The critical biological functions of membranes depend on their phospholipid composition. Consequently, bacteria maintain a nearly constant ratio of major membrane glycerophospholipids. Nevertheless, environmental signals can induce changes in the molar ratio of anionic to zwitterionic phospholipids, raising questions about the physiological roles of regulated changes in headgroup composition. The specific aims of this proposed research investigate whether regulated changes in phospholipid headgroup composition affect activities that involve macromolecular interactions with phospholipids in the Gram-negative pathogen Salmonella enterica using genetic and biochemical approaches. Aim 1 is to engineer Salmonella strains with adjustable phospholipid headgroup compositions. Aim 2 is to examine whether phospholipid composition affects the biochemical properties and sensing capabilities of PhoQ, an integral membrane sensor kinase that utilizes ligand-mediated interactions with anionic phospholipids to detect signals and to respond to low extracytoplasmic Mg2+, the presence of antimicrobial peptides, and acidic pH. Aim 3 is to investigate whether regulated changes in phospholipid composition mediate resistance to magainin 2, a cationic antimicrobial peptide that disrupts membranes to kill bacteria. Independent and parallel approaches will be adopted to address Aims 2 and 3: strains constructed in Aim 1 will be utilized for in vivo experiments, and reconstituted biochemical liposome systems will be utilized for in vitro experiments. Results from these proposed experiments will indicate whether regulated changes in phospholipid headgroup composition modulate the activities of an integral membrane sensor kinase or mediate resistance to a cationic antimicrobial peptide. Accomplishment of these aims will enable genetic investigations of alterations in phospholipid headgroup composition in a human pathogen and will illuminate novel physiological functions for an essential cellular component. PUBLIC HEALTH RELEVANCE: Biological membranes form barriers around cells, enclose compartments, and contain molecules that have essential functions for life. This proposal aims to understand whether changes in the composition of the inner membrane of the pathogenic bacterium Salmonella enterica, which causes gastroenteritis and typhoid fever in humans, affect the ability of Salmonella to adapt to new environments and to resist killing by antimicrobial peptides. Results from this proposed research will provide an understanding of the regulated cellular changes that enable Salmonella to prosper in different locales and to cause disease.