Overcoming the barriers that the cell envelope and efflux pumps provide to Gram-negative bacteria is a major bottleneck in the discovery and development of new antibiotics. To respond to this Call we are extending our current network on antibiotic translocation and propose an ambitious project ranging from identification of novel resistance mechanisms in clinical bacterial isolates to crystallization of molecular resistance components and all-atom modeling to determine the kinetics of permeation. Our particular strength lies in the identification and quantification of transport proteins involved in membrane permeation at the cellular level combined with our expertise in quantifying the rate limiting steps of translocation at atomic level. Based on our experience we intend to:1. develop multiple assays to determine intracellular concentrations of small molecules2. increase the overall structural knowledge of outer membrane proteins3. extend knowledge on active-uptake transport systems, such as iron transport4. identify novel targets whose modulation can alter the penetration of other small molecules5. obtain as complete a picture as possible of small molecule penetration into and efflux out of bacterial cellsCombining the research question at different levels we expect to understand the complexity of membrane permeability. In particular, we are convinced that our molecular approach is able to provide a general template for a guided design of antibiotics to achieve high permeability. In addition, we will create a data base to allow the sharing of antibacterial R&D information on an unprecedented level. Also, via careful analysis of this information we intend to provide learnings as best practices that will lead to an increase in the overall efficiency of antibacterial R&D.