Characterizing the regulation of cell envelope biosynthesis in Pseudomonas aeruginosa - Project Summary/Abstract The bacterial cell envelope is a multi-layered structure that performs a variety of critical functions such as providing protection from physical and chemical insults, including antibiotics. The cell envelope is essential to viability but how cell envelope biogenesis is regulated is poorly understood. Gram-negative bacteria are characterized by a cell envelope with three layers: an inner membrane composed of glycerophospholipids (GPLs), a cell wall made up of peptidoglycan (PG), and an asymmetric outer membrane in which the inner leaflet is comprised of GPLs and the outer leaflet is enriched in lipopolysaccharide (LPS). The biosynthetic pathways responsible for the production of LPS, PG, and GPLs rely on shared precursor pools. Specifically, LPS and GPLs both require acyl-acyl carrier proteins and LPS and PG both require UDP-N-acetylglucosamine. Thus, although it is important that enough LPS, PG, and GPLs are produced to support growth, it is critical that each biosynthetic pathway is tightly controlled to prevent runaway flux that could deplete the shared precursor and indirectly inhibit the production of another essential cell envelope component. Despite the importance of balanced cell envelope biosynthesis, little is known about the regulatory systems that control cell envelope biogenesis outside of classical model systems. Work in my laboratory is focused on identifying and characterizing the various processes that regulate cell envelope biosynthesis in Gram-negative bacteria, with a particular focus on the opportunistic pathogen Pseudomonas aeruginosa. I previously found that, in P. aeruginosa, the LPS and PG biosynthetic pathways are coordinated through a regulatory interaction between their committed enzymes, LpxC and MurA, respectively. Genetically uncoupling LPS and PG biogenesis resulted in measurable phenotypic changes such as loss of viability and alterations to cellular morphology, highlighting the importance of maintaining balanced cell envelope biosynthesis. Current studies in my laboratory have identified additional factors that appear to influence the equilibrium between LPS and PG production, indicating that the regulation of these pathways is multi-faceted. Over the next five years, my group will seek to further clarify the regulation of cell envelope biogenesis in P. aeruginosa using a combination of genetics, biochemistry, cell biology. The goals of this proposal are to (i) dissect the molecular basis of LPS and PG coordination along with the physiological consequences of uncoupling the two pathways, (ii) characterize two novel regulators of cell envelope biogenesis, and (iii) define the array of factors that control LPS, PG, and GPL production in P. aeruginosa. In addition to laying the groundwork for future therapeutic development in an organism infamous for antibiotic resistance, this work has the potential to provide insight into fundamental principles that govern bacterial physiology and cell envelope biosynthesis.
