Gram-negative bacteria are a major concern for public health, particularly due to the rise of antibiotic resistance. It is important to understand the biology and permeability of the outer membrane of these bacteria in order to increase the efficacy of antibiotics that have difficulty penetrating this structure. Here, we studied the genetic interactions of a subset of outer membrane-related gene deletions in the model Gram-negative bacterium
E. coli. We systematically combined these mutants with 3,985 nonessential gene and small RNA deletion mutations in the genome. We examined the viability of these double-deletion strains and probed their permeability characteristics using two antibiotics that have difficulty crossing the outer membrane barrier. An understanding of the genetic basis for outer membrane integrity can assist in the development of new antibiotics with favorable permeability properties and the discovery of compounds capable of increasing outer membrane permeability to enhance the activity of existing antibiotics.