The gram-negative outer membrane (OM) is asymmetric, with phospholipids on the inner leaflet and lipopolysaccharide (LPS) on the outer leaflet. A defining part of the gram-negative cell envelope, LPS is a major signal for infection in animals and is a target of last resort antibiotics, such as colistin. Normally essential, clinical use of colistin has selected for Acinetobacter baumannii strains that do not produce LPS. Although LPS has been implicated in diverse functions, its precise role in gram-negative biology is unclear. Recent work in E. coli suggests LPS is capable of contributing as much rigidity to the cell envelope as the cell wall, a function that requires polyionic interactions between negative moieties on LPS and divalent cations. Analysis of conditional E. coli mutants defective in LPS synthesis and transport revealed formation of filaments and cell chains, suggesting an additional role for LPS in cell division and separation. The essentiality of LPS in either process is unclear. To evaluate the essential role(s) of LPS in gram-negative biology, I will leverage a set of E. coli mutants to conditionally knockdown LPS synthesis and transport, manipulate LPS charge (and thereby its interactions with divalent cations to provide rigidity), and produce a minimal LPS structure. Using this collection in Aim 1, I will systematically characterize the effect(s) of LPS defects on cell growth and morphology to understand the contribution of LPS to both phenomena. In Aim 2, I will test my hypothesis that providing cell envelope rigidity is a primary, essential function of LPS. Aim 2.1 will evaluate the ability of hyperosmotic conditions (which reduce the force exerted by turgor pressure on the cell envelope) to compensate for LPS defects. Because the force of turgor pressure is spread between the cell wall and the OM, Aim 2.2 will test whether increasing rigidity of the cell envelope via cell wall crosslinking can compensate for LPS defects. In Aim 3, I will identify the steps in cell division and separation impacted by defects in LPS synthesis and transport, respectively. This effort will illuminate the mechanistic basis of filamentation and chaining in LPS mutants. Examination of A. baumannii LPS deletion mutants identified a correlation between division and cell survival. I am thus particularly interested in testing whether enhancing division using complementary genetic strategies promotes growth of LPS deficient E. coli. If enhancing cell envelope rigidity is an essential role of LPS, additional septa may offset lethality associated with LPS defects through a positive impact on the structural integrity of the cell as a whole. A major signaling molecule for the immune system and target for last resort antibiotics, a better understanding of the essential role of LPS in gram-negative biology will provide insights into mitigation and treatment of gram- negative pathogens. Through this F31 fellowship, I will develop expertise in microscopy, prote...