Reconstitution of bacterial lipopolysaccharide transport from purified components
Citation
Sherman, David Joseph. 2015. Reconstitution of bacterial lipopolysaccharide transport from purified components. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.Abstract
The surface of Gram-negative bacteria is largely composed of lipopolysaccharide (LPS), a complex glycolipid that contains multiple fatty acyl chains and up to hundreds of sugars. LPS is transported from its site of synthesis at the cytoplasmic inner membrane (IM), across an aqueous compartment (the periplasm), and through the outer membrane (OM) to the cell surface. LPS is essential for the survival of most Gram-negative bacteria. Seven essential and conserved lipopolysaccharide transport (Lpt) proteins in Escherichia coli are responsible for assembling LPS. These proteins form a continuous bridge from the IM to the OM, but it is unknown how they function in LPS transport.This dissertation describes the development of a reconstitution of LPS transport using purified Lpt proteins. The first two steps of this process are extraction of LPS from the IM by LptBFGC and transport along the periplasmic bridge comprising LptA. We obtained high-resolution crystal structures of the ATPase LptB, which taught us how to overexpress and purify LptBFGC as a stable complex that is highly active in proteoliposomes. Crystallographic snapshots of LptB bound to ATP and ADP provided insight into how energy is used to extract the fatty acyl chains of LPS from the IM. Using site-specific photocrosslinking, we monitored ATP-dependent extraction of LPS from proteoliposomes by LptC and release to LptA. This reconstitution of LptBFGC led to a hypothesis about a role for LptC in regulating the activity of the Lpt IM complex. We also developed methods to reconstitute the late steps of transport by monitoring LPS transit from LptA to the OM components, LptDE, contained in a different proteoliposome. One important observation from this reconstitution is that it appears as though the OM translocon can change activity depending on the amount of LPS in the OM. These studies provide us with the first clues as to how the cell might regulate LPS flux to the cell surface. This reconstitution will allow for mechanistic studies of LPS transport and will aid in the discovery and development of antibiotics targeting this essential process.
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