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Contact dependent inhibition (CDI) systems facilitate antagonistic toxin exchange between closely-related bacteria in a proximity-dependent manner. CDI+ bacteria use the large (~300 kDa) cell surface protein CdiA to intoxicate their neighbors by binding to an outer membrane (OM) receptor. Receptor binding occurs at a central 200-300 aminoacids (RBD) and initiates cleavage and translocation of the ~200 carboxy-terminal residues of CdiA (CdiA-CT). Delivery of CdiA-CT requires receptor proteins at each stage of membrane transport into the target cell. We have previously identified three OM receptors for CdiA in Enterobacteria: BamA, OmpC/F, and Tsx. Using the CDI locus from E. coli STEC_O31 as a model (cdiBCAI), in Chapter 2 I introduce a fourth class of CdiA (CdiA4) that exclusively binds to lipopolysaccharide to initiate CdiA-CT translocation. CdiA4 is also linked to an accessory gene, cdiC, which encodes a predicted lysine acyltransferase. Site-directed mutagenesis and HPLC-MS reveals CdiC and its close homologues form a subfamily of bacterial lysine acyltransferases within the toxin-activating acyltransferase (TAAT) family that modify their cognate CdiA proteins to promote LPS binding. In Chapter 3, I use this new CDI system to investigate the interaction between CdiA and O-antigen and find that O-antigen presents a barrier to toxin translocation at the outer membrane of target cells. Finally, in Ch. 4, I identify four new inner-membrane (IM) protein receptors that facilitate toxicity by two new members of the CDI ionophore family. I demonstrate that the native activity of these receptors is not required for CDI, but their presence in the IM is necessary for ionophore activity both from the periplasm or from the cytosol. Combined with a primary sequence analysis of these CdiA-CTs, the results in Chapter 4 offer a wealth of opportunities to explore the mechanism of CDI ionophore activity further. Together, the results of this work present a new physiological applica

Contact dependent inhibition (CDI) systems facilitate antagonistic toxin exchange between closely-related bacteria in a proximity-dependent manner. CDI+ bacteria use the large (~300 kDa) cell surface protein CdiA to intoxicate their neighbors by binding to an outer membrane (OM) receptor. Receptor binding occurs at a central 200-300 aminoacids (RBD) and initiates cleavage and translocation of the ~200 carboxy-terminal residues of CdiA (CdiA-CT). Delivery of CdiA-CT requires receptor proteins at each stage of membrane transport into the target cell. We have previously identified three OM receptors for CdiA in Enterobacteria: BamA, OmpC/F, and Tsx. Using the CDI locus from E. coli STEC_O31 as a model (cdiBCAI), in Chapter 2 I introduce a fourth class of CdiA (CdiA4) that exclusively binds to lipopolysaccharide to initiate CdiA-CT translocation. CdiA4 is also linked to an accessory gene, cdiC, which encodes a predicted lysine acyltransferase. Site-directed mutagenesis and HPLC-MS reveals CdiC and its close homologues form a subfamily of bacterial lysine acyltransferases within the toxin-activating acyltransferase (TAAT) family that modify their cognate CdiA proteins to promote LPS binding. In Chapter 3, I use this new CDI system to investigate the interaction between CdiA and O-antigen and find that O-antigen presents a barrier to toxin translocation at the outer membrane of target cells. Finally, in Ch. 4, I identify four new inner-membrane (IM) protein receptors that facilitate toxicity by two new members of the CDI ionophore family. I demonstrate that the native activity of these receptors is not required for CDI, but their presence in the IM is necessary for ionophore activity both from the periplasm or from the cytosol. Combined with a primary sequence analysis of these CdiA-CTs, the results in Chapter 4 offer a wealth of opportunities to explore the mechanism of CDI ionophore activity further. Together, the results of this work present a new physiological applica