Abstract
Colicins are natural protein antibiotics deployed by Escherichia coli to kill closely related bacterial competitors - they act by delivering a toxic protein domain intracellularly. A mechanistic understanding of how colicins manipulate endogenous protein-protein interactions to gain cell entry may lead to the development of novel therapeutics against antibiotic-resistant bacteria. By combining in vivo, in vitro and in silico biophysical techniques, we recently discovered that promiscuous interactions occur between the outer membrane proteins (OMPs) BtuB and OmpF, which are the receptor and translocator proteins for colicin E9 (ColE9), respectively. These promiscuous protein-protein interactions may contribute to the observed self-association of OMPs to form outer membrane (OM) islands. Super-resolution imaging, i.e. stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM), has been used to reveal the size and surface distribution of colicin-receptor complexes in these OM islands. Using confocal fluorescence recovery after photo-bleaching (FRAP) microscopy, we have for the first time detected in vivo assembly of the translocon complex observed as tethering of the inner membrane protein TolA by the N-terminus of ColE9 in a BtuB and TolB dependent manner. These in vivo studies have demonstrated that delivery of the intrinsically unstructured N-terminus of ColE9 to the periplasm is sufficient to assemble a periplasm-spanning translocon complex and that this occurs while the colicin molecule remains receptor-bound at the extracellular surface. Work is underway to elucidate the role of trans-periplasmic energy transduction in the formation and maintenance of this translocon complex.
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