GNPS - Induced Systemic Effects of Engineering the Cell Membrane Composition in Bacillus subtilis

Engineering bacterial cells to contain defined fatty acid compositions within their cell membranes establishes an in vivo experimental platform for membrane biophysics. Yet before fully realizing the potential of this system, it is prudent to understand the systemic changes induced by the labeling procedure itself. In this work, the systemic induced changes in cell are studied via analysis of cellular membrane compositions and shotgun proteomic analysis to compare expression levels of selected cellular proteins; in response to the labeling of Bacillus subtilis using cerulenin to inhibit fatty acid synthesis and subsequently providing selected fatty acids exogenously to be incorporated by the cell. Changes in the expression of enzymes involved in fatty acid biosynthesis and degradation are observed and consistent with expectations. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phophatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Beyond this, changes to cell wall enzymes and isoprenoid lipid production are also seen, which may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down regulated in the labeled cells, as was the actin-like protein mreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored between an enforced cell membrane fatty acid composition and lipid headgroup and raft cytoskeletal associated proteins.