Manipulating undecaprenyl phosphate levels to decipher mechanisms of competing cell envelope assembly pathways in Escherichia coli

PROJECT SUMMARY Bacteria are enveloped in sugary layers that maintain cell shape, protect against osmotic pressure, and resist environmental hazards. In Gram-negative bacteria, the cell envelope surrounds the cytoplasm and includes a cytoplasmic (inner) membrane, a thin peptidoglycan (PG) cell wall, and an outer membrane. The cell envelope is essential and is therefore a prominent target for antibacterials and vaccines. However, bacteria continue to develop new ways to evade these life-saving compounds. Thus, it is imperative that we understand the mechanisms of cell envelope assembly, ultimately to design new modes of interference. One poorly understood aspect of assembly revolves around the essential lipid carrier undecaprenyl phosphate (Und-P). Bacterial surface glycans, including PG and other clinically important polysaccharides, are assembled on and transported across the cytoplasmic membrane by Und-P. While decreasing the pool of Und-P induces cells to grow poorly and die, little to no effort has been applied to understand what effects occur when the pool of Und-P increases. Therefore, we will manipulate pathways that create Und-P to determine how maximizing the pool of Und-P affects Und-P-dependent processes. Similarly, pathways compete for a common pool of Und-P, but no one knows how this is done. Since such information is fundamental to disrupting the relationships among Und-P-utilizing pathways, we will manipulate pathways that use Und-P. Another poorly understood aspect of Und-P metabolism surrounds its utilization. Und-P inhibitors disrupt Und-P-dependent polymer formation, but how cells prioritize pathways when Und-P becomes limiting is not known. To that end, we will characterize Und-P pathways (including a potential new pathway) in Escherichia coli cells disrupted for Und-P metabolism. Finally, we will continue to pursue results from a genetic screen in a mutant defective for Und-P recycling, which has uncovered new and unexpected connections to Und-P metabolism that we propose to characterize in detail. In summary, results from this study will enable us to understand, in greater detail, the mysteries surrounding Und-P utilization and cell envelope assembly. At the same time, the tools and knowledge developed in the course of this work promise to provide signal benefits for those working on the design of antimicrobial therapies.