UC Santa Barbara

Bacteria occupy nearly every environmental niche on Earth, including extremities ofsalinity, temperature, pressure, and pH (1). In each of these settings, they share space and resources with other organisms and form a variety of symbiotic relationships (2). In some cases, bacteria have evolved to deliver toxic effectors into the environment (3), or directly into nearby organisms (4,5) to provide themselves with a growth advantage over their neighbors. A number of secretion systems have been identified which allow bacteria to translocate effectors across their membranes, as well as the membranes of targeted organisms (6). In Gram-negative bacteria, contact-dependent Type Vb secretion systems (T5SS) recognize specific surface epitopes on neighboring bacteria and deliver toxic proteins into their cytosol (7–9). Sibling cells are protected from intoxication by expression of an immunity protein which binds and inactivates cognate toxic effectors (10,11). This process, termed contact-dependent growth inhibition (CDI) is associated with kin recognition (8,12), genetic stability (13), biofilm formation (14,15) and pathogenesis (16). The secretory pathway of CDI systems across the membranes of toxinproducing cells appears highly conserved (17). However, the mechanisms underlying target cell recognition, traversal across target cell membranes, as well as the processes by which toxins exhibit their toxicity, are highly variable and in some cases not well understood (8,11,18,19). In chapter 1, I review the arsenal of systems used by Gram-negative bacteria to translocate proteins across their membranes, with special focus on Type Vb CDI systems. In chapter 2, I identify novel inner membrane proteins co-opted by the cytoplasm-entry domains of CDI systems. Additionally, I find that some unique entry domains the recognize same inner membrane proteins, suggesting differences in recognized epitopes. In chapters 3 and 4, I investigate potential growth inhibition mechanisms of two protein effectors found in CDI systems from Enterobacter cloacae. First, I cover an effector domain from E. cloacae S611 with homology to SAM-binding domains and propose that it could act as a toxic methyltransferase. Then, I investigate an effector domain from E. cloacae UCI49 with predicted homology to glutaminases and cysteine proteases, where I identify potential protein targets via mass spectrometry. Finally, in chapter 5 I discuss potential future directions in the field of CDI.