The influence of bacterial lipid composition variation on transporter function and folding.

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Bacterial species diversity is vast and the wide variety and distribution of lipids that reside in their membranes highlights this diversity. Lipids are hydrophobic chemicals that can vary in chain length, saturation, branching and head group charge; all of which result in changes in membrane thickness, net charge and fluidity. Lipid variation has can also be used as a way to classify and distinguish bacterial species from one another. Despite this knowledge, our understanding of how lipids influence the function of bacterial membranes remains poorly understood. ******A broad assortment of membrane proteins must function within diverse lipid membranes to perform essential functions for the cell. Membrane proteins such as transporters facilitate the movement of substances across these lipid barriers. How transporters fold and function in membranes composed of different lipid compositions is not well understood. ******The long-term objective of my research program is to examine the influence of bacterial lipid composition on transporter function and folding. To accomplish this, three short-term objectives will focus on the function of multi-ligand transporters found in the membranes of Gram-negative Escherichia coli and of Gram-positive Bacillus subtilis. Lipids that make up each species membrane differ significantly in fatty acid type, bilayer thickness and net charge. The unique differences of each species membrane are ideal to compare transporter function and folding in different lipid compositions by monitoring losses and gains in substrate transport that alter overall cell growth. ***Objective 1: Involves a comparison of natural lipid variation in E. coli and B. subtilis grown at different temperatures, pH and salinity; conditions which are known to alter lipid content and potentially regulate transporter folding and function. ***Objective 2: Develops an expression system in each species to deliberately vary the content of specific lipids and transporters by heterologous co-expression of particular lipid biosynthetic and transporter genes. The consequences of lipid variation will be monitored in vivo by observing changes in transporter folding and topology in bacterial membranes. ***Objective 3: Bioinformatic and statistical comparison of bacterial lipid compositions will be made with transporter protein sequence data to determine how lipid compositions influence amino acid conservation and evolution of regions that face lipids. ******The significance and impact of this program will provide insights into how lipids potentially regulate membrane protein activity and how growth conditions influence lipid content. Knowledge of how membrane proteins fold and function in diverse lipid environments not only furthers our fundamental knowledge of how these proteins work, but identifies better ways to bioengineer and isolate membrane proteins for industrial and academic applications. **