But how bacteria fill their cells with sacks of these water-insoluble polymers is still not well understood. At the American Chemical Society national meeting last month, chemistry professor JoAnne Stubbe of Massachusetts Institute of Technology revealed a few of the bacteria's tricks.

Many bacteria make such polyesters to store energy and carbon for a rainy day, just as glucose is stored as glycogen in humans and as starch in plants. Depending on the bacteria and the carbon source that's available, the type of polyhydroxyalkanoate produced can range from stiff and brittle plastics to rubbery polymers. The most common of these, polyhydroxybutyrate (PHB), is made from ß-hydroxybutyrate building blocks and can reach molecular weights of nearly 1 million daltons.

Bacteria make PHB and other polyesters the same way nature makes starch: by stringing together soluble monomers and storing the finished polymer product in water-insoluble granules. When needed, the polymer in these granules--which, in the case of PHB, can take up to a whopping 85% of the cell's dry weight--can be broken down quickly and the building blocks reused for energetic or synthetic purposes.

How nature makes, stores, and reuses PHB, starch, and related biopolymers "is an interesting--and quite common--unsolved problem in biology," Stubbe said at a symposium, sponsored by the Division of Biological Chemistry, in honor of her receipt of this year's Repligen Award for the chemistry of biological processes. "PHB could serve as the paradigm for these processes in general," she added.

Stubbe, who has been studying PHB in collaboration with MIT biologist Anthony J. Sinskey for more than a decade, noted that an enzyme known as PHB synthase makes PHB from ß-hydroxybutyrate monomers that have been activated by attachment to coenzyme A. When the normally soluble synthase enzyme is given the go-ahead to produce PHB, it somehow converts into an amphiphilic enzyme capable of doing interfacial catalysis. The polymer that the synthase produces is stored in protein-lined granules.

NOBODY KNOWS exactly how PHB granules form, Stubbe pointed out. A number of scientists have suggested that synthase enzymes might cluster together into micellelike structures with their growing PHB chains in the center, protected from the cytosol. The synthases could continue to string together monomers, pumping the PHB polymer into the central cavity to form the granule.

Stubbe recently offered another possible mechanism for granule formation. She proposed that the granules might have their origins in the lipid membrane: Perhaps synthases gather near the cell membrane's inner surface, where they can obtain soluble monomers from the cytoplasm and extrude the PHB polymer into the waxy lipid membrane. When a sufficiently large mass of polymer is produced, it could pinch off from the lipid membrane to form the granule. Indeed, previous labeling studies had placed the synthases on the surface of the granules, and electron microscopy (EM) studies had suggested that a single layer of lipid might surround the PHB granules, Stubbe noted.

But at the ACS meeting, Stubbe presented new electron microscopy results suggesting that neither of these models is correct. She and graduate student Jiamin Tian took EM images of bacteria that have just started to form small granules. "We expected that the granules would be near the plasma membrane," Stubbe said. Instead, the nascent granules are located on some kind of dark-stained scaffold in the middle of the cell. By examining thousands of different cells and watching the changes in cells over time, she and Tian concluded that each mysterious scaffold carries a few granules that grow bigger at about the same rate. They hope to figure out what the scaffolds are made of by isolating them and by using transcription profiling to check for increased lipid production.

A better understanding of PHB homeostasis is essential for successful metabolic engineering of plastics, Stubbe argued. A number of companies are on the verge of introducing commercial products made from bacterially produced PHB and related polyhydroxyalkanoates. In addition, some firms are trying to produce the polymers in plants. A clearer picture of how PHB is produced and packaged in bacteria could help those efforts, she said.