Illuminating protein interactions in the periplasm

Novel antibiotics are urgently needed to counteract the rise of resistant pathogens. Antibiotic regimens can fail not only due to the presence of specific antibiotic-resistance associated genes, but also due to pathogen escape niches. Fundamental research on how bacteria grow and divide is crucial for a better understanding of the problem and the development of solutions. Gram-negative bacteria are a special concern due to the emergence of strains resistant to a large number of antibiotics. As much as 30% of proteins from Gram-negative bacteria reside in the space between their inner- and outer-membrane termed the periplasm, including cell wall synthesis proteins, which are a major target of antibiotics. Due to cellular toxicity and protein folding issues, most fluorescent proteins do not function in the periplasm, impairing detailed analysis of the function and interaction partners of periplasmic proteins in live cells. This thesis describes the development and validation of the first assay to determine in vivo periplasmic protein interactions using Förster Resonance Energy Transfer. We also developed a novel fluorescent protein with increased periplasmic stability and reduced cellular toxicity that greatly improved our protein-protein interaction assay. Employing these assays, together with biochemical and molecular biology techniques, we established the interactions and elucidated aspects of the function of several essential cell division proteins. We also showed that ß-lactam inhibited proteins change their conformation and linked this to their enzymatic activity. Finally, we used the acquired knowledge to test several lead molecules on their ability to inhibit cell division proteins