Delivery and activity of toxic effector domains from contact-dependent growth inhibition systems in Escherichia coli

Bacteria are ubiquitous in nature and have evolved a variety of communication and competition systems to survive in dense, complex environments. Contact-dependent growth inhibition (CDI) is a microbial competition system that is widespread throughout Gram-negative bacteria. CDI is mediated by the CdiB/CdiA two-partner secretion system, which displays the large CdiA exoprotein on the surface of CDI+ inhibitor cells. The C-terminal domain of CdiA (CdiA-CT) is toxic and inhibits cell growth after delivery into target bacteria. CDI+ cells are protected from auto-inhibition by expression of a cognate immunity protein (CdiI), which binds to the CdiA-CT and inactivates toxicity. CdiA-CT/CdiI pairs are highly divergent across species, indicating that CDI systems are capable of deploying a variety of toxins.This thesis explores several aspects of CDI, including delivery of CdiA-CTs into target cells, the toxic activities that lead to growth inhibition by CdiA-CT domains, and target cell stress responses that may influence CDI populations in natural environments. In Chapter I, we provide a general introduction to both diffusible and contact-dependent bacterial competition systems. This provides an overview of our current knowledge of CDI systems and also highlights key features of other secretion systems that contribute to population dynamics within bacterial communities. We then present a genetic study characterizing pathways of CdiA-CT translocation into target cells in Chapter II. In Chapter III, we focus on one protein, YciB, which is required for translocation of CdiA-CTo11EC869, a DNase toxin from E. coli, and examine the role of YciB in E. coli physiology outside of CDI. Chapter IV explores genetic responses that occur inside target cells after delivery of CdiA-CTo11EC869; in Chapter V, we present a study characterizing CdiA-CT/CdiI modules related to CdiA-CT/CdiIo11EC869. In Chapter VI, we discuss unpublished work examining the role of the translation factor EF-Tu as a co-factor required for activity by numerous CdiA-CT toxins. Chapter VII describes a collaborative project that utilized principle components of CDI systems as synthetic biology tools. Finally, we discuss research questions of significant interest in the field of CDI in Chapter VIII