The robust barrier of the outer membrane is complemented by a seemingly thinner inner membrane that nonetheless poses significant resistance to antibiotic permeation and is a critical target for many AMPs. The inner membrane of Gramnegative bacteria resembles that of Gram-positive species, comprising mainly PE, PG, and CL. However, in Gram-negative bacteria, PE is the predominant lipid, followed by PG, with CL comprising approximately 5% and concentrated in polar and septal regions . Unlike Gram-positive bacteria with a significant number of aminoacylated PG, PG lipids in Gramnegative species are mostly unmodified. Gram-negative species also lack iso-and anteiso-branched acyl chains, though cyclopropanated and unsaturated chains are abundant. The PE:PG ratio in E. coli is around 4, with a higher ratio in the periplasmic leaflet and a lower one in the cytosolic leaflet . This composition indicates a greater abundance of PE in the Gram-negative inner membrane compared to the Gram-positive bacterial membrane. The preferential cytosolic orientation of PG lipids aligns with the "positive-inside" rule, where positively charged cytosolic loops of transmembrane proteins preferentially interact with PG or CL. As with Gram-positive bacteria, acyl chain composition in Gram-negative membranes is dynamic, changing throughout the life cycle. Cyclopropane modifications increase from early log to stationary phases, enhancing resistance to environmental stresses . This increase in cyclopropanated chains is accompanied by a decrease in unsaturated tails [241], leading to higher lipid ordering in the early log phase and a progressive membrane thinning during the stationary phase .