Many bacteria use Type IV Secretion Systems (T4SSs) to aid in pathogenesis by translocating virulence factors across the cell envelope and into eukaryotic cells. These systems are structurally and functionally diverse, but are often compared to the archetypal VirB/VirD4 T4SS of Agrobacterium tumefaciens. This system is composed of the VirD4 type IV coupling protein (T4CP) and 11 VirB subunits (VirB1-11) that assemble as the secretion channel and an extracellular pilus. The T4CP is an inner membrane ATPase that interacts with T4SS substrates and the secretion channel, and is thought to link substrates with the secretion channel and possibly energize transfer through the channel lumen. In this thesis, I sought to adapt T4SSs in the surrogate hosts A. tumefaciens and Escherichia coli for use in identification of novel T4SS effector proteins from genetically-intractable Rickettsial species. I first constructed chimeric T4SSs in A. tumefaciens by substituting native VirD4 with Rickettsial VirD4 homologs. However, I was unable to demonstrate transfer of the promiscuous IncQ plasmid pML122 or known A. tumefaciens effector proteins. I next tested the E. coli pKM101-encoded T4SS, which is known to transfer DNA substrates, for the capacity to deliver heterologous protein substrates to E. coli recipients. Using the Cre-recombinase reporter assay for translocation (CRAfT), I showed that pKM101 translocates effector proteins from A. tumefaciens and two Rickettsial species, Anaplasma phagocytophilum and Wolbachia pipientis. I next created chimeric T4CPs by joining the transmembrane domain (TMD) of pKM101-encoded TraJ with the soluble domains (SDs) of VirD4 homologs from A. tumefaciens and the Rickettsial species. I showed that all of these chimeric systems translocate protein substrates, although less efficiently than the native pKM101 T4SS. Finally, I demonstrated that a variable C-terminal extension (CTE) that is present on the A. tumefaciens and Rickettsial T4CPs plays a modulatory role for secretion of different protein substrates. My findings showed for the first time that a T4SS encoded by an E. coli conjugative plasmid is capable of translocating a variety of protein substrates from phylogenetically diverse alphaproteobacterial species, including A. tumefaciens, A. phagocytophilum, and W. pipientis
