The increased frequency of antibiotic resistant bacterial isolates is of great concern for public health. It is currently estimated that infections caused by multidrug resistant pathogens will surpass cancer as a leading cause of death by 2050. Of particular concern are gram-negative bacteria, due to their robust intrinsic resistance provided by a low permeability outer membrane barrier in combination with active efflux. Active efflux is mainly mediated by Resistance-Nodulation cell Division (RND) transporters, which associate as tripartite complexes that facilitate the export of substrates across the outer membrane. These two mechanisms of resistance work in synergy to efficiently limit intracellular accumulation of antibiotics. This directly results in very low hit rates during antimicrobial screenings and presents unique challenges in the development of antibiotics. In addition to providing resistance, RND transporters are also involved in the physiology of bacteria. Pseudomonas aeruginosa possesses twelve RND transporters of which three are indicated to be involved in its quorum sensing networks. Overexpressing or deleting the genes of these transporters has a great physiological impact and can result in a loss of virulence or extended lag phases during growth. In many cases, the endogenous substrates of these transporters are not known and their impact on the physiology of the cell is not well understood. This dissertation focuses on the interplay between the low permeability outer membrane and active efflux in drug resistance and their contribution to physiology of P. aeruginosa. Current methods to investigate the contributions of the outer membrane barrier in drug resistance, like the use of polymyxins to permeabilize the outer membrane, have many disadvantages. Here, we developed a novel approach to separate the contributions of efflux and the outer membrane in antimicrobial susceptibility by introducing a genetically modified pore that non-selectively increases the permeability of the outer membrane. In combination with the removal of efflux transporters, this hyperporination approach highlights the synergy of the outer membrane and efflux, and has potential implications for the development of new antibiotics. It could provide the means to discover new rules in drug design that predict the uptake of a compound into the gram-negative cell according to its structural features. Furthermore, this dissertation will characterize the contributions of the RND transporter MexGHI-OpmD from P. aeruginosa in the physiology and antimicrobial resistance of the bacterium. Our results suggest that the transporter is involved in establishing the steady-state concentrations of the important virulence factor pyocyanin. In addition, we showed that the unusual fourth component MexG physically interacts with the transporter and that its presence results in inhibition of the efflux activity towards certain substrates. Our results suggest that the activity of RND transporters are masked by the presence of the outer membrane and that hyperporination highlights the actual transport efficiencies of these pumps. RND transporters heavily rely on the outer membrane barrier, and this hyperporination approach could be used to re-evaluate their substrate specificities.