Summary Traditionally, studies of antibiotic resistance have focused on evolutionary and molecular mechanisms of resistance. However, many of our best antibiotics target the bacterial cell envelope, which is a mechanically robust, structural exoskeleton for the cell. Ultimately, these antibiotics cause cell death by weakening the envelope enough to cause explosion of the cell by the large, hydrostatic pressure within it. Despite the central mechanical importance of the cell envelope, we have little understanding of which molecules and moieties within it are critical for its load-bearing capacity. Addressing this question would transform our understanding of antibiotic resistance. A primary reason for this gap in our knowledge is the formidable challenge of applying mechanical forces to single bacterial cells while monitoring their physiology. The proposed research will address this obstacle by applying innovative, highly precise, high-throughput microfluidics and microscopy-based assays to measure the mechanical properties of two of the major cell envelope components in bacteria, the outer membrane and the cell wall. These assays will be combined with molecular and cell biological techniques, and biophysical theory, to explore an emerging paradigm within microbiology: that bacteria control antibiotic resistance by adaptively tuning the mechanical properties of their cell envelope. First, building on the recent landmark finding that the outer membrane confers antibiotic resistance to bacteria because of its mechanical strength, the dependence of outer membrane stiffness and mechanical antibiotic resistance on the fine-scale biochemical composition of the outer membrane will be systematically measured. Next, the mechanism of outer membrane vesiculation (a process underlying antibiotic resistance and pathogenesis) will be investigated by combining a theoretical mechanical model of vesiculation with novel microscopy assays to quantify vesiculation dynamics, while genetically tuning protein-protein interactions between the cell wall and outer membrane. Finally, the scope of these studies will be extended to Gram-positive bacteria by determining the dependence of antibiotic resistance on cell wall stiffness in these species, specifically focusing on the mechanical contributions of teichoic acids to resistance. Together, these studies will transform our understanding of bacterial pathogen survival and growth, and point to fresh strategies to circumvent antibiotic resistance and treat bacterial infections.